Abstract: Lactococcus lactis produces lactic acid and is widely used in the manufacturing of various fermented dairy products. However, the species is also frequently isolated from non-dairy niches, such as fermented plant material. Recently, these non-dairy strains have gained increasing interest, as they have been described to possess flavour-forming activities that are rarely found in dairy isolates and have diverse metabolic properties. We performed an extensive whole-genome diversity analysis on 39 L. lactis strains, isolated from dairy and plant sources. Comparative genome hybridization analysis with multi-strain microarrays was used to assess presence or absence of genes and gene clusters in these strains, relative to all L. lactis sequences in public databases, whereby chromosomal and plasmid-encoded genes were computationally analysed separately. Nearly 3900 chromosomal orthologous groups (chrOGs) were defined on basis of four sequenced chromosomes of L. lactis strains (IL1403, KF147, SK11, MG1363). Of these, 1268 chrOGs are present in at least 35 strains and represent the presently known core genome of L. lactis, and 72 chrOGs appear to be unique for L. lactis. Nearly 600 and 400 chrOGs were found to be specific for either the subspecies lactis or subspecies cremoris respectively. Strain variability was found in presence or absence of gene clusters related to growth on plant substrates, such as genes involved in the consumption of arabinose, xylan, α-galactosides and galacturonate. Further niche-specific differences were found in gene clusters for exopolysaccharides biosynthesis, stress response (iron transport, osmotolerance) and bacterial defence mechanisms (nisin biosynthesis). Strain variability of functions encoded on known plasmids included proteolysis, lactose fermentation, citrate uptake, metal ion resistance and exopolysaccharides biosynthesis. The present study supports the view of L. lactis as a species with a very flexible genome.
Abstract: A common bacterial strategy to cope with stressful conditions is the activation of alternative sigma factors that control specific regulons enabling targeted responses. In the human pathogen Bacillus cereus, activation of the major stress-responsive sigma factor σ(B) is controlled by a signalling route that involves the multi-sensor hybrid histidine kinase RsbK. RsbK-type kinases are not restricted to the B. cereus group, but occur in a wide variety of other bacterial species, including members of the the low-GC Gram-positive genera Geobacillus and Paenibacillus as well as the high-GC actinobacteria. Genome context and protein sequence analyses of 118 RsbK homologues revealed extreme variability in N-terminal sensory as well as C-terminal regulatory domains and suggested that RsbK-type kinases are subject to complex fine-tuning systems, including sensitization and desensitization via methylation and demethylation within the helical domain preceding the H-box. The RsbK-mediated stress-responsive sigma factor activation mechanism that has evolved in B. cereus and the other species differs markedly from the extensively studied and highly conserved RsbRST-mediated σ(B) activation route found in Bacillus subtilis and other low-GC Gram-positive bacteria. Implications for future research on sigma factor control mechanisms are presented and current knowledge gaps are briefly discussed.
Abstract: A novel 51-kb conjugative transposon of Lactococcus lactis, designated Tn6098, encoding the capacity to utilize α-galactosides such as raffinose and stachyose, was identified and characterized. Alpha-galactosides are a dominant carbon source in many plant-derived foods. Most dairy lactococcus strains are unable to use α-galactosides as a growth substrate, yet many of these strains are known to have beneficial industrial traits. Conjugal transfer of Tn6098 was demonstrated from the plant-derived donor strain L. lactis KF147 to the recipient L. lactis NZ4501, a derivative of the dairy model strain L. lactis MG1363. The integration of Tn6098 into the genome of the recipient strain was confirmed by Illumina sequencing of the transconjugant L. lactis NIZO3921. The molecular structure of the integration site was confirmed by a PCR product spanning the insertion site. A 15-bp direct repeat sequence (TTATACCATAATTAC) is present on either side of Tn6098 in the chromosome of L. lactis KF147. One copy of this sequence is also present in the L. lactis MG1363 chromosome and represents the sole integration site. Phenotypic characterization of all strains showed that the transconjugant has not only acquired the ability to grow well in soy milk, a substrate rich in α-galactosides, but also has retained the flavor-forming capabilities of the recipient strain L. lactis MG1363. This study demonstrates how (induced) conjugation can be used to exploit the beneficial industrial traits of industrial dairy lactic acid bacteria in fermentation of plant-derived substrates.
Abstract: The "Harlingen" IS6110 restriction fragment length polymorphism (RFLP) cluster has linked over hundred tuberculosis cases in The Netherlands since 1993. Four Mycobacterium tuberculosis isolates that were epidemiologically linked to this cluster had different spoligotype patterns as well as slightly divergent IS6110 profiles, compared to the majority of the isolates. Sequencing of the direct repeat (DR) locus revealed sequence polymorphisms at the putative deletion sites. These deletion footprints provided evidence for independent deletions of the central region of the DR locus in three isolates, while the different genotype of the fourth isolate was explained by transmission. Our finding suggests that convergent deletions in the DR locus occur frequently. However deletion footprints are not suitable to detect convergent deletions in the DR because they seem to be exceptional. Deletion footprints in the DR have not been described earlier and we did not observe them in any public M. tuberculosis complex sequences. We conclude that preferential deletions in the DR locus of closely related strains are usually an unnoted event that interferes with clustering of closely related strains.
Abstract: The Beijing genotype family is an epidemiologically important sub-group of Mycobacterium tuberculosis. It has been suggested that the high frequency of the Beijing isolates in some areas could be explained by selective advantages. Some evidence suggests that the emerging and most frequently isolated "Typical Beijing" lineage has the ability to circumvent BCG-induced immunity. To investigate the phylogeny of the Beijing genotype of M. tuberculosis, the genome of six Beijing strains from three different countries was sequenced with next-generation sequencing. The phylogeny of these strains was established using single nucleotide polymorphisms (SNPs). The three Typical Beijing strains clustered very tightly in the Beijing phylogeny suggesting that Typical Beijing strains represent a monophyletic lineage and resulted from recent diversification. Typing of 150 M. tuberculosis strains with a subset of the SNPs and comparison of the IS6110 restriction-fragment length polymorphism (RFLP) patterns of these strains to a database of 1522 Beijing RFLP patterns revealed that about 80% of all Beijing strains belong to the Typical Beijing subclone, which indicates clonal expansion. To identify the genomic changes that are characteristic for all Typical Beijing strains and to reconstruct their most recent common ancestor, the presence of SNPs was assayed in other Beijing strains. We identified 51 SNPs that define the minimal set of polymorphisms for all Typical Beijing strains. Nonsynonymous polymorphisms in genes coding for the regulatory network were over-represented in this set of mutations. We suggest that alterations in the response to environmental signals may have enabled Typical Beijing strains to develop the emerging phenotype.
Abstract: The genus Lactobacillus includes a diverse group of bacteria consisting of many species that are associated with fermentations of plants, meat or milk. In addition, various lactobacilli are natural inhabitants of the intestinal tract of humans and other animals. Finally, several Lactobacillus strains are marketed as probiotics as their consumption can confer a health benefit to host. Presently, 154 Lactobacillus species are known and a growing fraction of these are subject to draft genome sequencing. However, complete genome sequences are needed to provide a platform for detailed genomic comparisons. Therefore, we selected a total of 20 genomes of various Lactobacillus strains for which complete genomic sequences have been reported. These genomes had sizes varying from 1.8 to 3.3 Mb and other characteristic features, such as G+C content that ranged from 33% to 51%. The Lactobacillus pan genome was found to consist of approximately 14 000 protein-encoding genes while all 20 genomes shared a total of 383 sets of orthologous genes that defined the Lactobacillus core genome (LCG). Based on advanced phylogeny of the proteins encoded by this LCG, we grouped the 20 strains into three main groups and defined core group genes present in all genomes of a single group, signature group genes shared in all genomes of one group but absent in all other Lactobacillus genomes, and Group-specific ORFans present in core group genes of one group and absent in all other complete genomes. The latter are of specific value in defining the different groups of genomes. The study provides a platform for present individual comparisons as well as future analysis of new Lactobacillus genomes.
Abstract: Lactic acid bacteria (LAB) are a group of gram-positive, lactic acid producing Firmicutes. They have been extensively used in food fermentations, including the production of various dairy products. The proteolytic system of LAB converts proteins to peptides and then to amino acids, which is essential for bacterial growth and also contributes significantly to flavor compounds as end-products. Recent developments in high-throughput genome sequencing and comparative genomics hybridization arrays provide us with opportunities to explore the diversity of the proteolytic system in various LAB strains.
Abstract: A total of six polymorphisms were identified by comparing the genomes of the first and the last isolate of a well-characterized transmission chain of Mycobacterium tuberculosis involving five patients over a 12 and a half year period. The six polymorphisms consisted of four single nucleotide changes (SNPs), a tandem repeat polymorphism (TRP) and a previously identified IS6110 transposition event. These polymorphic sites were surveyed in each of the isolates from the five patients in the transmission chain. Surprisingly, five of the six polymorphisms accumulated in a single patient in the transmission chain; this patient had been non-compliant to tuberculosis treatment. This first insight into the tempo and mode of molecular evolution in M. tuberculosis at the patient-to-patient level suggests that the molecular evolution of the pathogen in vivo is characterized by periods of relative genomic stability followed by bursts of mutation. Whatever the mechanism for the accumulation of mutations, this observation may have profound consequences for the application of vaccines and therapeutic drugs, the management and treatment of disease outbreaks of M. tuberculosis, the most important bacterial pathogen of humans.
Abstract: A common bacterial strategy for monitoring environmental challenges is to use two-component systems, which consist of a sensor histidine kinase (HK) and a response regulator (RR). In the food-borne pathogen Bacillus cereus, the alternative sigma factor sigma(B) is activated by the RR RsbY. Here we present strong indications that the PP2C-type phosphatase RsbY receives its input from the multi-sensor hybrid kinase BC1008 (renamed RsbK). Genome analyses revealed that, across bacilli, rsbY and rsbK are located in a conserved gene cluster. A B. cereus rsbK deletion strain was shown to be incapable of inducing sigma(B) upon stress conditions and was impaired in its heat adaptive response. Comparison of the wild-type and rsbK mutant transcriptomes upon heat shock revealed that RsbK was primarily involved in the activation of the sigma(B)-mediated stress response. Truncation of the RsbK RR receiver domain demonstrated the importance of this domain for sigma(B) induction upon stress. The domain architecture of RsbK suggests that in the B. cereus group and in other bacilli, environmental and intracellular stress signalling routes are combined into one single protein. This strategy is markedly different from the sigma(B) activation pathway in other low-GC Gram-positives.
Abstract: Lactobacillus plantarum is a ubiquitous microorganism that is able to colonize several ecological niches, including vegetables, meat, dairy substrates and the gastro-intestinal tract. An extensive phenotypic and genomic diversity analysis was conducted to elucidate the molecular basis of the high flexibility and versatility of this species. First, 185 isolates from diverse environments were phenotypically characterized by evaluating their fermentation and growth characteristics. Strains clustered largely together within their particular food niche, but human fecal isolates were scattered throughout the food clusters, suggesting that they originate from the food eaten by the individuals. Based on distinct phenotypic profiles, 24 strains were selected and, together with a further 18 strains from an earlier low-resolution study, their genomic diversity was evaluated by comparative genome hybridization against the reference genome of L. plantarum WCFS1. Over 2000 genes were identified that constitute the core genome of the L. plantarum species, including 121 unique L. plantarum-marker genes that have not been found in other lactic acid bacteria. Over 50 genes unique for the reference strain WCFS1 were identified that were absent in the other L. plantarum strains. Strains of the L. plantarum subspecies argentoratensis were found to lack a common set of 24 genes, organized in seven gene clusters/operons, supporting their classification as a separate subspecies. The results provide a detailed view on phenotypic and genomic diversity of L. plantarum and lead to a better comprehension of niche adaptation and functionality of the organism.
Abstract: Lactococcus lactis is a lactic acid bacterium used in the production of many fermented dairy products. We report the complete genome sequence of L. lactis subsp. lactis KF147, a nondairy strain isolated from mung bean sprouts. The circular chromosome of 2,598,144 bp, the largest among the sequenced lactococcal strains, encodes many properties related to adaptation to the plant environment.
Abstract: Anaerobic ammonium-oxidizing (anammox) bacteria perform a key step in global nitrogen cycling. These bacteria make use of an organelle to oxidize ammonia anaerobically to nitrogen (N2) and so contribute approximately 50% of the nitrogen in the atmosphere. It is currently unknown which proteins constitute the organellar proteome and how anammox bacteria are able to specifically target organellar and cell-envelope proteins to their correct final destinations. Experimental approaches are complicated by the absence of pure cultures and genetic accessibility. However, the genome of the anammox bacterium Candidatus "Kuenenia stuttgartiensis" has recently been sequenced. Here, we make use of these genome data to predict the organellar sub-proteome and address the molecular basis of protein sorting in anammox bacteria.
Abstract: A pangenome is the total of genes present in strains of the same species. Pangenome microarrays allow determining the genomic content of bacterial strains more accurately than conventional comparative genome hybridization microarrays. PanCGHweb is the first tool that effectively calls genotype based on pangenome microarray data. AVAILABILITY: PanCGHweb, the web tool is accessible from: http://bamics2.cmbi.ru.nl/websoftware/pancgh/.
Abstract: In Lactic Acid Bacteria (LAB), the extracellular and surface-associated proteins can be involved in processes such as cell wall metabolism, degradation and uptake of nutrients, communication and binding to substrates or hosts. A genome-scale comparative study of these proteins (secretomes) can provide vast information towards the understanding of the molecular evolution, diversity, function and adaptation of LAB to their specific environmental niches.
Abstract: To investigate whether genome sequencing yields more useful markers than those currently used to study the epidemiology of tuberculosis, it was applied to three Mycobacterium tuberculosis isolates of the Harlingen outbreak. Our findings suggest that single nucleotide polymorphisms can be used to identify transmission chains in restriction fragment length polymorphism clusters.
Abstract: Lactobacilli belong to the lactic acid bacteria, which play a key role in industrial and artisan food raw-material fermentation, including a large variety of fermented dairy products. Next to their role in fermentation processes, specific strains of Lactobacillus are currently marketed as health-promoting cultures or probiotics. The last decade has witnessed the completion of a large number of Lactobacillus genome sequences, including the genome sequences of some of the probiotic species and strains. This development opens avenues to unravel the Lactobacillus-associated health-promoting activity at the molecular level. It is generally considered likely that an important part of the Lactobacillus effector molecules that participate in the proposed health-promoting interactions with the host (intestinal) system resides in the bacterial cell envelope. For this reason, it is important to accurately predict the Lactobacillus exoproteomes. Extensive annotation of these exoproteomes, combined with comparative analysis of species- or strain-specific exoproteomes, may identify candidate effector molecules, which may support specific effects on host physiology associated with particular Lactobacillus strains. Candidate health-promoting effector molecules of lactobacilli can then be validated via mutant approaches, which will allow for improved strain selection procedures, improved product quality control criteria and molecular science-based health claims.
Abstract: Gene regulatory networks can be reconstructed by combining transcriptome data from many different experiments to elucidate relations between the activity of certain transcription factors and the genes they control. To obtain insight in the regulatory network of Lactobacillus plantarum, microarray transcriptome data from more than 70 different experimental conditions were combined and the expression profiles of the transcriptional units (TUs) were compared. The TUs that displayed correlated expression were used to identify putative cis-regulatory elements by searching the upstream regions of the TUs for conserved motifs. Predicted motifs were extended and refined by searching for motifs in the upstream regions of additional TUs with correlated expression. In this way, cis-acting elements were identified for 41 regulons consisting of at least four TUs (correlation > 0.7). This set of regulons included the known regulons of CtsR and LexA, but also several novel ones encompassing genes with coherent biological functions. Visualization of the regulons and their connections revealed a highly interconnected regulatory network. This network contains several subnetworks that encompass genes of correlated biological function, such as sugar and energy metabolism, nitrogen metabolism and stress response.
Abstract: MOTIVATION: Pangenome arrays contain DNA oligomers targeting several sequenced reference genomes from the same species. In microbiology, these can be employed to investigate the often high genetic variability within a species by comparative genome hybridization (CGH). The biological interpretation of pangenome CGH data depends on the ability to compare strains at a functional level, particularly by comparing the presence or absence of orthologous genes. Due to the high genetic variability, available genotype-calling algorithms can not be applied to pangenome CGH data. RESULTS: We have developed the algorithm PanCGH that incorporates orthology information about genes to predict the presence or absence of orthologous genes in a query organism using CGH arrays that target the genomes of sequenced representatives of a group of microorganisms. PanCGH was tested and applied in the analysis of genetic diversity among 39 Lactococcus lactis strains from three different subspecies (lactis.cremoris, hordniae) and isolated from two different niches (dairy and plant). Clustering of these strains using the presence/absence data of gene orthologs revealed a clear separation between different subspecies and reflected the niche of the strains.
Abstract: Carnein is an 80 kDa subtilisin-like serine protease from the latex of the plant Ipomoea carnea which displays an exceptional resistance to chemical and thermal denaturation. In order to obtain the first crystal structure of a plant subtilisin and to gain insight into the structural determinants underlying its remarkable stability, carnein was isolated from I. carnea latex, purified and crystallized by the hanging-drop vapour-diffusion method. A data set was collected to 2.0 A resolution in-house from a single crystal at 110 K. The crystals belonged to the trigonal space group P3(1)21 or P3(2)21, with unit-cell parameters a = b = 126.9, c = 84.6 A, alpha = beta = 90, gamma = 120 degrees. Assuming the presence of one molecule per asymmetric unit, the Matthews coefficient is 2.46 A(3) Da(-1), corresponding to a solvent content of 50%. Structure determination of the enzyme is in progress.
Abstract: Twenty-four Lactobacillus plantarum supermotifs (LPSMs) with lengths from approximately 800 to 1,000 nucleotides were identified in the L. plantarum genome. LPSMs were conserved in other L. plantarum strains but not in other species. Secondary structure analysis predicted that LPSMs may fold into cruciform-like structures. Preliminary experiments indicate that the LPSMs are transcribed.
Abstract: Lactobacillus bulgaricus and Streptococcus thermophilus, used in yogurt starter cultures, are well known for their stability and protocooperation during their coexistence in milk. In this study, we show that a close interaction between the two species also takes place at the genetic level. We performed an in silico analysis, combining gene composition and gene transfer mechanism-associated features, and predicted horizontally transferred genes in both L. bulgaricus and S. thermophilus. Putative horizontal gene transfer (HGT) events that have occurred between the two bacterial species include the transfer of exopolysaccharide (EPS) biosynthesis genes, transferred from S. thermophilus to L. bulgaricus, and the gene cluster cbs-cblB(cglB)-cysE for the metabolism of sulfur-containing amino acids, transferred from L. bulgaricus or Lactobacillus helveticus to S. thermophilus. The HGT event for the cbs-cblB(cglB)-cysE gene cluster was analyzed in detail, with respect to both evolutionary and functional aspects. It can be concluded that during the coexistence of both yogurt starter species in a milk environment, agonistic coevolution at the genetic level has probably been involved in the optimization of their combined growth and interactions.
Abstract: In bacteria, environmental challenges are often translated into a transcriptional response via the cognate response regulators (RRs) of specialized two-component systems (TCSs). A phylogenetic footprinting/shadowing approach was designed and used to identify many novel RR-specific operators for species of the Bacillus cereus group and related Gram-positives. Analysis of the operator sequences revealed characteristic traits for each RR subfamily. For instance, operators related to the largest subfamily (OmpR) typically consisted of direct repeats (e.g. TTAAGA-N5-TTAAGA), whereas operators related to the second largest family (NarL) consisted of inverted repeats (e.g. ATGACA-N2-TGTCAT). This difference indicates a fundamentally different organization of the bound RR dimers between the two subfamilies. Moreover, the identification of the specific operator motifs allowed relating several RRs to a minimal regulon and thereby to a characteristic transcriptional response. Mostly, these regulons comprised genes encoding transport systems, suggesting a direct coupling of stimulus perception to the transport of target compounds. New biological roles could be attributed to various TCSs, including roles in cytochrome c biogenesis (HssRS), transport of carbohydrates, peptides and/or amino acids (YkoGH, LytSR), and resistance to toxic ions (LiaSR), antimicrobial peptides (BceRS) and beta-lactam antibiotics (BacRS, YcbLM). As more and more bacterial genome sequences are becoming available, the use of comparative analyses such as the approach applied in this study will further increase our knowledge of bacterial signal transduction mechanisms and provide directions for the assessment of their role in bacterial performance and survival strategies.
Abstract: Most Gram-positive bacteria inhabiting the gastrointestinal tract are capable of hydrolysing bile salts. Bile salt hydrolysis is thought to play an important role in various biological processes in the host. Therefore, correct annotation of bacterial bile salt hydrolases (Bsh) in public databases (EC 3.5.1.24) is of importance, especially for lactobacilli, which are considered to play a major role in bile salt hydrolysis in vivo. In the present study, all enzymes listed in public databases that belong to the Bsh family and the closely related penicillin V acylase (Pva; EC 3.5.1.11) family were compared with the sequences annotated as Bsh in Lactobacillus plantarum WCFS1, as an example. In Gram-positive bacteria, a clear distinction was made between the two families using sequence alignment, phylogenetic clustering, and protein homology modelling. Biochemical and structural data on experimentally verified Bsh and Pva enzymes were used for validation of function prediction. Hidden Markov models were constructed from the sequence alignments to enable a more accurate prediction of Bsh-encoding genes, and their distinction from those encoding members of the Pva family. Many Pva-related sequences appeared to be annotated incorrectly as Bsh in public databases. This refinement in the annotation of Bsh family members influences the prediction of the function of bsh-like genes in species of the genus Lactobacillus, and it is discussed in detail.
Abstract: BACKGROUND: T-box anti-termination is an elegant and sensitive mechanism by which many bacteria maintain constant levels of amino acid-charged tRNAs. The amino acid specificity of the regulatory element is related to a so-called specifier codon and can in principle be used to guide the functional annotation of the genes controlled via the T-box anti-termination mechanism. RESULTS: Hidden Markov Models were defined to search the T-box regulatory element and were applied to all completed prokaryotic genomes. The vast majority of the genes found downstream of the retrieved elements encoded functionalities related to transport and synthesis of amino acids and the charging of tRNA. This is completely in line with findings reported in literature and with the proposed biological role of the regulatory element. For several species, the functional annotation of a large number of genes encoding proteins involved in amino acid transport could be improved significantly on basis of the amino acid specificity of the identified T-boxes. In addition, these annotations could be extrapolated to a larger number of orthologous systems in other species. Analysis of T-box distribution confirmed that the element is restricted predominantly to species of the phylum Firmicutes. Furthermore, it appeared that the distribution was highly species specific and that in the case of amino acid transport some boxes seemed to "pop-up" only recently. CONCLUSION: We have demonstrated that the identification of the molecular specificity of a regulatory element can be of great help in solving notoriously difficult annotation issues, e.g. by defining the substrate specificity of genes encoding amino acid transporters on basis of the amino acid specificity of the regulatory T-box. Furthermore, our analysis of the species-dependency of the occurrence of specific T-boxes indicated that these regulatory elements propagate in a semi-independent way from the genes that they control.
Abstract: To what extent can modes of gene regulation be explained by systems-level properties of metabolic networks? Prior studies on co-regulation of metabolic genes have mainly focused on graph-theoretical features of metabolic networks and demonstrated a decreasing level of co-expression with increasing network distance, a naïve, but widely used, topological index. Others have suggested that static graph representations can poorly capture dynamic functional associations, e.g., in the form of dependence of metabolic fluxes across genes in the network. Here, we systematically tested the relative importance of metabolic flux coupling and network position on gene co-regulation, using a genome-scale metabolic model of Escherichia coli. After validating the computational method with empirical data on flux correlations, we confirm that genes coupled by their enzymatic fluxes not only show similar expression patterns, but also share transcriptional regulators and frequently reside in the same operon. In contrast, we demonstrate that network distance per se has relatively minor influence on gene co-regulation. Moreover, the type of flux coupling can explain refined properties of the regulatory network that are ignored by simple graph-theoretical indices. Our results underline the importance of studying functional states of cellular networks to define physiologically relevant associations between genes and should stimulate future developments of novel functional genomic tools.
Abstract: Microarrays are used to profile transcriptional activity, providing global cell biology insight. Particularly for plants, interpretation of transcriptional profiles is challenging because many genes have unknown functions. Furthermore, many plant gene sequences do not have clear homologs in other model organisms. Fortunately, over the past five years, various tools that assist plant scientists have been developed. Here, we evaluate the currently available in silico tools for reconstruction of cellular (metabolic, biochemical and signal transduction) pathways based on plant gene expression datasets. Furthermore, we show how expression-profile comparison at the level of these various cellular pathways contributes to the postulation of novel hypotheses which, after experimental verification, can provide further insight into decisive elements that have roles in cellular processes.
Abstract: Lactococcus lactis is a primary constituent of many starter cultures used for the manufacturing of fermented dairy products, but the species also occurs in various nondairy niches such as (fermented) plant material. Three genome sequences of L. lactis dairy strains (IL-1403, SK11, and MG1363) are publicly available. An extensive molecular and phenotypic diversity analysis was now performed on two L. lactis plant isolates. Diagnostic sequencing of their genomes resulted in over 2.5 Mb of sequence for each strain. A high synteny was found with the genome of L. lactis IL-1403, which was used as a template for contig mapping and locating deletions and insertions in the plant L. lactis genomes. Numerous genes were identified that do not have homologs in the published genome sequences of dairy L. lactis strains. Adaptation to growth on substrates derived from plant cell walls is evident from the presence of gene sets for the degradation of complex plant polymers such as xylan, arabinan, glucans, and fructans but also for the uptake and conversion of typical plant cell wall degradation products such as alpha-galactosides, beta-glucosides, arabinose, xylose, galacturonate, glucuronate, and gluconate. Further niche-specific differences are found in genes for defense (nisin biosynthesis), stress response (nonribosomal peptide synthesis and various transporters), and exopolysaccharide biosynthesis, as well as the expected differences in various mobile elements such as prophages, plasmids, restriction-modification systems, and insertion sequence elements. Many of these genes were identified for the first time in Lactococcus lactis. In most cases good correspondence was found with the phenotypic characteristics of these two strains.
Abstract: BACKGROUND: A key problem in the sequence-based reconstruction of regulatory networks in bacteria is the lack of specificity in operator predictions. The problem is especially prominent in the identification of transcription factor (TF) specific binding sites. More in particular, homologous TFs are abundant and, as they are structurally very similar, it proves difficult to distinguish the related operators by automated means. This also holds for the LacI-family, a family of TFs that is well-studied and has many members that fulfill crucial roles in the control of carbohydrate catabolism in bacteria including catabolite repression. To overcome the specificity problem, a comprehensive footprinting approach was formulated to identify TF-specific operator motifs and was applied to the LacI-family of TFs in the model gram positive organism, Lactobacillus plantarum WCFS1. The main premise behind the approach is that only orthologous sequences that share orthologous genomic context will share equivalent regulatory sites. RESULTS: When the approach was applied to the 12 LacI-family TFs of the model species, a specific operator motif was identified for each of them. With the TF-specific operator motifs, potential binding sites were found on the genome and putative minimal regulons could be defined. Moreover, specific inducers could in most cases be linked to the TFs through phylogeny, thereby unveiling the biological role of these regulons. The operator predictions indicated that the LacI-family TFs can be separated into two subfamilies with clearly distinct operator motifs. They also established that the operator related to the 'global' regulator CcpA is not inherently distinct from that of other LacI-family members, only more degenerate. Analysis of the chromosomal position of the identified putative binding sites confirmed that the LacI-family TFs are mostly auto-regulatory and relate mainly to carbohydrate uptake and catabolism. CONCLUSION: Our approach to identify specific operator motifs for different TF-family members is specific and in essence generic. The data infer that, although the specific operator motifs can be used to identify minimal regulons, experimental knowledge on TF activity especially is essential to determine complete regulons as well as to estimate the overlap between TF affinities.
Abstract: BACKGROUND: In the past decades, various protein subcellular-location (SCL) predictors have been developed. Most of these predictors, like TMHMM 2.0, SignalP 3.0, PrediSi and Phobius, aim at the identification of one or a few SCLs, whereas others such as CELLO and Psortb.v.2.0 aim at a broader classification. Although these tools and pipelines can achieve a high precision in the accurate prediction of signal peptides and transmembrane helices, they have a much lower accuracy when other sequence characteristics are concerned. For instance, it proved notoriously difficult to identify the fate of proteins carrying a putative type I signal peptidase (SPIase) cleavage site, as many of those proteins are retained in the cell membrane as N-terminally anchored membrane proteins. Moreover, most of the SCL classifiers are based on the classification of the Swiss-Prot database and consequently inherited the inconsistency of that SCL classification. As accurate and detailed SCL prediction on a genome scale is highly desired by experimental researchers, we decided to construct a new SCL prediction pipeline: LocateP. RESULTS: LocateP combines many of the existing high-precision SCL identifiers with our own newly developed identifiers for specific SCLs. The LocateP pipeline was designed such that it mimics protein targeting and secretion processes. It distinguishes 7 different SCLs within Gram-positive bacteria: intracellular, multi-transmembrane, N-terminally membrane anchored, C-terminally membrane anchored, lipid-anchored, LPxTG-type cell-wall anchored, and secreted/released proteins. Moreover, it distinguishes pathways for Sec- or Tat-dependent secretion and alternative secretion of bacteriocin-like proteins. The pipeline was tested on data sets extracted from literature, including experimental proteomics studies. The tests showed that LocateP performs as well as, or even slightly better than other SCL predictors for some locations and outperforms current tools especially where the N-terminally anchored and the SPIase-cleaved secreted proteins are concerned. Overall, the accuracy of LocateP was always higher than 90%. LocateP was then used to predict the SCLs of all proteins encoded by completed Gram-positive bacterial genomes. The results are stored in the database LocateP-DB http://www.cmbi.ru.nl/locatep-db1. CONCLUSION: LocateP is by far the most accurate and detailed protein SCL predictor for Gram-positive bacteria currently available.
Abstract: Noroviruses (NoVs) are considered to be a major cause of acute nonbacterial gastroenteritis in humans. The NoV genus is genetically diverse, and genotype GII.4 has been most commonly identified worldwide in recent years. In this study we analyzed the complete capsid gene of NoV strains belonging to the less prevalent genotype GII.2. We compared a total of 36 complete capsid sequences of GII.2 sequences obtained from the GenBank (n = 5) and from outbreaks or sporadic cases that occurred in The Netherlands (n = 10) and in Osaka City, Japan (n = 21), between 1976 and 2005. Alignment of all capsid sequences did not show fixation of amino acid substitutions over time as an indication for genetic drift. In contrast, when strains previously recognized as recombinants were excluded from the alignment, genetic drift was observed. Substitutions were found at five informative sites (two in the P1 subdomain and three in the P2 subdomain), segregating strains into five genetic groups (1994 to 1997, 1999 to 2000, 2001 to 2003, 2004, and 2005). Only one amino acid position changed consistently between each group (position 345). Homology modeling of the GII.2 capsid protein showed that the five amino acids were located on the surface of the capsid and close to each other at the interface of two monomers. The data suggest that these changes were induced by selective pressure, driving virus evolution. Remarkably, this was observed only for nonrecombinant genomes, suggesting differences in behavior with recombinant strains.
Abstract: Noroviruses are the causative agents of the majority of viral gastroenteritis outbreaks in humans. During the past 15 years, noroviruses of genotype GGII.4 have caused four epidemic seasons of viral gastroenteritis, during which four novel variants (termed epidemic variants) emerged and displaced the resident viruses. In order to understand the mechanisms and biological advantages of these epidemic variants, we studied the genetic changes in the capsid proteins of GGII.4 strains over this period. A representative sample was drawn from 574 GGII.4 outbreak strains collected over 15 years of systematic surveillance in The Netherlands, and capsid genes were sequenced for a total of 26 strains. The three-dimensional structure was predicted by homology modeling, using the Norwalk virus (Hu/NoV/GGI.1/Norwalk/1968/US) capsid as a reference. The highly significant preferential accumulation and fixation of mutations (nucleotide and amino acid) in the protruding part of the capsid protein provided strong evidence for the occurrence of genetic drift and selection. Although subsequent new epidemic variants differed by up to 25 amino acid mutations, consistent changes were observed in only five positions. Phylogenetic analyses showed that each variant descended from its chronologic predecessor, with the exception of the 2006b variant, which is more closely related to the 2002 variant than to the 2004 variant. The consistent association between the observed genetic findings and changes in epidemiology leads to the conclusion that population immunity plays a role in the epochal evolution of GGII.4 norovirus strains.
Abstract: In silico identification criteria were defined to predict if genes encoding histidine protein kinases (HPKs) and response regulators (RRs) could be part of peptide-based quorum sensing (QS) two-component regulatory systems (QS-TCSs) in Firmicutes. These criteria were used to screen HPKs and RRs annotated on the completed genome sequences of Lactobacillus species, and several (putative) QS-TCSs were identified in this way. The five peptide-based QS-TCSs that were predicted on the Lactobacillus plantarum WCFS1 genome were further analysed to test their (QS) functionality. Four of these systems contained an upstream gene encoding a putative autoinducing peptide (AIP), of which two were preceded by a double-glycine-type leader peptide. One of these was identical to the plnABCD regulatory system of L. plantarum C11 and was shown to regulate plantaricin production in L. plantarum WCFS1. The third TCS was designated lamBDCA for Lactobacillus agr-like module, where the lamD gene was shown to encode a cyclic thiolactone peptide. The fourth TCS was paralogous to the lam system and contained a putative AIP-encoding gene but lacked the lamB gene. Finally, a genetically separated orphan HPK and RR that showed clear peptide-based QS characteristics could form a fifth peptide-based QS-TCS. The predicted presence of multiple (peptide-based) QS-TCSs in some lactobacilli and in particular in L. plantarum might be a reflection of the ability of these species to persist in a diverse range of ecological niches.
Abstract: Subtilisin-like serine proteases (subtilases) are a very diverse family of serine proteases with low sequence homology, often limited to regions surrounding the three catalytic residues. Starting with different Hidden Markov Models (HMM), based on sequence alignments around the catalytic residues of the S8 family (subtilisins) and S53 family (sedolisins), we iteratively searched all ORFs in the complete genomes of 313 eubacteria and archaea. In 164 genomes we identified a total of 567 ORFs with one or more of the conserved regions with a catalytic residue. The large majority of these contained all three regions around the "classical" catalytic residues of the S8 family (Asp-His-Ser), while 63 proteins were identified as S53 (sedolisin) family members (Glu-Asp-Ser). More than 30 proteins were found to belong to two novel subsets with other evolutionary variations in catalytic residues, and new HMMs were generated to search for them. In one subset the catalytic Asp is replaced by an equivalent Glu (i.e. Glu-His-Ser family). The other subset resembles sedolisins, but the conserved catalytic Asp is not located on the same helix as the nucleophile Glu, but rather on a beta-sheet strand in a topologically similar position, as suggested by homology modeling. The Prokaryotic Subtilase Database (www.cmbi.ru.nl/subtilases) provides access to all information on the identified subtilases, the conserved sequence regions, the proposed family subdivision, and the appropriate HMMs to search for them. Over 100 proteins were predicted to be subtilases for the first time by our improved searching methods, thereby improving genome annotation.
Abstract: Cis-acting elements in Lactobacillus plantarum were predicted by comparative analysis of the upstream regions of conserved genes and predicted transcriptional units (TUs) in different bacterial genomes. TUs were predicted for two species sets, with different evolutionary distances to L.plantarum. TUs were designated 'cluster of orthologous transcriptional units' (COT) when >50% of the genes were orthologous in different species. Conserved DNA sequences were detected in the upstream regions of different COTs. Subsequently, conserved motifs were used to scan upstream regions of all TUs. This method revealed 18 regulatory motifs only present in lactic acid bacteria (LAB). The 18 LAB-specific candidate regulatory motifs included 13 that were not described previously. These LAB-specific different motifs were found in front of genes encoding functions varying from cold shock proteins to RNA and DNA polymerases, and many unknown functions. The best-described LAB-specific motif found was the CopR-binding site, regulating expression of copper transport ATPases. Finally, all detected motifs were used to predict co-regulated TUs (regulons) for L.plantarum, and transcriptome profiling data were analyzed to provide regulon prediction validation. It is demonstrated that phylogenetic footprinting using different species sets can identify and distinguish between general regulatory motifs and LAB-specific regulatory motifs.
Abstract: A genome-scale metabolic model of the lactic acid bacterium Lactobacillus plantarum WCFS1 was constructed based on genomic content and experimental data. The complete model includes 721 genes, 643 reactions, and 531 metabolites. Different stoichiometric modeling techniques were used for interpretation of complex fermentation data, as L. plantarum is adapted to nutrient-rich environments and only grows in media supplemented with vitamins and amino acids. (i) Based on experimental input and output fluxes, maximal ATP production was estimated and related to growth rate. (ii) Optimization of ATP production further identified amino acid catabolic pathways that were not previously associated with free-energy metabolism. (iii) Genome-scale elementary flux mode analysis identified 28 potential futile cycles. (iv) Flux variability analysis supplemented the elementary mode analysis in identifying parallel pathways, e.g. pathways with identical end products but different co-factor usage. Strongly increased flexibility in the metabolic network was observed when strict coupling between catabolic ATP production and anabolic consumption was relaxed. These results illustrate how a genome-scale metabolic model and associated constraint-based modeling techniques can be used to analyze the physiology of growth on a complex medium rather than a minimal salts medium. However, optimization of biomass formation using the Flux Balance Analysis approach, reported to successfully predict growth rate and by product formation in Escherichia coli and Saccharomyces cerevisiae, predicted too high biomass yields that were incompatible with the observed lactate production. The reason is that this approach assumes optimal efficiency of substrate to biomass conversion, and can therefore not predict the metabolically inefficient lactate formation.
Abstract: The predicted extracellular proteins of the bacterium Lactobacillus plantarum were analysed to gain insight into the mechanisms underlying interactions of this bacterium with its environment. Extracellular proteins play important roles in processes ranging from probiotic effects in the gastrointestinal tract to degradation of complex extracellular carbon sources such as those found in plant materials, and they have a primary role in the adaptation of a bacterium to changing environmental conditions. The functional annotation of extracellular proteins was improved using a wide variety of bioinformatics methods, including domain analysis and phylogenetic profiling. At least 12 proteins are predicted to be directly involved in adherence to host components such as collagen and mucin, and about 30 extracellular enzymes, mainly hydrolases and transglycosylases, might play a role in the degradation of substrates by L. plantarum to sustain its growth in different environmental niches. A comprehensive overview of all predicted extracellular proteins, their domains composition and their predicted function is provided through a database at http://www.cmbi.ru.nl/secretome which could serve as a basis for targeted experimental studies into the function of extracellular proteins.
Abstract: Members of the Bacillus cereus group are ubiquitously present in the environment and can adapt to a wide range of environmental fluctuations. In bacteria, these adaptive responses are generally mediated by two-component signal transduction systems (TCSs), which consist of a histidine kinase (HK) and its cognate response regulator (RR). With the use of in silico techniques, a complete set of HKs and RRs was recovered from eight completely sequenced B. cereus group genomes. By applying a bidirectional best-hits method combined with gene neighbourhood analysis, a footprint of these proteins was made. Around 40 HK-RR gene pairs were detected in each member of the B. cereus group. In addition, each member contained many HK and RR genes not encoded in pairs ("orphans"). Classification of HKs and RRs based on their enzymic domains together with the analysis of two neighbour-joining trees of these domains revealed putative interaction partners for most of the "orphans". Putative biological functions, including involvement in virulence and host-microbe interactions, were predicted for the B. cereus group HKs and RRs by comparing them with those of B. subtilis and other micro-organisms. Remarkably, B. anthracis appeared to lack specific HKs and RRs and was found to contain many truncated, putatively non-functional, HK and RR genes. It is hypothesized that specialization of B. anthracis as a pathogen could have reduced the range of environmental stimuli to which it is exposed. This may have rendered some of its TCSs obsolete, ultimately resulting in the deletion of some HK and RR genes.
Abstract: BACKGROUND: The genomic information of a species allows for the genome-scale reconstruction of its metabolic capacity. Such a metabolic reconstruction gives support to metabolic engineering, but also to integrative bioinformatics and visualization. Sequence-based automatic reconstructions require extensive manual curation, which can be very time-consuming. Therefore, we present a method to accelerate the time-consuming process of network reconstruction for a query species. The method exploits the availability of well-curated metabolic networks and uses high-resolution predictions of gene equivalency between species, allowing the transfer of gene-reaction associations from curated networks. RESULTS: We have evaluated the method using Lactococcus lactis IL1403, for which a genome-scale metabolic network was published recently. We recovered most of the gene-reaction associations (i.e. 74 - 85%) which are incorporated in the published network. Moreover, we predicted over 200 additional genes to be associated to reactions, including genes with unknown function, genes for transporters and genes with specific metabolic reactions, which are good candidates for an extension to the previously published network. In a comparison of our developed method with the well-established approach Pathologic, we predicted 186 additional genes to be associated to reactions. We also predicted a relatively high number of complete conserved protein complexes, which are derived from curated metabolic networks, illustrating the potential predictive power of our method for protein complexes. CONCLUSION: We show that our methodology can be applied to accelerate the reconstruction of genome-scale metabolic networks by taking optimal advantage of existing, manually curated networks. As orthology detection is the first step in the method, only the translated open reading frames (ORFs) of a newly sequenced genome are necessary to reconstruct a metabolic network. When more manually curated metabolic networks will become available in the near future, the usefulness of our method in network prediction is likely to increase.
Abstract: Lactic acid bacteria (LAB) are frequently encountered inhabitants of the human intestinal tract. A protective layer of mucus covers the epithelial cells of the intestine, offering an attachment site for these bacteria. In this study bioinformatics tools were used to identify and characterize proteins containing one type of mucus-binding domain, called MUB, that is postulated to play an important role in the adherence of LAB to this mucus layer. By searching in all protein databases 48 proteins containing at least one of these MUB domains in nine LAB species were identified. These MUB domains varied in size, ranging from approximately 100 to more than 200 residues per domain. Complete MUB domains were found exclusively in LAB. The number of MUB domains present in a single protein varied from 1 to 15. In some cases, orthologous proteins in closely related species contained a different number of domains, indicating that repeats of the domain undergo rapid duplication and deletion. Proteins containing the MUB domain were often encoded by gene clusters that encode multiple extracellular proteins. In addition to one or more copies of the MUB domain, many of these proteins contained other domains that are predicted to be involved in binding to and degradation of extracellular components. These findings strongly suggest that the MUB domain is an LAB-specific functional unit that performs its task in various domain contexts and could fulfil an important role in host-microbe interactions in the gastrointestinal tract.
Abstract: Lactococcus lactis is the primary model organism for lactic acid bacteria (LAB) and is widely used in the production of fermented dairy products. In recent years there has been increasing interest in strains isolated from non-dairy environments, as these exhibit a high metabolic diversity and have unique flavour-forming activities. Recent progress has been made in understanding the natural diversity and adaptive responses of L. lactis from dairy and non-dairy origins. Genome sequencing and comparative genomics have also had an impact on understanding natural diversity within the species, and have provided new opportunities for industrial strain development.
Abstract: The complete genome sequences of the lactic acid bacteria (LAB), Lactobacillus plantarum, Lactococcus lactis, and Lactobacillus johnsonii were used to compare location, sequence, organisation, and regulation of the ribosomal RNA (rrn) operons. All rrn operons of the examined LAB diverge from the origin of replication, which is compatible with their efficient expression. All operons show a common organisation of 5'-16S-23S-5S-3' structure, but differ in the number, location and specificity of the tRNA genes. In the 16S-23S intergenic spacer region, two of the five rrn operons of Lb. plantarum and three of the six of Lb. johnsonii contain tRNA-ala and tRNA-ile genes, while L. lactis has a tRNA-ala gene in all six operons. The number of tRNA genes following the 5S rRNA gene ranges up to 14, 16, and 21 for L. lactis, Lb. johnsonii and Lb. plantarum, respectively. The tRNA gene complements are similar to each other and to those of other bacteria. Micro-heterogeneity was found within the rRNA structural genes and spacer regions of each strain. In the rrn operon promoter regions of Lb. plantarum and L. lactis marked differences were found, while the promoter regions of Lb. johnsonii showed a similar tandem promoter structure in all operons. The rrn promoters of L. lactis show either a single or a tandem promoter structure. All promoters of Lb. plantarum contain two or three -10 and -35 regions, of which either zero to two were followed by an UP-element. The Lb. plantarum rrnA, rrnB, and rrnC promoter regions display similarity to the rrn promoter structure of Esherichia coli. Differences in regulation between the five Lb. plantarum promoters were studied using a low copy promoter-probe plasmid. Taking copy number and growth rate into account, a differential expression over time was shown. Although all five Lb. plantarum rrn promoters are significantly different, this study shows that their activity was very similar under the circumstances tested. An active promoter was also identified within the Lb. plantarum rrnC operon preceding a cluster of 17 tRNA genes.
Abstract: Surface proteins of gram-positive bacteria often play a role in adherence of the bacteria to host tissue and are frequently required for virulence. A specific subgroup of extracellular proteins contains the cell wall-sorting motif LPxTG, which is the target for cleavage and covalent coupling to the peptidoglycan by enzymes called sortases. A comprehensive set of putative sortase substrates was identified by in silico analysis of 199 completely sequenced prokaryote genomes. A combination of detection methods was used, including secondary structure prediction, pattern recognition, sequence homology, and genome context information. With the hframe algorithm, putative substrates were identified that could not be detected by other methods due to errors in open reading frame calling, frameshifts, or sequencing errors. In total, 732 putative sortase substrates encoded in 49 prokaryote genomes were identified. We found striking species-specific variation for the LPxTG motif. A hidden Markov model (HMM) based on putative sortase substrates was created, which was subsequently used for the automatic detection of sortase substrates in recently completed genomes. A database was constructed, LPxTG-DB (http://bamics3.cmbi.kun.nl/sortase_substrates), containing for each genome a list of putative sortase substrates, sequence information of these substrates, the organism-specific HMMs based on the consensus sequence of the sortase recognition motif, and a graphic representation of this consensus.
Abstract: Lactobacillus plantarum is a versatile and flexible species that is encountered in a variety of niches and can utilize a broad range of fermentable carbon sources. To assess if this versatility is linked to a variable gene pool, microarrays containing a subset of small genomic fragments of L. plantarum strain WCFS1 were used to perform stringent genotyping of 20 strains of L. plantarum from various sources. The gene categories with the most genes conserved in all strains were those involved in biosynthesis or degradation of structural compounds like proteins, lipids, and DNA. Conversely, genes involved in sugar transport and catabolism were highly variable between strains. Moreover, besides the obvious regions of variance, like prophages, other regions varied between the strains, including regions encoding plantaricin biosynthesis, nonribosomal peptide biosynthesis, and exopolysaccharide biosynthesis. In many cases, these variable regions colocalized with regions of unusual base composition. Two large regions of flexibility were identified between 2.70 and 2.85 and 3.10 and 3.29 Mb of the WCFS1 chromosome, the latter being close to the origin of replication. The majority of genes encoded in these variable regions are involved in sugar metabolism. This functional overrepresentation and the unusual base composition of these regions led to the hypothesis that they represented lifestyle adaptation regions in L. plantarum. The present study consolidates this hypothesis by showing that there is a high degree of gene content variation among L. plantarum strains in genes located in these regions of the WCFS1 genome. Interestingly, based on our genotyping data L. plantarum strains clustered into two clearly distinguishable groups, which coincided with an earlier proposed subdivision of this species based on conventional methods.
Abstract: Lactobacillus plantarum WCFS1 harbors three plasmids, pWCFS101, pWCFS102, and pWCFS103, with sizes of 1,917, 2,365, and 36,069 bp, respectively. The two smaller plasmids are of unknown function and contain replication genes that are likely to function via the rolling-circle replication mechanism. The host range of the pWCFS101 replicon includes Lactobacillus species and Lactococcus lactis, while that of the pWCFS102 replicon also includes Carnobacterium maltaromaticum and Bacillus subtilis. The larger plasmid is predicted to replicate via the theta-type mechanism. The host range of its replicon seems restricted to L. plantarum. Cloning vectors were constructed based on the replicons of all three plasmids. Plasmid pWCFS103 was demonstrated to be a conjugative plasmid, as it could be transferred to L. plantarum NC8. It confers arsenate and arsenite resistance, which can be used as selective markers.
Abstract: The prospect of understanding the relationship between the genome and the physiology of an organism is an important incentive to reconstruct metabolic networks. The first steps in the process can be automated and it does not take much effort to obtain an initial metabolic reconstruction from a genome sequence. However, such a reconstruction is certainly not flawless and correction of the many imperfections is laborious. It requires the combined analysis of the available information on protein sequence, phylogeny, gene-context and co-occurrence but is also aided by high-throughput experimental data. Simultaneously, the reconstructed network provides the opportunity to visualize the "omics" data within a relevant biological functional context and thus aids the interpretation of those data.
Abstract: A key complication in comparative genomics for reliable gene function prediction is the existence of duplicated genes. To study the effect of gene duplication on function prediction, we analyze orthologs between pairs of genomes where in one genome the orthologous gene has duplicated after the speciation of the two genomes (i.e. inparalogs). For these duplicated genes we investigate whether the gene that is most similar on the sequence level is also the gene that has retained the ancestral gene-neighborhood. Although the majority of investigated cases show a consistent pattern between sequence similarity and gene-neighborhood conservation, a substantial fraction, 29-38%, is inconsistent. The observation of inconsistency is not the result of a chance outcome owing to a lack of divergence time between inparalogs, but rather it seems to be the result of a chance outcome caused by very similar rates of sequence evolution of both inparalogs relative to their ortholog. If one-to-one orthologous relationships are required, it is advisable to combine contextual information (i.e. gene-neighborhood in prokaryotes and co-expression in eukaryotes) with protein sequence information to predict the most probable functional equivalent ortholog in the presence of inparalogs.
Abstract: Lactococcus lactis strains are known to carry plasmids encoding industrially important traits. L. lactis subsp. cremoris SK11 is widely used by the dairy industry in cheese making. Its complete plasmid complement was sequenced and found to contain the plasmids pSK11A (10,372 bp), pSK11B (13,332 bp), pSK11L (47,165 bp), and pSK11P (75,814 bp). Six highly homologous repB-containing replicons were found, all belonging to the family of lactococcal theta-type replicons. Twenty-three complete insertion sequence elements segment the plasmids into numerous modules, many of which can be identified as functional units or containing functionally related genes. Plasmid-encoded functions previously known to reside on L. lactis SK11 plasmids were now mapped in detail, e.g., lactose utilization (lacR-lacABCDFEGX), the proteolytic system (prtM-prtP, pepO, pepF), and the oligopeptide permease system (oppDFBCA). Newly identified plasmid-encoded functions could facilitate the uptake of various cations, while the pabA and pabB genes could be essential for folate biosynthesis. A competitive advantage could be obtained by using the putative flavin adenine dinucleotide-dependent d-lactate dehydrogenase and oxalate:formate antiporter for enhanced ATP synthesis, while the activity of the predicted alpha-acetolactate decarboxylase may contribute to the formation of an additional electron sink. Various stress response proteins are plasmid encoded, which could enhance strain robustness. A substantial number of these "adaptation" genes have not been described before on L. lactis plasmids. Moreover, several genes were identified for the first time in L. lactis, possibly reflecting horizontal gene transfer.
Abstract: On the basis of the annotated genome we reconstructed the metabolic pathways of the lactic acid bacterium Lactobacillus plantarum WCFS1. After automatic reconstruction by the Pathologic tool of Pathway Tools (http://bioinformatics.ai.sri.com/ptools/), the resulting pathway-genome database, LacplantCyc, was manually curated extensively. The current database contains refinements to existing routes and new gram-positive bacterium-specific reactions that were not present in the MetaCyc database. These reactions include, for example, reactions related to cell wall biosynthesis, molybdopterin biosynthesis, and transport. At present, LacplantCyc includes 129 pathways and 704 predicted reactions involving some 670 chemical species and 710 enzymes. We tested vitamin and amino acid requirements of L. plantarum experimentally and compared the results with the pathways present in LacplantCyc. In the majority of cases (32 of 37 cases) the experimental results agreed with the final reconstruction. LacplantCyc is the most extensively curated pathway-genome database for gram-positive bacteria and is open to the microbiology community via the World Wide Web (www.lacplantcyc.nl). It can be used as a reference pathway-genome database for gram-positive microbes in general and lactic acid bacteria in particular.
Abstract: Genome sequences are now available for many of the microbes that cause food-borne diseases. The information contained in pathogen genome sequences, together with the development of themed and whole-genome DNA microarrays and improved proteomics techniques, might provide tools for the rapid detection and identification of such organisms, for assessing their biological diversity and for understanding their ability to respond to stress. The genomic information also provides insight into the metabolic capacity and versatility of microbes; for example, specific metabolic pathways might contribute to the growth and survival of pathogens in a range of niches, such as food-processing environments and the human host. New concepts are emerging about how pathogens function, both within foods and in interactions with the host. The future should bring the first practical benefits of genome sequencing to the field of microbial food safety, including strategies and tools for the identification and control of emerging pathogens.
Abstract: The first comprehensive comparative analysis of lactobacilli was done by comparing the genomes of Lactobacillus plantarum (3.3 Mb) and Lactobacillus johnsonii (2.0 Mb). L. johnsonii is predominantly found in the gastrointestinal tract, while L. plantarum is also found on plants and plant-derived material, and is used in a variety of industrial fermentations. The L. plantarum and L. johnsonii chromosomes have only 28 regions with conservation of gene order, totalling about 0.75 Mb; these regions are not co-linear, indicating major chromosomal rearrangements. Metabolic reconstruction indicates many differences between L. johnsonii and L. plantarum: numerous enzymes involved in sugar metabolism and in biosynthesis of amino acids, nucleotides, fatty acids and cofactors are lacking in L. johnsonii. Major differences were seen in the number and types of putative extracellular proteins, which are of interest because of their possible role in host-microbe interactions. The differences between L. plantarum and L. johnsonii, both in genome organization and gene content, are exceptionally large for two bacteria of the same genus, emphasizing the difficulty in taxonomic classification of lactobacilli.
Abstract: SUMMARY: A Web-based visualization tool, the Microbial Genome Viewer, is presented that allows the user to combine complex genomic data in a highly interactive way. This Web tool enables the interactive generation of chromosome wheels and linear genome maps from genome annotation data stored in a MySQL database. The generated images are in scalable vector graphics (SVG) format, which is suitable for creating high-quality scalable images and dynamic Web representations. Gene-related data such as transcriptome and time-course microarray experiments can be superimposed on the maps for visual inspection. AVAILABILITY: The Microbial Genome Viewer 1.0 is freely available at http://www.cmbi.kun.nl/MGV
Abstract: Lactic acid bacteria (LAB) have been widely used in food fermentations and, more recently, as probiotics in health-promoting food products. Genome sequencing and functional genomics studies of a variety of LAB are now rapidly providing insights into their diversity and evolution and revealing the molecular basis for important traits such as flavor formation, sugar metabolism, stress response, adaptation and interactions. Bioinformatics plays a key role in handling, integrating and analyzing the flood of 'omics' data being generated. Reconstruction of metabolic potential using bioinformatics tools and databases, followed by targeted experimental verification and exploration of the metabolic and regulatory network properties, are the present challenges that should lead to improved exploitation of these versatile food bacteria.
Abstract: The Lactobacillus plantarum commensal WCFS1 contains four prophage elements in its genome. Lp1 and Lp2 are two about 40-kb-long uninducible prophages that share closely related DNA packaging, head and tail genes defining a second lineage of pac-site Siphoviridae in L. plantarum, distinct from L. plantarum phage phig1e, but related to Bacillus phage SPP1 and Lactococcus phage TP901-1. Northern analysis revealed transcribed prophage genes exclusively near both attachment sites. Comparative genomics identified candidate lysogenic conversion genes (LCG) downstream of the lysis cassette and within the lysogeny module. Notable are genes with sequence similarities to putative LCG from Streptococcus pyogenes prophages and to a Bacillus plasmid. Both prophages harbored tRNA genes. R-Lp3 and R-Lp4 represent short prophage remnants; R-Lp3 abuts Lp2 and displays sequence links to cos-site Siphoviridae.
Abstract: The 3,308,274-bp sequence of the chromosome of Lactobacillus plantarum strain WCFS1, a single colony isolate of strain NCIMB8826 that was originally isolated from human saliva, has been determined, and contains 3,052 predicted protein-encoding genes. Putative biological functions could be assigned to 2,120 (70%) of the predicted proteins. Consistent with the classification of L. plantarum as a facultative heterofermentative lactic acid bacterium, the genome encodes all enzymes required for the glycolysis and phosphoketolase pathways, all of which appear to belong to the class of potentially highly expressed genes in this organism, as was evident from the codon-adaptation index of individual genes. Moreover, L. plantarum encodes a large pyruvate-dissipating potential, leading to various end-products of fermentation. L. plantarum is a species that is encountered in many different environmental niches, and this flexible and adaptive behavior is reflected by the relatively large number of regulatory and transport functions, including 25 complete PTS sugar transport systems. Moreover, the chromosome encodes >200 extracellular proteins, many of which are predicted to be bound to the cell envelope. A large proportion of the genes encoding sugar transport and utilization, as well as genes encoding extracellular functions, appear to be clustered in a 600-kb region near the origin of replication. Many of these genes display deviation of nucleotide composition, consistent with a foreign origin. These findings suggest that these genes, which provide an important part of the interaction of L. plantarum with its environment, form a lifestyle adaptation region in the chromosome.
Abstract: EXProt is a non-redundant protein database containing a selection of entries from genome annotation projects and public databases, aimed at including only proteins with an experimentally verified function. In EXProt release 2.0 we have collected entries from the Pseudomonas aeruginosa community annotation project (PseudoCAP), the Escherichia coli genome and proteome database (GenProtEC) and the translated coding sequences from the Prokaryotes division of EMBL nucleotide sequence database, which are described as having an experimentally verified function. Each entry in EXProt has a unique ID number and contains information about the species, amino acid sequence, functional annotation and, in most cases, links to references in MEDLINE/PubMed and to the entry in the original database. EXProt is indexed in SRS at CMBI (http://www.cmbi.kun.nl/srs/) and can be searched with BLAST and FASTA through the EXProt web page (http://www.cmbi.kun.nl/EXProt/).
Abstract: EXProt (database for EXPerimentally verified Protein functions) is a new non-redundant database containing protein sequences for which the function has been experimentally verified. It is a selection of 3976 entries from the Prokaryotes section of the EMBL Nucleotide Sequence Database, Release 66, and 375 entries from the Pseudomonas Community Annotation Project (PseudoCAP). The entries in EXProt all have a unique ID number and provide information about the organism, protein sequence, functional annotation, link to entry in original database, and if known, gene name and link to references in PubMed/Medline. The EXProt web page (http://www.cmbi.nl/EXProt) provides further details of the database and a link to a BLAST search (blastp & blastx) of the database. The EXProt entries are indexed in SRS (http://www.cmbi.nl/srs/) and can be searched by means of keywords. Authors can be reached by email (exprot(cmbi.kun.nl).
Abstract: The fls gene encoding fervidolysin, a keratin-degrading proteolytic enzyme from the thermophilic bacterium Fervidobacterium pennivorans, was isolated using degenerate primers combined with Southern hybridization and inverse polymerase chain reaction. Further sequence characterization demonstrated that the 2.1-kb fls gene encoded a 699-amino-acid preproenzyme showing high homology with the subtilisin family of the serine proteases. It was cloned into a pET9d vector, without its signal sequence, and expressed in Escherichia coli. The heterologously produced fervidolysin was purified by heat incubation followed by ion exchange chromatography and emerged in the soluble fraction as three distinct protein bands, as judged from sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Amino-terminal-sequence analysis of these bands and their comparison with that determined from biochemically purified keratinase and its predicted protein sequence, identified them as a 73-kDa fervidolysin precursor, a 58-kDa mature fervidolysin, and a 14-kDa fervidolysin propeptide. Using site-directed mutagenesis, the active-site histidine residue at position 79 was replaced by an alanine residue. The resulting fervidolysin showed a single protein band corresponding in size to the 73-kDa fervidolysin precursor, indicating that its proteolytic cleavage resulted from an autoproteolytic process. Knowledge-based modeling experiments showed a distinctive binding region for subtilases, in which binding of the propeptide could take place prior to autoproteolysis. Assays using keratin and other proteinaceous substrates did not display fervidolysin activity, perhaps because of the tight binding of the propeptide in the substrate-binding site, where it could then function as an inhibitor.
Abstract: Sulfur metabolism in gram-positive bacteria is poorly characterized. Information on the molecular mechanisms of regulation of genes involved in sulfur metabolism is limited, and no regulator genes have been identified. Here we describe the regulation of the lactococcal metC-cysK operon, encoding a cystathionine beta-lyase (metC) and cysteine synthase (cysK). Its expression was shown to be negatively affected by high concentrations of cysteine, methionine, and glutathione in the culture medium, while sulfur limitation resulted in a high level of expression. Other sulfur sources tested showed no significant effect on metC-cysK gene expression. In addition we found that O-acetyl-l-serine, the substrate of cysteine synthase, was an inducer of the metC-cysK operon. Using a random mutagenesis approach, we identified two genes, cmbR and cmbT, involved in regulation of metC-cysK expression. The cmbT gene is predicted to encode a transport protein, but its precise role in regulation remains unclear. Disruption of cmbT resulted in a two- to threefold reduction of metC-cysK transcription. A 5.7-kb region containing the cmbR gene was cloned and sequenced. The encoded CmbR protein is homologous to the LysR family of regulator proteins and is an activator of the metC-cysK operon. In analogy to CysB from Escherichia coli, we propose that CmbR requires acetylserine to be able to bind the activation sites and subsequently activate transcription of the metC-cysK operon.
Abstract: BACKGROUND: The genome of Mycobacterium tuberculosis H37Rv has five copies of a cluster of genes known as the ESAT-6 loci. These clusters contain members of the CFP-10 (lhp) and ESAT-6 (esat-6) gene families (encoding secreted T-cell antigens that lack detectable secretion signals) as well as genes encoding secreted, cell-wall-associated subtilisin-like serine proteases, putative ABC transporters, ATP-binding proteins and other membrane-associated proteins. These membrane-associated and energy-providing proteins may function to secrete members of the ESAT-6 and CFP-10 protein families, and the proteases may be involved in processing the secreted peptide. RESULTS: Finished and unfinished genome sequencing data of 98 publicly available microbial genomes has been analyzed for the presence of orthologs of the ESAT-6 loci. The multiple duplicates of the ESAT-6 gene cluster found in the genome of M. tuberculosis H37Rv are also conserved in the genomes of other mycobacteria, for example M. tuberculosis CDC1551, M. tuberculosis 210, M. bovis, M. leprae, M. avium, and the avirulent strain M. smegmatis. Phylogenetic analyses of the resulting sequences have established the duplication order of the gene clusters and demonstrated that the gene cluster known as region 4 (Rv3444c-3450c) is ancestral. Region 4 is also the only region for which an ortholog could be found in the genomes of Corynebacterium diphtheriae and Streptomyces coelicolor. CONCLUSIONS: Comparative genomic analysis revealed that the presence of the ESAT-6 gene cluster is a feature of some high-G+C Gram-positive bacteria. Multiple duplications of this cluster have occurred and are maintained only within the genomes of members of the genus Mycobacterium.
Abstract: The gene encoding the major intracellular tributyrin esterase of Lactococcus lactis was cloned using degenerate DNA probes based on 19 known N-terminal amino acid residues of the purified enzyme. The gene, named estA, was sequenced and found to encode a protein of 258 amino acid residues. The transcription start site was mapped 233 nucleotides upstream of the start codon, and a canonical promoter sequence was identified. The deduced amino acid sequence of the estA product contained the typical GXSXG motif found in most lipases and esterases. The protein was overproduced up to 170-fold in L. lactis by use of the nisin-controlled expression system recently developed for lactic acid bacteria. The estA gene was inactivated by chromosomal integration of a temperature-sensitive integration vector. This resulted in the complete loss of esterase activity, which could then be recovered after complementation of the constructed esterase-deficient strain with the wild-type estA gene. This confirms that EstA is the main enzyme responsible for esterase activity in L. lactis. Purified recombinant enzyme showed a preference for short-chain acyl esters, surprisingly also including phospholipids. Medium- and long-acyl-chain lipids were also hydrolyzed, albeit less efficiently. Intermediate characteristics between esterases and lipases make intracellular lactococcal EstA difficult to classify in either of these two groups of esterolytic enzymes. We suggest that, in vivo, EstA could be involved in (phospho)lipid metabolism or cellular detoxification or both, as its sequence showed significant similarity to S-formylglutathione hydrolase (FGH) of Paracoccus denitrificans and human EstD (or FGH), which are part of a universal formaldehyde detoxification pathway.
Abstract: The Lactococcus lactis SK11 cell envelope proteinase is an extracellular, multidomain protein of nearly 2,000 residues consisting of an N-terminal serine protease domain, followed by various other domains of largely unknown function. Using a strategy of deletion mutagenesis, we have analyzed the function of several C-terminal domains of the SK11 proteinase which are absent in cell envelope proteinases of other lactic acid bacteria. The various deletion mutants were functionally expressed in L. lactis and analyzed for enzyme stability, activity, (auto)processing, and specificity toward several substrates. C-terminal deletions of first the cell envelope W (wall) and AN (anchor) domains and then the H (helix) domain leads to fully active, secreted proteinases of unaltered specificity. Gradually increasing the C-terminal deletion into the so-called B domain leads to increasing instability and autoproteolysis and progressively less proteolytic activity. However, the mutant with the largest deletion (838 residues) from the C terminus and lacking the entire B domain still retains proteolytic activity. All truncated enzymes show unaltered proteolytic specificity toward various substrates. This suggests that the main role played by these domains is providing stability or protection from autoproteolysis (B domain), spacing away from the cell (H domain), and anchoring to the cell envelope (W and AN domains). In addition, this study allowed us to more precisely map the main C-terminal autoprocessing site of the SK11 proteinase and the epitope for binding of group IV monoclonal antibodies.
Abstract: The multi-domain, cell-envelope proteinases encoded by the genes prtB of Lactobacillus delbrueckii subsp. bulgaricus, prtH of Lactobacillus helveticus, prtP of Lactococcus lactis, scpA of Streptococcus pyogenes and csp of Streptococcus agalactiae have been compared using multiple sequence alignment, secondary structure prediction and database homology searching methods. This comparative analysis has led to the prediction of a number of different domains in these cell-envelope proteinases, and their homology, characteristics and putative function are described. These domains include, starting from the N-terminus, a pre-pro-domain for secretion and activation, a serine protease domain (with a smaller inserted domain), two large middle domains A and B of unknown but possibly regulatory function, a helical spacer domain, a hydrophilic cell-wall spacer or attachment domain, and a cell-wall anchor domain. Not all domains are present in each cell-envelope proteinase, suggesting that these multi-domain proteins are the result of gene shuffling and domain swapping during evolution.
Abstract: Nisin is an amphiphilic peptide with a strong antimicrobial activity against various Gram-positive bacteria. Its activity results from permeabilization of bacterial membranes, causing efflux of cytoplasmic compounds. To get information on the molecular mechanism of membrane permeabilization, a mutant of nisin Z containing the C-terminal extension Asp-(His)6 was produced. The biological and anionic lipid-dependent membrane activity of this peptide was very similar to that of nisin Z. Analysis of the pH dependence of model membrane interactions with the elongated peptide indicated the importance of electrostatic interactions of the C-terminus with the target membrane for membrane permeabilization. Most importantly, the membrane topology of the C-terminus of the molecule could be determined by trypsin digestion experiments, in which trypsin was encapsulated in the lumen of large unilamellar vesicles. The results show that the C-terminal part of the peptide translocates across model membranes. The pH and anionic lipid dependence of translocation closely paralleled the results of membrane permeabilization studies. Binding of nickel ions to the histidines blocked translocation of the C-terminus and concomitantly resulted in a 4-fold reduced capacity to induce K+ leakage. The results demonstrate for the first time that pore formation of nisin involves translocation of the C-terminal region of the molecule across the membrane.
Abstract: Nisin is a 34 residue long peptide belonging to the group A lantibiotics with antimicrobial activity against Gram-positive bacteria. The antimicrobial activity is based on pore formation in the cytoplasmic membrane of target organisms. The mechanism which leads to pore formation remains to be clarified. We studied the orientation of nisin via site-directed tryptophan fluorescence spectroscopy. Therefore, we engineered three nisin Z variants with unique tryptophan residues at positions 1, 17, and 32, respectively. The activity of the tryptophan mutants against Gram-positive bacteria and in model membrane systems composed of DOPC or DOPG was established to be similar to that of wild type nisin Z. The tryptophan fluorescence emission maximum showed an increasing blue-shift upon interaction with vesicles containing increased amounts of DOPG, with the largest effect for the 1W peptide. Studies with the aqueous quencher acrylamide showed that all tryptophans became inaccessible from the aqueous phase in the presence of negatively charged lipids in the vesicles. From these results it is concluded that anionic lipids mediate insertion of the tryptophan residues in at least three positions of the molecule into the lipid bilayer. The depth of insertion of the tryptophan residues was determined via quenching of the tryptophan fluorescence by spin-labeled lipids. The results showed that the depth of insertion was dependent on the amount of negatively charged lipids. In membranes containing 50% DOPG, the distances from the bilayer center were determined to be 15.7, 15.0, and 18.4 A for the tryptophan at position 1, 17, and 32, respectively. In membranes containing 90% DOPG, these distances were calculated to be 10.8, 11.5, and 13.1 A, respectively. These results suggest an overall parallel average orientation of nisin in the membrane, with respect to the membrane surface, with the N-terminus more deeply inserted than the C-terminus. These data were used to model the orientation of nisin in the membrane.
Abstract: Subtilases are members of the clan (or superfamily) of subtilisin-like serine proteases. Over 200 subtilases are presently known, more than 170 of which with their complete amino acid sequence. In this update of our previous overview (Siezen RJ, de Vos WM, Leunissen JAM, Dijkstra BW, 1991, Protein Eng 4:719-731), details of more than 100 new subtilases discovered in the past five years are summarized, and amino acid sequences of their catalytic domains are compared in a multiple sequence alignment. Based on sequence homology, a subdivision into six families is proposed. Highly conserved residues of the catalytic domain are identified, as are large or unusual deletions and insertions. Predictions have been updated for Ca(2+)-binding sites, disulfide bonds, and substrate specificity, based on both sequence alignment and three-dimensional homology modeling.
Abstract: The interaction of nisin Z and a nisin Z mutant carrying a negative charge in the C-terminus ([Glu-32]-nisin Z) with anionic lipids was characterized in model membrane systems, and bacterial membrane systems. We focused on three possible steps in the mode of action of nisin, i.e., binding, insertion, and pore formation of nisin Z. Increasing amounts of anionic lipids in both model and natural membranes were found to strongly enhance the interaction of nisin Z with the membranes at all stages. The results reveal a good correlation between the anionic lipid dependency of the three stages of interaction, of which the increased binding is probably the major determinant for antimicrobial activity. Maximal nisin Z activity could be observed for negatively charged lipid concentrations exceeding 50-60%, both in model membrane systems as well as in bacterial membrane systems. We propose that the amount of negatively charged lipids of the bacterial target membrane is a major determinant for the sensitivity of the organism for nisin. Nisin Z induced leakage of the anionic carboxyfluorescein was more efficient as compared to the leakage of the potassium cation. This lead to the conclusion that an anion-selective pore is formed. In contrast to the results obtained for nisin Z, the binding of [Glu-32]-nisin Z to vesicles remained low even in the presence of high amounts of negatively charged lipids. The insertion and pore-forming ability of [Glu-32]-nisin Z were also decreased. These results demonstrate that the C-terminus of nisin is responsible for the initial interaction of nisin, i.e., binding to the target membrane.
Abstract: Three mutants of the antibiotic nisin Z, in which the Val32 residue was replaced by a Glu, Lys or Trp residue, were produced and characterized for the purpose of establishing the role of charge differences in the C-terminal part of nisin on antimicrobial activity and signaling properties. 1H-NMR analyses showed that all three mutants harbor an unmodified serine residue at position 33, instead of the usual dehydroalanine. Apparently, the nature of the residue preceding the serine to be dehydrated, strongly affects the efficiency of modification. Cleavage of [Glu32,Ser33]nisin Z by endoproteinase Glu-C yielded [Glu32]nisin Z(1-32)-peptide, which has a net charge difference of -2 relative to wild-type nisin Z. The activity of [Lys32,Ser33]nisin Z against Micrococcus flavus was similar to that of wild-type nisin, while [Trp32,Ser33]nisin Z, [Glu32,Ser33]nisin Z and [Glu32]nisin Z(1-32)-peptide exhibited 3-5-fold reduced activity, indicating that negative charges in the C-terminal part of nisin Z are detrimental for activity. All variants showed significant loss of activity against Streptococcus thermophilus. The potency of the nisin variants to act as signaling molecules for auto-induction of biosynthesis was significantly reduced. To obtain mutant production, extracellular addition of (mutant) nisin Z to the lactococcal expression strains was essential.
Abstract: The hyperthermophilic archaeon Pyrococcus furiosus produces an extracellular, glycosylated hyperthermostable subtilisin-like serine protease, termed pyrolysin (Voorhorst,W.G.B., Eggen,R.I.L., Geerling,A.C.M., Platteeuw,C., Siezen,R.J. and de Vos,W.M. (1996) J. Biol. Chem., 271, 20426-20431). Based on the pyrolysin coding sequence, a pyrolysin-like gene fragment was cloned and characterized from the extreme thermophilic archaeon Thermococcus stetteri. Like pyrolysin, the deduced sequence of this serine protease, designated stetterlysin, contains a catalytic domain with high homology with other subtilases, allowing homology modelling starting from known crystal structures. Comparison of the predicted three-dimensional models of the catalytic domain of stetterlysin and pyrolysin with the crystal structure of subtilases from mesophilic and thermophilic origin, i.e. subtilisin BPN' and thermitase, and the homology model of subtilisin S41 from psychrophilic origin, led to the identification of features that could be related to protein stabilization. Higher thermostability was found to be correlated with an increased number of residues involved in pairs and networks of charge-charge and aromatic-aromatic interactions. These highly thermostable proteases have several extra surface loops and inserts with a relatively high frequency of aromatic residues and Asn residues. The latter are often present in putative N-glycosylation sites. Results from modelling of known substrates in the substrate-binding region support the broad substrate range and the autocatalytic activation previously suggested for pyrolysin.
Abstract: The antimicrobial membrane-interacting polypeptide nisin is a prominent member of the lantibiotic family, the members of which contain thioether-bridged residues called lanthionines. To gain insight into the complex biosynthesis and the structure/function relationship of lantibiotics, the final intermediate in the biosynthesis of nisin A was studied by nuclear magnetic resonance spectroscopy. In aqueous solution the leader peptide part of this precursor adopts predominantly a random coil structure, as does the synthetic leader peptide itself. The spatial structure of the fully modified nisin part of the precursor is similar to that of nisin in water. The leader peptide part does not interact with the nisin part of the precursor molecule. Thus, these two parts of the precursor do not influence each other's conformation significantly. The conformation of the precursor was also studied while complexed to micelles of dodecylphosphocholine, mimicking the primary target of the antimicrobial activity of nisin, i.e. the cytoplasmic membrane. The location of the molecule relative to the micelles was investigated by using micelle-inserted spin-labeled 5-doxylstearic acid. It was observed that the N-terminal half of the nisin part of the precursor interacts in a different way with micelles than does the corresponding part of mature nisin, whereas no significant differences were found for the C-terminal half of the nisin part. In this model system the leader peptide is in contact with the micelles. It is concluded that the strongly reduced in vivo activity of the precursor molecule relative to that of nisin is not caused by a difference in the spatial structure of nisin and of the corresponding part of precursor nisin in water or by a shielding of the membrane interaction surface of the nisin part of the precursor by the leader peptide. Probably a different interaction of the N-terminal part of the nisin region with membranes contributes to the low activity by preventing productive insertion. The residues of the leader peptide part just next to the nisin part are likely to contribute most to the low activity of the precursor.
Abstract: Clinical isolates of Enterococcus faecalis more commonly produce a cytolysin than do commensal isolates. Epidemiologic evidence and animal-model studies have established a role for the cytolysin in the pathogenesis of enterococcal disease. The cytolysin consists of two structural subunits, CylLL and CylLS, that are activated by a third component, CylA. Genetic and biochemical characterization of CylA indicate that it is a serine protease, and that activation putatively results from cleavage of one or both cytolysin subunits. Genetic evidence also suggests that the cytolysin subunits are related to the rapidly growing class of bacteriocins termed lantibiotics. However, unlike lantibiotics, the cytolysin is lytic for eukaryotic as well as prokaryotic cells, and it consists of two structural subunits. This report describes the purification and characterization of the cytolysin subunits and detection of lanthionine-type post-translational modifications within their structures. Furthermore, the cleavage specificity of the CylA activator is reported and it is shown that proteolytic activation of both subunits is essential for activity.
Abstract: Nisin, a 34-residue peptide bacteriocin, contains the less common amino acids lanthionine, beta-methyl-lanthionine, dehydroalanine (Dha), and dehydrobutyrine (Dhb). Several chemically modified nisin A species were purified by reverse-phase HPLC and characterized by two-dimensional NMR and electrospray mass spectrometry. Five constituents, [2-hydroxy-Ala5]nisin, [Ile4-amide,pyruvyl-Leu6]des-Dha5-nisin, [Met(O)21]nisin, [Ser33]nisin, and nisin-(1-32)-peptide amide, were found in a commercial nisin sample. A further species, [2-hydroxy-Ala5]nisin-(1-32)-peptide amide, was obtained by freeze drying an acidic nisin solution. These compounds are formed by chemical modification of nisin: the addition of a water molecule to the dehydroalanine residues, which can lead to the cleavage of the polypeptide chain, or the oxidation of methionine residues. The 2-hydroxyalanine-containing products have a limited stability; they are spontaneously converted into the corresponding des-dehydroalanine derivatives. The growth-inhibiting activity of the modified nisins towards different bacteria was determined. The 2-hydroxyalanine-containing species and the des-dehydroalanine derivative show a strong reduction in biological activity as compared to native nisin. [Met(O)21]nisin and [Ser33]nisin show moderate or no reduction in biological activity.
Abstract: The hyperthermostable serine protease pyrolysin from the hyperthermophilic archaeon Pyrococcus furiosus was purified from membrane fractions. Two proteolytically active fractions were obtained, designated high (HMW) and low (LMW) molecular weight pyrolysin, that showed immunological cross-reaction and identical NH2-terminal sequences in which the third residue could be glycosylated. The HMW pyrolysin showed a subunit mass of 150 kDa after acid denaturation. Incubation of HMW pyrolysin at 95 degrees C resulted in the formation of LMW pyrolysin, probably as a consequence of COOH-terminal autoproteolysis. The 4194-base pair pls gene encoding pyrolysin was isolated and characterized, and its transcription initiation site was identified. The deduced pyrolysin sequence indicated a prepro-enzyme organization, with a 1249-residue mature protein composed of an NH2-terminal catalytic domain with considerable homology to subtilisin-like serine proteases and a COOH-terminal domain that contained most of the 32 possible N-glycosylation sites. The archaeal pyrolysin showed highest homology with eucaryal tripeptidyl peptidases II on the amino acid level but a different cleavage specificity as shown by its endopeptidase activity toward caseins, casein fragments including alphaS1-casein and synthetic peptides.
Abstract: The lantibiotic lacticin 481 is a bacteriocin produced by Lactococcus lactis ssp. lactis. This polypeptide contains 27 amino acids, including the unusual residues dehydrobutyrine and the thioether-bridging lanthionine and 3-methyllanthionine. Lacticin 481 belongs to a structurally distinct group of lantibiotics, which also include streptococcin A-FF22, salivaricin A and variacin. Here we report the first complete structure of this type of lantibiotic. The exact location of the thioether bridges in lacticin 481 was determined by a combination of peptide chemistry, mass spectrometry and NMR spectroscopy, showing connections between residues 9 and 14, 11 and 25, and 18 and 26.
Abstract: Homology modelling was used to predict enzyme-substrate interactions in three entirely different subtilisin-like enzymes of unknown three-dimensional structure. i.e. (a) cell-envelope proteinase of Lactococcus lactis, (b) putative leader peptidase for pre-nisin from L. lactis, and (c) human furin. Models were based on known three-dimensional structures of subtilisins and thermitase in complex with inhibitors. Detailed analysis of interactions of the P1-P4 residues of model substrates with the S1-S4 binding sites in each enzyme suggest that electrostatic interactions at all four binding sites can contribute to binding and hence to specificity. In particular, one or more negative charges in the S1 or S4 pockets can lead to a high selectivity for Arg residues in the substrate. Many of the predicted interactions have been confirmed by engineering of either enzyme, substrate or both.
Abstract: Monomolecular layers of lipids at the air/water interface have been used as a model membrane to study membrane interactions of the lantibiotic nisin. The natural lantibiotics nisin A and nisin Z proved to have a high affinity for the anionic lipids phosphatidylglycerol and bis(phosphatidyl)glycerol (cardiolipin). The interaction with zwitterionic phopholipids or neutral lipids is very low at surface pressures higher than 32 mN/m. Nisin, nisin mutants and lacticin 481 show a remarkable correlation between anti-microbial activity and anionic lipid interaction. The results indicate that primarily the N-terminal part (residues 1-22) penetrates into the lipid phase. Reduction of the flexibility at positions 20-21 has a negative effect on monolayer interaction and activity. The C-terminal part is probably responsible for ionic interactions of nisin in monomeric or oligomeric form with anionic lipids. In mixtures of anionic and zwitterionic lipids maximal interactions are found at approximately 70 mol/100 mol anionic lipid. Gram-positive bacteria, which form the main target for nisin, are characterized by a high content of anionic lipids in the membrane. Monolayers formed of lipid extracts of bacteria sensitive to nisin were more strongly penetrated than those of bacteria relatively insensitive to nisin.
Abstract: Lantibiotics form a group of modified peptides with unique structures, containing post-translationally modified amino acids such as dehydrated and lanthionine residues. In the gram-positive bacteria that secrete these lantibiotics, the gene clusters flanking the structural genes for various linear (type A) lantibiotics have recently been characterized. The best studied representatives are those of nisin (nis), subtilin (spa), epidermin (epi), Pep5 (pep), cytolysin (cyl), lactocin S (las) and lacticin 481 (lct). Comparison of the lantibiotic gene clusters shows that they contain conserved genes that probably encode similar functions. The nis, spa, epi and pep clusters contain lanB and lanC genes that are presumed to code for two types of enzymes that have been implicated in the modification reactions characteristic of all lantibiotics, i.e. dehydration and thio-ether ring formation. The cyl, las and lct gene clusters have no homologue of the lanB gene, but they do contain a much larger lanM gene that is the lanC gene homologue. Most lantibiotic gene clusters contain a lanP gene encoding a serine protease that is presumably involved in the proteolytic processing of the prelantibiotics. All clusters contain a lanT gene encoding an ABC transporter likely to be involved in the export of (precursors of) the lantibiotics. The lanE, lanF and lanG genes in the nis, spa and epi clusters encode another transport system that is possibly involved in self-protection. In the nisin and subtilin gene clusters two tandem genes, lanR and lanK, have been located that code for a two-component regulatory system. Finally, non-homologous genes are found in some lantibiotic gene clusters. The nisI and spaI genes encode lipoproteins that are involved in immunity, the pepI gene encodes a membrane-located immunity protein, and epiD encodes an enzyme involved in a post-translational modification found only in the C-terminus of epidermin. Several genes of unknown function are also found in the las gene cluster. A database has been assembled for all putative gene products of type A lantibiotic gene clusters. Database searches, multiple sequence alignment and secondary structure prediction have been used to identify conserved sequence segments in the LanB, LanC, LanE, LanF, LanG, LanK, LanM, LanP, LanR and LanT gene products that may be essential for structure and function. This database allows for a rapid screening of newly determined sequences in lantibiotic gene clusters.
Abstract: Whereas protein engineering of enzymes and structural proteins nowadays is an established research tool for studying structure-function relationships of polypeptides and for improving their properties, the engineering of posttranslationally modified peptides, such as the lantibiotics, is just coming of age. The engineering of lantibiotics is less straightforward than that of unmodified proteins, since expression systems should be developed not only for the structural genes but also for the genes encoding the biosynthetic enzymes, immunity protein and regulatory proteins. Moreover, correct posttranslational modification of specific residues could in many cases be a prerequisite for production and secretion of the active lantibiotic, which limits the number of successful mutations one can apply. This paper describes the development of expression systems for the structural lantibiotic genes for nisin A, nisin Z, gallidermin, epidermin and Pep5, and gives examples of recently produced site-directed mutants of these lantibiotics. Characterization of the mutants yielded valuable information on biosynthetic requirements for production. Moreover, regions in the lantibiotics were identified that are of crucial importance for antimicrobial activity. Eventually, this knowledge will lead to the rational design of lantibiotics optimally suited for fighting specific undesirable microorganisms. The mutants are of additional value for studies directed towards the elucidation of the mode of action of lantibiotics.
Abstract: A model is presented for the 3-D structure of the catalytic domain of the putative leader peptidase NisP of Lactococcus lactis, and the interaction with its specific substrate, the precursor of the lantibiotic nisin. This homology model is based on the crystal structures of subtilisin BPN' and thermitase in complex with the inhibitor eglin. Predictions are made of the general protein fold, inserted loops, Ca2+ binding sites, aromatic interactions and electrostatic interactions of NisP. Cleavage of the leader peptide from precursor nisin by NisP is the last step in maturation of nisin. A detailed prediction of the substrate binding site attempts to explain the basis of specificity of NisP for precursor nisin. Specific acidic residues in the S1 subsite of the substrate binding region of NisP appear to be of particular importance for electrostatic interaction with the P1 Arg residue of precursor nisin after which cleavage occurs. The hydrophobic S4 subsite of NisP may also contribute to substrate binding as it does in subtilisins. Predictions of enzyme-substrate interaction were tested by protein engineering of precursor nisin and determining susceptibility of mutant precursors to cleavage by NisP. An unusual property of NisP predicted from this catalytic domain model is a surface patch near the substrate binding region which is extremely rich in aromatic residues.(ABSTRACT TRUNCATED AT 250 WORDS)
Abstract: Lantibiotics are bacteriocins that contain unusual amino acids such as lanthionines and alpha, beta-didehydro residues generated by posttranslational modification of a ribosomally synthesized precursor protein. The structural gene encoding the novel lantibiotic epilancin K7 from Staphylococcus epidermidis K7 was cloned and its nucleotide sequence was determined. The gene, which was named elkA, codes for a 55-residue preprotein, consisting of an N-terminal 24-residue leader peptide, and a C-terminal 31-residue propeptide which is posttranslationally modified and processed to yield mature epilancin K7. In common with the type-A lantibiotics nisin A and nisin Z, subtilin, epidermin, gallidermin and Pep5, pre-epilancin K7 has a so-called class-Al leader peptide. Downstream and upstream of the elkA gene, the starts of two open-reading-frames, named elkP and elkT, were identified. The elkP and elkT genes presumably encode a leader peptidase and a translocator protein, respectively, which may be involved in the processing and export of epilancin K7. The amino acid sequence of the unmodified pro-epilancin K7, deduced from the elkA gene sequence, is in full agreement with the amino acid sequence of mature epilancin K7, determined previously by means of NMR spectroscopy [van de Kamp, M., Horstink, L. M., van den Hooven, M. W., Konings, R. N. M., Hilbers, C. W., Sahl, H.-G., Metzger, J. W. & van de Ven, F. J. M. (1995) Eur. J. Biochem. 227, 757-771]. The first residue of mature epilancin K7 appears to be modified in a way that has not been described for any other lantibiotic so far. NMR experiments show that the elkA-encoded serine residue at position +1 of pro-epilancin K7 is modified to a 2-hydroxypropionyl residue in the mature protein.
Abstract: Lantibiotics form a family of highly modified peptides which are secreted by several Gram-positive bacteria. They exhibit antimicrobial activity, mainly against other Gram-positive bacteria, by forming pores in the cellular membrane. These antimicrobial peptides are ribosomally synthesized and contain leader peptides which do not show the characteristics of signal sequences. Several amino acid residues of the precursor lantibiotic are enzymatically modified, whereafter secretion and processing of the leader peptide takes place, yielding the active antimicrobial substance. For several lantibiotics the gene clusters encoding biosynthetic enzymes, translocator proteins, self-protection proteins, processing enzymes and regulatory proteins have been identified. This MicroReview describes the current knowledge about the biosynthetic, immunity and regulatory processes leading to lantibiotic production. Most of the attention is focused on the lantibiotic nisin, which is produced by the food-grade bacterium Lactococcus lactis and is widely used as a preservative in the food industry.
Abstract: Nisin is a 3.4-kDa antimicrobial peptide that, as a result of posttranslational modifications, contains unsaturated amino acids and lanthionine residues. It is applied as a preservative in various food products. The solubility and stability of nisin and nisin mutants have been studied. It is demonstrated that nisin mutants can be produced with improved functional properties. The solubility of nisin A is highest at low pH values and gradually decreases by almost 2 orders of magnitude when the pH of the solution exceeds a value of 7. At low pH, nisin Z exhibits a decreased solubility relative to that of nisin A; at neutral and higher pH values, the solubilities of both variants are comparable. Two mutants of nisin Z, which contain lysyl residues at positions 27 and 31, respectively, instead of Asn-27 and His-31, were produced with the aim of reaching higher solubility at neutral pH. Both mutants were purified to homogeneity, and their structures were confirmed by one- and two-dimensional 1H nuclear magnetic resonance. Their antimicrobial activities were found to be similar to that of nisin Z, whereas their solubilities at pH 7 increased by factors of 4 and 7, respectively. The chemical stability of nisin A was studied in the pH range of 2 to 8 and at a 20, 37, and 75 degrees C. Optimal stability was observed at pH 3.0. Nisin Z showed a behavior similar to that of nisin A. A mutant containing dehydrobutyrine at position 5 instead of dehydroalanine had lower activity but was significantly more resistant to acid-catalyzed chemical degradation than wild-type nisin Z.
Abstract: The catalytic domain of the cell-envelope proteinase from Lactococcus lactis SK11 has various inserts, situated in external loops of the catalytic domain, compared with the related subtilisins. Protein engineering was employed to analyse the necessity and function of one of these extra loops (residues 205-219), that is predicted to be located in close proximity to the substrate-binding region and is susceptible to autoproteolysis. We constructed a deletion mutant which lacks 14 residues of this surface loop and subsequently introduced various insertion cassettes coding either for the original loop with three mutations (E205S/E218T/M219S: triple-mutant proteinase) or for neutral spacers (1, 4, 7 and 16 serine residues). Engineered proteinases were analysed for activity, (auto)processing, and cleavage specificity. The presence of residues 205-219 is shown to be essential for proteolytic activity, as only triple-mutant proteinase retained activity towards casein substrates. The triple-mutant proteinase was found to be defective in C-terminal autoprocessing, and subsequent release from the lactococcal cell envelope in a calcium-free medium, indicative of either an altered proteolytic specificity or altered accessibility of the processing site. The specificity change appears to be subtle, as only small differences were found between wild-type and triple-mutant proteinase in the breakdown of casein substrates.
Abstract: The Lactococcus lactis SK11 cell-envelope proteinase contains various inserts, located in external loops of the catalytic domain compared with related subtilisins. In this study, protein engineering was employed to determine the function of the largest loop insertion (residues 238-388) relative to the subtilisin structure. By site-directed mutagenesis we have deleted the fragment of the proteinase gene encoding these 151 residues and analyzed the mutant delta 238-388 proteinase for activity, (auto)processing and cleavage specificity. This extra segment is found to be inessential for activity, and its removal does not inhibit folding as the mutant proteinase is still active. In addition, the N- and C-terminal autoprocessing of the delta 238-388 proteinase appears to be unchanged. However, removal of residues 238-388 altered substantially the caseinolytic specificity of the enzyme, indicating that this extra segment influences substrate specificity. Residues 238-388 were shown to contain a specific epitope for a monoclonal antibody.
Abstract: Structural genes for small lanthionine-containing antimicrobial peptides, known as lantibiotics, encode N-terminal leader sequences which are not present in the mature peptide, but are cleaved off at some stage in the maturation process. Leader sequences of the different lantibiotics share a number of identical amino acid residues, but they are clearly different from sec-dependent protein export signal sequences. We studied the role of the leader sequence of the lantibiotic nisin, which is produced and secreted by Lactococcus lactis, by creating site-directed mutations at various positions in the leader peptide sequence. Mutations at Arg-1 and Ala-4, but not at the conserved Pro-2, strongly affected the processing of the leader sequence and resulted in the extracellular accumulation of a biologically inactive precursor peptide. Amino acid analysis and 1H NMR studies indicated that the precursor peptide with an Ala-4-->Asp mutation contained a modified nisin structural part with the (mutated) unmodified leader sequence still attached to it. The Ala-4-->Asp precursor peptide could be activated in vitro by enzymatic cleavage with trypsin, liberating nisin. These results confirmed that cleavage of the leader peptide is the last step in nisin maturation and is necessary to generate a biologically active peptide. Several mutations, i.e. Pro-2-->Gly,Pro-2-->Val, Asp-7-->Ala,Lys-9-->Leu,Ser-10-->Ala/Ser-12-->Ala and Val-11-->Asp/Val-13-->Glu in the leader peptide did not have any detectable effect on nisin production and secretion, although some of them affected highly conserved residues. When mutations were created in the -18 to -15 region of the nisin leader peptide (i.e. Phe-18-->Leu,Leu-16-->Lys,Asp-15-->Ala), no secretion or intracellular accumulation could be detected of nisin or its precursors. This suggested that these conserved residues are involved in the maturation process and may interact with lantibiotic-specific modifying enzymes.
Abstract: A model is presented for the three-dimensional structure of the catalytic domain of the human serine proteinase furin and its interaction with model substrates. This homology model is based on the crystal structures of subtilisin BPN' and thermitase in complex with the inhibitor eglin, and it also applies to other members of the eukaryotic subtilisin-like proprotein convertases. Predictions are made of the general protein fold, inserted loops, disulfide bonds, Ca(2+)-binding sites and salt bridges. A detailed prediction of the substrate-binding region attempts to explain the basis of specificity for multiple basic residues preceding the cleavage site. Specific acidic residues in the S1, S2 and S4 subsites of the substrate-binding region of furin are identified which appear to be of particular importance, while residues of the S2', S3, S5 and S6 subsites may also contribute to substrate binding. Based on this model, protein engineering can be employed not only to test the predicted enzyme-substrate interactions, as demonstrated for human furin, but, equally importantly, to design proprotein convertases with a desired specificity, or to design novel substrates or inhibitors.
Abstract: The DNA sequence encoding the leader peptide of the lantibiotic subtilin from Bacillus subtilis was fused to the sequence encoding pronisin Z, and this hybrid gene was expressed in a Lactococcus lactis strain that produces nisin A. This strain simultaneously secreted nisin A and a protein of approximately 6 kDa. Amino acid sequencing of the purified 6 kDa protein and structural analysis of its main tryptic fragment by two-dimensional 1H-NMR showed that it consists of the unmodified leader peptide of subtilin, without the N-terminal methionine residue, linked to a fully matured nisin Z part. The hybrid protein and its main tryptic fragment [ITPQ]-nisin Z, showed at least 200-fold lower antimicrobial activities than nisin Z against three different indicator strains.
Abstract: A cDNA clone containing the entire coding region for bovine beta-casein A3 flanked by 53 base pairs of 5' non-coding and 358 base pairs of 3' non-coding sequences was isolated from a bovine mammary cDNA phagemid library. The coding segment for mature beta-casein was subcloned into the T7 expression system, in which the expression of recombinant beta-casein was controlled by the T7 gene 10 promoter and ribosome binding site. High level expression of Met-beta-casein to approximately 20% of the total soluble proteins was obtained in Escherichia coli within 2 h after induction of T7 RNA-polymerase synthesis. In an attempt to induce secretion the coding segment for mature beta-casein was coupled to the ompA translational initiation signal and signal peptide coding sequence but no secretion of the fusion protein and no processing of the signal peptide from the fusion protein was observed. Instead, the Met-beta-casein could be isolated in a soluble form from E.coli cells after an osmotic shock, indicative of a periplasmic location. This procedure did not lyse the cells. The protein was purified to homogeneity after a pH 4.8 isoelectric precipitation followed by reversed-phase high-performance liquid chromatography. The beta-casein cDNA was altered to change the main chymosin cleavage site in beta-casein at position 192-193 in two ways, namely from Leu-Tyr to Pro-Pro and to Leu-stop.(ABSTRACT TRUNCATED AT 250 WORDS)
Abstract: The proprotein processing activity of mutants of the subtilisin-like enzyme furin was studied in transfected mammalian cells. Our studies indicate that the three residues of the catalytic triad of furin, Asp46, His87, and Ser261, are critical not only for substrate processing but also for maturation of furin. Furthermore, evidence is provided that maturation of furin occurs through an intramolecular autocatalytic process. Substitution of the asparagine residue (Asn188) of the oxyanion hole by an alanine residue appears to block substrate processing but not furin maturation. Analysis of carboxyl-terminal deletion mutants revealed that the segment encompassing residues Glu449 to Glu469 of the "middle" domain, which is more than 100 residues downstream of the predicted catalytic domain, contains residues that seem to be critical for processing activity but that the more carboxyl-terminal cysteine-rich region, the transmembrane region, and the cytosolic tail are dispensable. Finally, we made mutants in the substrate binding region of human furin and studied their ability to process von Willebrand factor (pro-vWF) substrates, including wild-type pro-vWF as well as pro-vWF mutants in which the P1 (vWFR-1G), P2 (vWFK-2A), or P4 (vWFR-4A) basic residue with respect to the pro region cleavage site had been mutated. It is demonstrated that particular negatively charged residues in or near the substrate binding region of furin are critical for cleavage activity and specificity of the enzyme for multiple basic residues in the substrate. Furthermore, substrate binding region mutants of furin were obtained, which cleaved either the pro-vWFK-2A or pro-vWFR-4A mutant of pro-vWF more efficiently than wild-type pro-vWF.
Abstract: The nisin gene cluster nisABTCIPR of Lactococcus lactis, located on a 10-kbp DNA fragment of the nisin-sucrose transposon Tn5276, was characterized. This fragment was previously shown to direct nisin-A biosynthesis and to contain the nisP and nisR genes, encoding a nisin leader peptidase and a positive regulator, respectively [van der Meer, J. R., Polman, J., Beerthuyzen, M. M., Siezen, R. J., Kuipers, O. P. & de Vos, W. M. (1993) J. Bacteriol. 175, 2578-2588]. Further sequence analysis revealed the presence of four open-reading frames, nisB, nisT, nisC and nisI, downstream of the structural gene nisA. The nisT, nisC and nisI genes were subcloned and expressed individually in Escherichia coli, using the T7-RNA-polymerase system. This resulted in the production of radiolabelled proteins with sizes of 45 kDa (NisC) and 32 kDa (NisI). The nisT gene product was not detected, possibly because of protein instability. The deduced amino acid sequence of NisI contained a consensus lipoprotein signal sequence, suggesting that this protein is a lipid-modified extracellular membrane-anchored protein. Expression of nisI in L. lactis provided the cells with a significant level of protection against exogenously added nisin, indicating that NisI plays a role in the immunity mechanism. In EDTA-treated E. coli cells, expression of nisI conferred up to a 170-fold increase in immunity against nisin A compared to controls. Moreover, a lactococcal strain deficient in nisin-A production, designated NZ9800, was created by gene replacement of nisA by a truncated nisA gene and was 10-fold less resistant to nisin A than the wild-type strain. A wild-type immunity level to nisin and production of nisin was obtained in strain NZ9800 harboring complementing nisA and nisZ plasmids. Transcription analyses of several L. lactis strains indicated that an expression product of the nisA gene, together with NisR, is required for the activation of nisA transcription.
Abstract: The substrate-binding region of the cell-envelope proteinase of Lactococcus lactis strain SK11 was modelled, based on sequence homology of the catalytic domain with the serine proteinases subtilisin and thermitase. Substitutions, deletions and insertions were introduced, by site-directed and cassette mutagenesis of the prtP gene encoding this enzyme, based on sequence comparison both with subtilisin and with the homologous L.lactis strain Wg2 proteinase, which has different proteolytic properties. The engineered enzymes were investigated for thermal stability, proteolytic activity and cleavage specificity towards small chromogenic peptide substrates and the peptide alpha s1-casein(1-23). Mutations in the subtilisin-like substrate-binding region showed that Ser433 is the active site residue, and that residues 138 and 166 at either side of the binding cleft play an important role in substrate specificity, particularly when these residues and the substrate are oppositely charged. The K748T mutation in a different domain also affected specificity and stability, suggesting that this residue is in close proximity to the subtilisin-like domain and may form part of the substrate-binding site. Several mutant SK11 proteinases have novel properties not previously encountered in natural variants. Replacements of residues 137-139AKT along one side of the binding cleft produced the 137-139GPP mutant proteinase with reduced activity and narrowed specificity, and the 137-139GLA mutant with increased activity and broader specificity. Furthermore, the 137-139GDT mutant had a specificity towards alpha s1-casein(1-23) closely resembling that of L.lactis Wg2 proteinase. Mutants with an additional negative charge in the binding region were more stable towards autoproteolysis.
Abstract: Biosynthesis of the lantibiotic peptide nisin by Lactococcus lactis NIZO R5 relies on the presence of the conjugative transposon Tn5276 in the chromosome. A 12-kb DNA fragment of Tn5276 including the nisA gene and about 10 kb of downstream DNA was cloned in L. lactis, resulting in the production of an extracellular nisin precursor peptide. This peptide reacted with antibodies against either nisin A or the synthetic leader peptide, suggesting that it consisted of a fully modified nisin with the nisin leader sequence still attached to it. This structure was confirmed by N-terminal sequencing and 1H-nuclear magnetic resonance analysis of the purified peptide. Deletion studies showed that the nisR gene is essential for the production of this intermediate. The deduced amino acid sequence of the nisR gene product indicated that the protein belongs to the family of two-component regulators. The deduced amino acid sequence of NisP, the putative product of the gene upstream of nisR, showed an N-terminal signal sequence, a catalytic domain with a high degree of similarity to those of subtilisin-like serine proteases, and a putative C-terminal membrane anchor. Cell extracts of Escherichia coli overexpressing nisP were able to cleave the nisin precursor peptide, producing active, mature nisin. A similar activation was obtained with whole cells but not with membrane-free extracts of L. lactis strains carrying Tn5276 in which the nisA gene had been inactivated. The results indicate that the penultimate step in nisin biosynthesis is secretion of precursor nisin without cleavage of the leader peptide, whereas the last step is the cleavage of the leader peptide sequence from the fully maturated nisin peptide.
Abstract: Two natural variants of the lantibiotic nisin that are produced by Lactococcus lactis are known. They have a similar structure but differ in a single amino acid residue at position 27; histidine in nisin A and asparagine in nisin Z (J.W.M. Mulders, I.J. Boerrigter, H.S. Rollema, R.J. Siezen, and W.M. de Vos, Eur. J. Biochem, 201:581-584, 1991). The nisin variants were purified to apparent homogeneity, and their biological activities were compared. Identical MICs of nisin A and nisin Z were found with all tested indicator strains of six different species of gram-positive bacteria. However, at concentrations above the MICs, with nisin Z the inhibition zones obtained in agar diffusion assays were invariably larger than those obtained with nisin A. This was observed with all tested indicator strains. These results suggest that nisin Z has better diffusion properties than nisin A in agar. The distribution of the nisin variants in various lactococcal strains was determined by amplification of the nisin structural gene by polymerase chain reaction followed by direct sequencing of the amplification product. In this way, it was established that the nisZ gene for nisin Z production is widely distributed, having been found in 14 of the 26 L. lactis strains analyzed.
Abstract: The small antimicrobial peptide nisin, produced by Lactococcus lactis, contains the uncommon amino acid residues dehydroalanine and dehydrobutyrine and five thio ether bridges. Since these structures are posttranslationally formed from Ser, Thr, and Cys residues, it is feasible to study their role in nisin function and biosynthesis by protein engineering. Here we report the development of an expression system for mutated nisin Z (nisZ) genes, using nisin A producing L. lactis as a host. Replacement by site-directed mutagenesis of the Ser-5 codon in nisZ by a Thr codon, led to a mutant with a dehydrobutyrine instead of a dehydroalanine residue at position 5, as shown by NMR. Its antimicrobial activity was 2-10-fold lower relative to wild-type nisin Z, depending on the indicator strain used. In another mutagenesis study a double mutation was introduced in the nisZ gene by replacing the codons for Met-17 and Gly-18 by codons for Gln and Thr, respectively, as in the third lanthionine ring of the related antimicrobial peptide subtilin from Bacillus subtilis. This resulted in the simultaneous production of two mutant species, one containing a Thr residue and the other containing a dehydrobutyrine residue at position 18, both having different bacteriocidal properties.
Abstract: The nucleotide sequence of the pepN gene from Lactococcus lactis encoding a zinc-metallo aminopeptidase has been determined. The open reading frame of 2,538 base pairs encodes a protein with a calculated M(r) of 95,368, which agrees with the apparent M(r) of 95,000 of the gene product which was identified by polyclonal antibodies raised against the purified aminopeptidase. The amino acid sequence of the aminopeptidase of L. lactis was found to be similar to the corresponding enzymes of human, rat and mouse, with almost 30% of the residues identical. Also, a highly conserved area was identified which has similarity with the active site of thermolysin. A zinc-binding site, as well as the catalytic site for PepN, is predicted to lie within this conserved stretch. Putative promoter regions upstream of PepN were confirmed by primer extension analysis.
Abstract: Plasmids containing wild-type and hybrid proteinase genes were constructed from DNA fragments of the prtP genes of Lactococcus lactis strains Wg2 and SK11. These plasmids were introduced into the plasmid-free strain L. lactis MG1363. The serine proteinases produced by these L. lactis strains were isolated, and their cleavage specificity and rate towards alpha s1- and beta-casein was investigated. The catalytic properties of both the SK11 and Wg2 proteinases, which differ in 44 out of 1902 amino acid residues, could be changed dramatically by the reciprocal exchange of specific fragments between the two enzymes. As a result, various L. lactis strains were constructed having new proteolytic properties that differ from those of the parental strains. Furthermore, two segments in the proteinase could be identified that contribute significantly to the cleavage specificity towards casein; within these two segments, several amino acid residues were identified that are important for substrate cleavage rate and specificity. The results also indicate that the lactococcal proteinase has an additional domain involved in substrate binding compared with the related subtilisins. This suggests that the 200 kd L. lactis proteinase may be the representative of a new subclass of subtilisin-like enzymes.
Abstract: Lactococcus lactis strain NIZO 22186 produces an extracellular, lanthionine-containing 3.5-kDa polypeptide with antimicrobial activity. Its retention time on reversed-phase (RP) HPLC and its amino acid composition showed high similarities but no complete identity to nisin. The gene for this lantibiotic, designated nisZ, has been cloned and its nucleotide sequence was found to be identical to that of the precursor nisin gene apart from a single mutation resulting in the substitution His27Asn in the mature polypeptide. NMR studies of the natural nisin variant, which has been designated nisin Z, confirmed the His27Asn substitution and indicated that it has a similar structure to nisin.
Abstract: Subtilases are members of the family of subtilisin-like serine proteases. Presently, greater than 50 subtilases are known, greater than 40 of which with their complete amino acid sequences. We have compared these sequences and the available three-dimensional structures (subtilisin BPN', subtilisin Carlsberg, thermitase and proteinase K). The mature enzymes contain up to 1775 residues, with N-terminal catalytic domains ranging from 268 to 511 residues, and signal and/or activation-peptides ranging from 27 to 280 residues. Several members contain C-terminal extensions, relative to the subtilisins, which display additional properties such as sequence repeats, processing sites and membrane anchor segments. Multiple sequence alignment of the N-terminal catalytic domains allows the definition of two main classes of subtilases. A structurally conserved framework of 191 core residues has been defined from a comparison of the four known three-dimensional structures. Eighteen of these core residues are highly conserved, nine of which are glycines. While the alpha-helix and beta-sheet secondary structure elements show considerable sequence homology, this is less so for peptide loops that connect the core secondary structure elements. These loops can vary in length by greater than 150 residues. While the core three-dimensional structure is conserved, insertions and deletions are preferentially confined to surface loops. From the known three-dimensional structures various predictions are made for the other subtilases concerning essential conserved residues, allowable amino acid substitutions, disulphide bonds, Ca(2+)-binding sites, substrate-binding site residues, ionic and aromatic interactions, proteolytically susceptible surface loops, etc. These predictions form a basis for protein engineering of members of the subtilase family, for which no three-dimensional structure is known.
Abstract: We have cloned usp45, a gene encoding an extracellular secretory protein of Lactococcus lactis subsp. lactis strain MG1363. Unidentified secreted 45-kDa protein (Usp45) is secreted by every mesophilic L. lactis strain we tested so far and it is chromosomally encoded. The nucleotide sequence of the usp45 gene revealed an open reading frame of 1383 bp encoding a protein of 461 amino acids (aa), composed of a 27-aa signal peptide and a mature protein initiated at Asp28. The gene contains a consensus promoter sequence and a weak ribosome-binding site; the latter is rather uncommon for Gram-positive bacteria. Expression studies in Escherichia coli showed efficient synthesis and secretion of the protein. Usp45 has an unusual aa composition and distribution, and it is predicted to be structurally homologous with P54 of Enterococcus faecium. Up to now, no biological activity could be postulated for this secreted protein.
Abstract: The structural bovine eye lens protein alpha-crystallin was dissociated in 7 M urea and its four subunits, A1, A2, B1, and B2, were separated by means of ion-exchange chromatography. Homopolymeric reaggregates of these subunits were prepared by removal of the denaturant via dialysis. It was found that subunits were exchanged upon incubation of mixtures of two homopolymers under native conditions. New hybrid species were formed within 24 h as demonstrated by isoelectric focusing. Moreover, native alpha-crystallin molecules also exchanged subunits when incubated with homopolymeric aggregates of B2 subunits. Subunit exchange between native alpha-crystallin molecules is postulated, and a "dynamic quaternary structure" is presented that allows the polydisperse protein to adapt to changes in cytoplasmic conditions upon aging of the lens tissue.
Abstract: The human fur gene encodes a protein, designated furin, the C-terminal half of which contains a transmembrane and a cysteine-rich receptor-like domain. The N-terminal half of furin exhibits striking primary amino acid sequence similarity to the catalytic domains of members of the subtilisin family of serine proteases. We here report characteristics of the furin protein and propose a three-dimensional model for its presumptive catalytic domain with characteristics, that predict furin to exhibit an endoproteolytic cleavage selectivity at paired basic residues. This prediction is substantiated by transfection and cotransfection experiments, using COS-1 cells. Full length fur cDNA evokes the specific synthesis of two polypeptides of about 100 kDa and 90 kDa as appeared from Western blot analysis of transfected COS-1 cells using a polyclonal anti-furin antiserum. Functional analysis of furin was performed by cotransfection of fur cDNA with cDNA encoding the 'wild type' precursor of von Willebrand factor (pro-vWF) and revealed an increased proteolytic processing of provWF. In contrast, cotransfection of fur cDNA with a recombinant derivative (provWFgly763), having the arginine residue adjacent to the proteolytic cleavage site (arg-ser-lys-arg) replaced by glycine, revealed that provWFgly763 is not processed by the fur gene product. We conclude that in higher eukaryotes, furin is the prototype of a subtilisin-like class of proprotein processing enzymes with substrate specificity for paired basic residues.
Abstract: We have determined the complete nucleotide sequence of the gene for the cell envelope-located proteinase of Lactococcus lactis SK11. The gene contains a very AT-rich promoter region followed by the coding sequence of a protein of 1962 amino acids. Comparison of the NH2-terminal amino acid sequence of the mature proteinase and the expected primary translation product of the proteinase gene indicates that the enzyme is probably synthesized as a pre-pro-protein. This is confirmed by expression studies of the proteinase gene in Escherichia coli. The amino acid sequence of the proteinase shows significant homology to a number of serine proteinases of the subtilisin family. Compared with the related proteinase of L. lactis Wg2, the proteinase of L. lactis SK11 contains a 60-amino acids duplication and a total of 44-amino acid substitutions, some of which may account for the different cleavage specificity of both enzymes. Furthermore, a region was identified in the Lactococcus proteinase, which shows homology to the membrane-anchoring domains of a number of proteins from other Gram-positive bacteria.
Abstract: Soluble extracts from young bovine, human, rat and rabbit lenses were fractionated by high resolution size-exclusion chromatography to demonstrate the existence of three discrete size-classes of monomeric crystallins in each species. These were identified by ion exchange chromatography, amino acid analysis, SDS electrophoresis and isoelectric focusing as the beta s-, gamma A- and gamma B-crystallins. Conventional SDS electrophoretic analysis of these proteins revealed apparent Mr values of about 23kD, 22kD and 19kD, respectively. Similar analysis in the presence of 6 M urea showed the proteins all co-migrated with an apparent Mr of about 20,500, which is far more consistent with the molecular weights calculated from beta s- and gamma-crystallin sequence data. Amino acid compositions of all the beta s samples indicate a high degree of homology to the bovine protein, whose sequence is known. The different species beta s-crystallins showed other general similarities in size, charge, thiol content and secondary structural properties. On the other hand, near UV CD and fluorescence emission and energy transfer measurements indicate that these proteins have subtle yet significant differences in their tertiary structures. Unlike the gamma-crystallins, the secondary structure of all of the beta s samples is completely denatured in the presence of 8 M urea at 20 degrees C.
Abstract: Calf lenses which are incubated in solutions of 1-150 mM H2O2 for 24 hr remain clear at 20 degrees C. While insoluble lens protein increases by at most 2-3%, we find extensive oxidation of exposed protein thiols, major shifts in the size distribution of crystallins, and progressive generation of more acidic polypeptides. Some of these oxidative modifications are reversible with reducing agent. beta H-Crystallins are particularly susceptible to oxidation: disulfide-bonded soluble aggregates form at low H2O2 levels, while irreversible dissociation to beta L-crystallins occurs at high H2O2 concentration. The gamma-crystallins are particularly prone to charge modification. In contrast, the size and charge distributions of alpha-crystallins appear to be virtually unaffected by H2O2.
Abstract: Measurements were made of lens wet weight, body length and spine diameter of male and female spiny dogfish Squalus acanthias. Age was estimated from body length and spine diameter. It appears that the dogfish eye lens grows continuously during life. Relationships were established between lens weight and age, which allow minimal age to be estimated from lens weight.
Abstract: Mammalian lenses contain multiple gamma-crystallin gene products, which are differentially synthesized during lens development. We now report the isolation and characterization of multiple gamma-crystallins from lenses of adult spiny dogfish (Squalus acanthias) aged about 20-30 years. About 50% of total lens protein solubilized in 50 mM phosphate, pH 7.0; about 25% of this soluble fraction consists of gamma-crystallins as determined by gel filtration. These gamma-crystallins appear homogeneous with respect to molecular weight (approximately equal to 20,000) on SDS-polyacrylamide gels, but their isoelectric points range from below pH 6 to above 10. Preparative cation-exchange chromatography on SP-Sephadex at pH 4.8 resolves four major subfractions, while anion-exchange on DEAE-cellulose at pH 9.5 resolves seven subfractions. Although these procedures separate basic from acidic polypeptides, most of these gamma-crystallin subfractions still consist of polypeptide mixtures, as determined by ion-exchange HPLC and isoelectric focusing. Analytical cation-exchange HPLC on SynChropak CM300 at pH 6.0 resolves at least 10 different gamma-crystallin components. Amino acid compositions of all the subfractions are similar, yet distinct in the sense that three subclasses can be distinguished. Sulfhydryl residues range from three to six per chain, most of which are buried. The large heterogeneity of gamma-crystallins in adult lens may result from different gene products in combination with post-translational modification.
Abstract: We have isolated, purified and characterized six individual gamma-crystallin polypeptides present in the rat lens. Comparison of their amino acid compositions with the known structure of the six gamma-crystallin genes permits a one-to-one correspondence to be made between each protein synthesized and the encoding gene. This demonstrates that each of the six genes is actually expressed in vivo. Two classes of three gamma-crystallins each, which we have designated classes gamma ABC and gamma DEF, are known to exist, on the basis of internal sequence homology. We have measured the temperature-dependent phase-separation characteristics of solutions of the six purified gamma-crystallins, and find that the three members of the gamma DEF class (gamma 2-2, gamma 3-1 and gamma 4-1) are all cryo-proteins with relatively high phase-separation temperatures, whereas the three gamma ABC crystallins (gamma 1-1, gamma 1-2 and gamma 2-1) do not show phase separation above -7 degrees C. We have measured the spatial distribution in rat lens of each of the alpha-, beta- and gamma-crystallins as a function of age from 1 to 420 days, using size-exclusion and ion-exchange high-pressure liquid chromatography (HPLC). Our findings in the cortical layer permit us to establish the differential synthesis of each of the crystallins during lens development. Particular attention has been devoted to the spatial and temporal distribution of the six individual gamma-crystallins. Up to birth, synthesis of the three components of the gamma DEF class predominates, and in particular that of gamma 2-2. In subsequent development the three components of the gamma ABC class assume a greater proportion of monomeric crystallins synthesized, while beta s-crystallin synthesis predominates in late development. Our analysis of different layers within single lenses provides novel information on spatial gradients of the water-soluble and water-insoluble protein fractions as a function of age. We consider the consequences of these findings for lens transparency and opacity in both rat and mouse lens. We show that the high concentrations of gamma DEF-crystallins appear to be responsible for the opacity known to occur in young rat lenses. We conclude from these observations that close control of the differential synthesis of gamma-crystallins plays an important role in maintaining lens transparency during development.
Abstract: Cation-exchange high-performance liquid chromatography on SynChropak CM300 in Tris-acetate buffers of pH 5-7, using sodium acetate gradients, produces an excellent separation of the various gamma-crystallin gene products and their post-synthetically modified forms from eye lens. With a single analysis of total lens extract, the gamma-crystallins can be resolved, quantified and collected for amino acid analysis. Experimental conditions are presented for optimal resolution of individual human, rat, bovine and dogfish shark gamma-crystallins. Applications presented include determinations of different synthesis of gamma-crystallins and chemical modification (oxidation by hydrogen peroxide) in situ.
Abstract: The denaturation behavior of bovine lens gamma-crystallin fractions II, III, and IV and their susceptibility to proteolysis in vitro was compared to determine whether differences in their stability could play a role in cataract formation. Tertiary and secondary structure changes induced by increasing concentrations of urea, guanidine hydrochloride, and sodium dodecyl sulfate and by increasingly alkaline pH were followed by near-UV and far-UV circular dichroism, Trp fluorescence emission, and exposure of sulfhydryl groups. Major differences were found in the denaturation and proteolysis behavior of the three gamma-crystallin fractions. In general, the unfolding of gamma-II and gamma-III crystallins is rather gradual, suggesting the presence of intermediate unfolding states; in contrast, the order-disorder transition of gamma-IV crystallin is abrupt. The gamma-IV crystallin fraction is the most stable in urea and guanidine hydrochloride, but is most susceptible to nonspecific proteolysis and alkaline pH denaturation. Differences in denaturation and proteolysis behavior are attributed to the inherent differences in the tertiary structures of these crystallins.
Abstract: The gamma-crystallin fractions II, III and IV from calf eye lens were treated with the thiol-specific fluorescent probe 2-(4'-maleimidylanilino)naphthalene-6-sulfonate (MIANS), in order to determine the reactivity of the seven (gamma-II) or six (gamma-III, gamma-IV) cysteine residues. Two classes of reactive cysteines were distinguished by variations in fluorescence intensity with increasing molar excess of the probe, and approximately three cysteines were nonreactive in each gamma-crystallin. From the position of the emission maximum, it is apparent that MIANS-labeled cysteines of gamma-IV are in the least hydrophobic environment. Fluorescence energy transfer was observed from tryptophan to MIANS-labeled cysteines in both gamma-II and gamma-III crystallins, with efficiencies of 86% and 89%, respectively, but not in gamma-IV crystallin. We suggest that the spatial arrangements and microenvironments of cysteine residues of gamma-crystallins are sufficiently different from each other to account for the variations in fluorescence characteristics of the MIANS-labeled proteins and the lack of energy transfer in gamma-IV crystallins.
Abstract: We have isolated the individual gamma-crystallins expressed in young human lenses and identified with which of the six known human gamma-crystallin genes they each correspond. We find that at least 90% of the gamma-crystallins synthesized in the young human lens are the products of genes gamma G3 and gamma G4. We demonstrate that gamma G4-crystallin undergoes a temperature-dependent phase separation, and we have measured the low-concentration branch of its coexistence curve (phase separation temperature vs. concentration) up to about 40 mg/ml. By comparison, we found no evidence of gamma G3-crystallin phase separating, even at lower temperatures and higher concentrations. This is consistent with predictions based on sequence homology between human and rat gamma-crystallins. The implications of these findings for human inherited and senile cataracts are considered.
Abstract: The concentration-dependence of beta-crystallin aggregation was studied by both high- and low-pressure size exclusion chromatography of calf lens cortical extract (0.5-249 mg ml-1), nuclear extract (0.2-304 mg ml-1) and purified beta-crystallins (0.4-52 mg ml-1). A reversible equilibrium exists between beta H(igh)-crystallins (predominantly hexamers) and a portion of the beta L(ow)-crystallins (predominantly dimers). Association to beta H-crystallin is more extensive in the nucleus than in the cortex. Moreover, at physiological protein concentrations, the weight percentage of beta H-crystallins is greater than that of beta L-crystallins, in both the cortex and the nucleus. beta H-Crystallins can be fully dissociated to beta L-crystallin at low protein concentration. On the other hand, not all of the beta L species are competent to associate to beta H at high concentrations. This association appears to be directly dependent on the presence of beta B1 chains. We therefore propose that the concentration and spatial distribution of beta H-crystallin in vivo is actually regulated by differential synthesis of beta B1 polypeptides.
Abstract: A novel procedure is presented for the rapid quantitative analysis of eye lens gamma-crystallins and beta s-crystallin by ion-exchange high-performance liquid chromatography on Synchropak CM300. At least six different gamma-crystallin gene products can be resolved from the soluble fraction of calf lens extract. This method is applicable to the analysis of microsections from individual lenses, and can be used to rapidly characterize spatial variations in gamma-crystallin composition which occur with aging and cataractogenesis.
Abstract: To determine the molecular mechanisms for cold cataract formation in the nucleus of the young mammalian lens, we have investigated the thermally reversible opacification of gamma-crystallin solutions isolated from calf lens. Coexistence curves (plots of opacification temperature Tc versus protein concentration) were determined for the individual gamma-crystallin fractions II, III, and IV as well as for the unfractionated gamma-crystallin mixtures isolated from the nucleus and cortex. The coexistence curve of gamma IV-crystallin is remarkably elevated above those of gamma II- and gamma III-crystallin and the gamma-crystallin mixtures. The gamma IV-crystallin fraction is the major determinant of the opacification temperature within the whole lens or isolated cytoplasm. Quasielastic light-scattering spectroscopy of gamma IV-crystallin solutions indicates that above Tc there are two populations of protein aggregates of distinctly different mean size. As the temperature is lowered towards Tc, both populations increase in size. Opacification occurs when the population of large scatterers, which is composed of less than 0.1% protein by weight, reaches an average radius of about 20,000 A.
Abstract: In the bovine lens the gamma IV-crystallin fraction is a principal determinant of the phase separation and opacification temperature, Tc (Siezen et al, Proc. Natl. Acad. Sci. USA 82, 1985, 1701). We have now measured the effect on Tc of purified gamma IV-crystallin solutions produced by a variety of reagents which affect protein-protein, protein-water and water-water interactions. Ionic strengths less than physiological increase Tc dramatically, while higher ionic strength has very little effect. Calcium ion concentrations up to 8 mM produce no change in Tc. Glycerol and acrylamide both depress Tc linearly with reagent concentrations; Tc depression of gamma IV-crystallin by these compounds is quantitatively the same as for whole lens. Sulfhydryl reducing agents such as glutathione and dithiothreitol lower Tc, while hydrogen peroxide increases Tc. Changes in opacification temperature of gamma IV-crystallin produced by oxidizing and reducing agents are time-dependent and highly non-linear with reagent concentration. Our results clearly show that bovine gamma IV-crystallin is an important target protein for various reagents which are known perturbants of the opacification temperature of whole lens. The relevance of these findings to human diabetic and senile cataract formation is discussed.
Abstract: The three major bovine gamma-crystallin fractions (gamma-II, gamma-III and gamma-IV) are known to have closely related (80-90%) amino acid sequences and three-dimensional folding of the polypeptide backbone. Their chiroptical and emission properties, as measured by circular dichroism (CD) and fluorescence, are now shown to differ distinctly. The far-ultraviolet CD spectra indicate that all three gamma-crystallins have predominantly beta-sheet conformation (45-60%) with only subtle differences in secondary structure. The fluorescence emission maxima of gamma-II, gamma-III and gamma-IV, due to the four tryptophan residues, appear at 324, 329 and 334 nm, respectively, suggesting that tryptophan residues are buried in environments of decreasing hydrophobicity. Corresponding differences in quantum yield may be due to fluorescence quenching by neighboring sulfur-containing residues. Titratable tyrosines are maximal for gamma-III, as manifested from difference absorption spectra at alkaline pH. The near-ultraviolet CD spectra differ in position, magnitude and sign of tryptophan and tyrosine transitions. In addition, a characteristic CD maximum at 235 nm, presumably due to tyrosine-tyrosine exciton interactions, differs in magnitude for each gamma-crystallin. This study shows that the environment and interactions of the aromatic residues of the individual gamma-crystallin fractions are quite different. These variations in tertiary structure may be significant, in terms of stability of gamma-crystallins towards aggregation and denaturation, for understanding lens transparency and cataract formation in general.
Abstract: Low temperature induced opacification (cold cataract) of the nucleus of young mammalian lenses is associated with a phase separation of proteins in the lens cell cytoplasm. Calf lenses were treated with a variety of imido-esters and N-hydroxysuccinimide-esters, which react specifically with amino groups. Many potent inhibitors of phase separation cataract were identified which lower the opacification temperature by 6 degrees C or more. Lenses generally remain clear, colorless and soft. Furthermore, suppression of the cold cataract temperature is permanent upon removal of excess reagent.
Abstract: The 'microheterogeneity model' (R.J. Siezen and R. van Driel, Biochim. Biophys. Acta 295 (1973) 131) and the 'incompetent whole molecule model' (G. Kegeles, Arch. Biochem. Biophys. 180 (1977) 530) for the dissociation of Helix pomatia alpha-hemocyanin whole molecules to half molecules were tested experimentally, using ultracentrifugation and stopped-flow dilution analysis. Results of differential sedimentation experiments followed by stopped flow analysis of separated fractions of 60 S and 100 S molecules were not entirely as predicted by the incompetent model, the agreement depending on the pH and ionic strength of analysis. A considerable amount of stopped-flow dilution response could be attributed to material sedimenting between 60 and 100 S. This material appears to be the main equilibrating fraction, and its amount is considerably larger than that predicted by the microheterogeneity model. Increased hydrostatic pressure was found to enhance this fraction, whereas fixation or low ionic strength reduced or eliminated this fraction. Nonequilibrium components of 30, 50 and 80 S were detected and partially purified by differential sedimentation.
Abstract: Crystallins from calf lens were subjected to ultrafiltration through an Amicon XM-300 membrane to determine whether specific interactions between identical proteins (self-association) or different proteins (mixed-association) could be detected and quantified. Single crystallins at different concentrations, simple mixtures and total lens extracts were studied separately. alpha-Crystallin (Mr 800 000) is nearly fully retained (greater than 95%) by XM-300. Retention of beta-crystallins (Mr 50 000-200 000) is found to be much higher than expected from their molecular weights. Ultrafiltration of gamma-crystallin (Mr 20 000) solutions of 1.0-22.6 g/l shows that retention increases as a function of protein concentration. In solutions of single crystallins, self-association effects could not be separated from concentration polarization effects at the membrane surface. In mixtures of crystallins, mixed-association could not be separated from self-association, concentration polarization and excluded volume effects on self-association.
Abstract: A competitive radioimmunoassay was used to determine the reactivities of alpha-crystallins from 13 species with antibodies directed toward calf alpha-crystallin. The results indicate that species as diverse as human and dogfish share the same number of crossreacting antigenic determinants. The various alpha-crystallins can be distinguished only on the basis of their differing affinities for the antiserum. Hydrophilicity profiles for alpha A and alpha B polypeptides of all species were found to be remarkably similar. On the basis of these, four major sequential determinants could be predicted for each polypeptide. The location and sequence of these determinants were found to be essentially conserved in all alpha-crystallins examined. These results are in agreement with the observed crossreactivities. However, there was little obvious correlation between substitutions in determinants and observed variations in respective alpha-crystallin/antibody affinities. Conservation of antigenic determinants over such a wide evolutionary range may reflect stringent constraints on the overall surface and three-dimensional structure of vertebrate alpha-crystallins.
Abstract: The X-ray crystallographic structure of bovine gamma-crystallin shows four similar folding motifs each composed of about 42 residues arranged as four topologically sequential, anti-parallel beta-strands. Since the beta and gamma-crystallin sequences show good homology, proposals for a four-motif beta-crystallin model have been made. The other bovine eye-lens protein species, alpha-crystallins, are not homologous to beta or gamma-crystallin in primary structure. In the present work, smoothed plots of amino acid sequence number versus a residue characteristic (e.g. hydrophobicity) were calculated for the various crystallins. Cross-correlation coefficients were then determined between pairs of crystallin plots for various registers of the curves. The correlation plots were then combined for several characteristics and for pairwise comparisons between beta or gamma-crystallin and the alpha-crystallins. The resulting plots showed four peaks separated by about 42 residues for the alpha-crystallins, suggesting that they also possess a four-motif beta-barrel structure. The physical parameter comparison technique appears generally applicable in suggesting a structural and functional relationship amongst proteins that show no primary sequence homology.
Abstract: Bovine lens alpha A- and alpha B-crystallin polypeptides show extensive sequence homology with each other, but apparently none with beta Bp- and gamma 2-crystallin. Despite only 30% sequence homology, the latter two proteins are assumed to have a strong correspondence in tertiary structure, consisting of four structurally similar folding units of antiparallel beta-sheet. We have tested for internal structural repeats in all crystallins, and structural homology between crystallins, by comparing various physical properties of the amino acid residues, such as bulkiness and propensity to form beta-sheet and beta-turn structure. Two procedures used a combination of five physical parameters to calculate correlation coefficients. The 4-fold structural repeat in gamma 2-crystallin and the internal duplication in beta Bp-crystallin were readily detectable, as was also the strong structural homology between corresponding folding units in beta Bp- and gamma 2-crystallin. However, for alpha-crystallin polypeptides, no conclusive support was obtained for either a four-unit or a six-unit folding, the two models previously considered by us. The third procedure compared smoothened hydropathy plots, representing hydrophilic and hydrophobic regions along the polypeptide sequences. Hydropathy profiles were found to show strong correspondence, particularly between alpha B-crystallin and beta Bp-crystallin. These observations support a similar 4-fold folding pattern for all bovine crystallins. A possible role in subunit interactions of the N-terminal folding unit, which has hydrophobic surface characteristics in both alpha- and beta-crystallin polypeptides, is proposed.
Abstract: Concentrated solutions of calf alpha-crystallin (up to 45 g/l) and gamma-crystallin (up to 67 g/l) were subjected to frontal exclusion chromatography at pH 7.3, ionic strength 0.17 and 20 degrees C. The experimental concentration dependence of the weight-average partition coefficient was compared with theoretical expressions, which include considerations of thermodynamic non-ideality effects, for the concentration dependence of a single solute and of a solute undergoing reversible self-association. Two types of association pattern were examined, discrete dimerization and indefinite self-association. The partition chromatography results are consistent with an indefinite self-association of gamma-crystallin, governed by an isodesmic association constant of 6.7 X 10(-3) l/g. alpha-Crystallin appears to self-associate either very weakly, with a maximal association constant of 0.9 X 10(-3) l/g, or not at all; the distinction depends on the assessment of the non-ideality coefficients. The consequences of excluded volume effects on these self-association equilibria at high total protein concentration are discussed. Mixtures of alpha-crystallin and gamma-crystallin were analyzed by frontal exclusion chromatography (up to 14 g/l) and sedimentation velocity (up to 115 g/l): no interaction was observed.
Abstract: Calf lens nuclear alpha-crystallin was separated into five molecular weight subpopulations by exclusion chromatography on Bio-Gel A-5m. These subpopulations were compared by amino acid analysis, ultraviolet absorption analysis, fluorescence, far- and near-ultraviolet circular dichroism, isoelectric focusing, SDS-polyacrylamide gel electrophoresis and sedimentation velocity analysis. Although only minor differences were detectable in most physicochemical properties, progressive changes were found in the near-ultraviolet circular dichroism spectra and in pellet hardness after centrifugation. Minute amounts of beta-crystallin polypeptides and a 43 kDa component were present in all five subpopulations. In addition, the highest molecular weight aggregates contain some gamma-crystallin polypeptides. A slow re-equilibration of separated subpopulations towards the initial distribution was observed by rechromatography.
Abstract: Circular dichroism spectra (196-240 nm) of calf alpha-, beta H-, beta L- and gamma-crystallins were measured and analyzed over the entire wavelength range with five curve-fitting procedures for estimating protein secondary structure. For gamma-crystallin the estimates are in good agreement with the X-ray structure. For all four crystallins the estimates are very similar: 0-9% alpha-helix and 51-68% beta-sheet. This is in accordance with the three-dimensional homology of beta Bp- and gamma 2-crystallin polypeptide chains as postulated from their 30% sequence homology, and suggests that alpha A- and alpha B-crystallin chains may also have a corresponding structure. Secondary structure elements in the four amino acid sequences were predicted using two different comprehensive prediction methods. For gamma 2-crystallin the predictions of beta-sheet are in good agreement with the X-ray structure and with circular dichroism estimates. For beta Bp-crystallin only the C-terminal domain secondary structure predictions are considered satisfactory, which possibly relates to the proposed role of the N-terminal domain in subunit interactions. The combined predictions for alpha A- and alpha B-chains (3% helix, 49% sheet) are in excellent agreement with circular dichroism. Moreover, the good alignment of predicted beta-sheet segments in alpha-crystallin chains with known beta-sheet strands in gamma 2- (and presumably beta Bp-) crystallin strongly supports a similar 4-motif folding pattern in all four calf crystallin chains.
Abstract: The main water-soluble proteins of squid lens (S-crystallins) have a molecular weight of 60 000, a sedimentation coefficient s020,w of 5.2 S, 20-30% alpha-helical secondary structure, and an unusually high methionine content (12%). The subunits of Mr 30 000 (major) and Mr 27 000 (minor) have related N-terminal amino acid sequences, but a very heterogeneous charge distribution with predominantly basic isoelectric points. Higher-Mr aggregates have similar secondary/tertiary structure and amino acid composition, but contain additional acidic subunits and Mr 35 000-40 000 subunits. S-crystallins resemble vertebrate beta-crystallins in their quaternary structure, and their N-terminal sequence shows analogy with the first 19 residues of calf beta/gamma-crystallin folding units. In the urea-soluble and urea-insoluble lens fractions polypeptides of Mr 58 000 and 80 000, respectively, predominate, which presumably correspond to the main cytoskeleton and membrane proteins. Water-soluble lens components of less than Mr 2000 were isolated which have ultraviolet absorption maxima at 327 and 370 nm.
Abstract: Cytoskeletal and membrane proteins were isolated from respectively the urea-soluble and urea-insoluble fractions of the squid lens. The main cytoskeletal polypeptide has a molecular weight (63000) and an amino acid composition similar to those of vertebrate intermediate filament proteins, including mammalian lens vimentins. Intermediate filaments, and bundles thereof, were regenerated from the squid lens urea-soluble fraction upon removal of urea. The main membrane polypeptide of 140000 Mr has an amino acid composition entirely different from that of the main intrinsic membrane protein of 26000 Mr which is found in all vertebrates. Although non-EDTA-extractable, the 140000 Mr squid membrane polypeptide is best classified as extrinsic, since it has a high polarity (mainly acidic residues) and can be degraded completely upon trypsin treatment of squid lens membranes.
Abstract: Theory pertaining to the interpretation of partition chromatography results obtained with self-associating protein systems studied at high total concentrations is extended to permit consideration of situations in which both monomeric and dimeric states partition. This development, which includes considerations of thermodynamic nonideality effects, permits a quantitative correlation of human oxyhemoglobin results reported previously and obtained in this work employing a different stationary matrix of controlled-pore glass beads. The two sets of results, obtained at pH 7.3 and 20 degree C, indicate that the alpha 2 beta 2 species of oxyhemoglobin self-associates. Two types of association pattern, discrete dimerization and an indefinite self-association, are examined. This is done for a realistic range of values for the radius, r. of the effective hard sphere appropriate to the calculation of the covolume of the alpha 2 beta 2 species in the assessment of the thermodynamic nonideality contribution. Assessed values of the isodesmic association constant range from 66 +/- 23 M-1 (r - 2.84 nm) to 154 +/- 26 M-1 (r = 3.13 nm). This mode of indefinite association is marginally favored over dimerization when the larger value of r is considered, the two patterns becoming virtually indistinguishable for the lower value of r. Partition chromatography results are also presented for human deoxyhemoglobin up to a total concentration of 225 g/l, and are analyzed in a similar fashion to show that the indefinite self-association pattern is favored, governed by an isodesmic constant in the range 91 +/- 9 M-1 (r = 2.84 nm) to 223 +/- 84 M-1 (r = 3.13 nm). Comparison of the constants assessed for the oxy and deoxy systems permits discussion of the concept that oxygen binds preferentially to the alpha 2 beta 2 species of deoxyhemoglobin in comparison with its polymers.
Abstract: Reaction of calf alpha-crystallin, which consists of about 30 A and 10 B subunits, with various bisimido esters at pH 8.0-8.5 and room temperature leads to inter-subunit crosslinking. Little difference is observed in the efficiency of crosslinking by reagents of a homologous series with different lengths (from C6 to C12). At any stage of the reaction an exponential decrease is found in the amounts of crosslinked dimer, trimer, tetramer etc., with no preference for the formation of any particular oligomer, suggesting that all subunits on the surface of the spherical alpha-crystallin molecule are in quasi-equivalent positions. Molecular weight analysis by docdecylsulfate gel electrophoresis and reversible crosslinking show that both A and B subunits occur in an apparently constant ratio in the crosslinked oligomers, from which we infer that both types of subunits are randomly arranged on the surface of the alpha-crystallin molecule. Not all of the subunits can form inter-chain crosslinks, as a limiting value of about 60% of the subunits is found in oligomeric form. A simple explanation could be a core of subunits which is inaccessible to certain chemical reagents, although other alternatives are also discussed.
Abstract: An improved iterative method for computing association constants from sedimentation equilibrium results obtained with self-interacting protein systems is presented which accounts for the composition-dependence of the activity coefficients of all oligomeric species. The method is based on the calculation of viral coefficients from covolume and charge considerations, the statistical mechanical basis of which is discussed in relation to the DLVO theory. The method is applied to results obtained with lysozyme in diethylbarbiturate buffer of pH 8.0 and ionic strength 0.15 at 15 degrees C. It is shown that these results, encompassing a range of total solute concentration up to 19.7 g/liter are consistent with self-association patterns comprising either a monomer--dimer--trimer system or an isodesmic indefinite self-association of the monomer, the latter being favored. A firmer distinction between these possibilities is sought on the basis of the dependence of the weight-average partition coefficient, determined by frontal gel chromatography, on total solute concentration (up to 56.6/liter). This analysis accounts for the composition-dependence of the ratio of the activity coefficients of partitioning monomer in mobile and stationary phases. It is concluded that all results are consistent with an indefinite self-association of lysozyme governed by a single association constant of 4.61 x 10(2) liter/mole.
Abstract: The stability of the native quaternary structure of bovine alpha-crystallin was studied, by sedimentation analysis and electron microscopy, as a function of pH (7--11), ionic strength (0.01--0.5), temperature (6--60 degrees C) and calcium ion concentration (0 and 10 mM). Three successive transitions are distinguished at 20 degrees C. Firstly, a slow transconformation step, which is independent of pH, ionic strength or calcium ions. Secondly, an irreversible primary dissociation step, favoured by increasing pH above 8 and/or a lower ionic strength, with formation of 'alkali-modified alpha-crystallin', which is spherically shaped like the native protein but has a smaller average diameter, sedimentation coefficient and molecular weight. Thirdly, with further increase of pH above 9, a rapidly reversible dissociation of alkali-modified alpha-crystallin characterized by a single reaction boundary in sedimentation velocity analysis. In the presence of calcium ions the quaternary structure is stabilized to the extent that no dissociation is observed up to at least pH 10.3. Upon increase of temperature, at pH 7.3, a slow irreversible dissociation and swelling run parallel until a limit is reached around 37 degrees C with formation of 'temperature-modified alpha-crystallin', which is indistinguishable from the native protein by electron microscopy, but has a higher relative viscosity and lower sedimentation coefficient and molecular weight. Calcium ions have little or no effect on this transition. Above 37 degrees C a reversal of this transition or aggregation is indicated. These findings, together with previous structural data on microheterogeneity, reassociation from urea, and aging of alpha-crystallin in vivo, are incorporated into a hypothetical scheme of transitions, based on a three-layer model for the quaternary structure.
Abstract: Expressions that take into account the effects of thermodynamic non-ideality, described in terms of a high-order virial expansion, are derived for the concentration-dependence of the weight-average partition coefficient in exclusion chromatography of a single solute and of a solute undergoing reversible self-association. Comparison of the concentration-dependences predicted by those expressions with results obtained for bovine and human oxyhemoglobins on CPG-10-120 porous glass beads in 0.156 I phosphate-chloride buffer, pH 7.3, shows that neither oxyhemoglobin conforms with the concept of it being a single alpha 2 beta 2 entity with Stokes radius of 3.13 nm, the experimental value. Previously published osmotic pressure and sedimentation equilibrium results are also shown to be inconsistent with this concept. On the other hand, both sets of exclusion chromatography results are consistent with the joint operation of thermodynamic non-ideality and reversible association of the alpha 2 beta 2 species. From the magnitude of the equilibrium constant, derived for either of two possible modes of association, it is calculated that only half of the oxyhemoglobin would be in the alpha 2 beta 2 states under conditions of oxygen saturation and a concentration of 320 g/liter, that pertaining in the red blood cell. The consequences of this association phenomenon are discussed in relation to the oxygen binding curves obtained by others in the presence and absence of 2,3-diphosphoglycerate (DPG). An explanation is provided of the observed dependence on hemoglobin concentration of oxygen-binding in the presence of DPG, and of the absence of such an effect in DPG-free solutions. It is concluded that the control of oxygen binding to hemoglobin in the physiological situation involves the joint operation of self-association and allosteric effects.
Abstract: alpha-Crystallin from calf eye lens is found to have 30 sulfhydryl groups per 800 000 daltons by modification with 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) and 4,4'-dithiopyridine in 6 M urea. These -SH groups can be divided into three different classes in native alpha-crystallin from their reactivity with DTNB, 4,4'-dithiopyridine, iodoacetamide and ethylenimine. Results obtained with these reagents point to the presence of 7 +/- 1 thiol groups (Class I) which are likely to be surface exposed, with a concomitant 40--45% quenching of tryptophan fluorescence in DTNB-modified alpha-crystallin. Another 10 +/- 1 thiol groups (Class II) must be in a hydrophobic environment since they react only with the hydrophobic reagents, causing a further decrease in fluorescence intensity. Class III, 13 +/- 2 thiol groups, is inaccesible to any of these reagents. Introduction of up to 15 negatively charged thionitrobenzoate groups or seven positively charged aminoethyl groups in the alpha-crystallin molecule at pH 7.5--8.0 did not change the state of aggregation as judged from the sedimentation coefficients.
Abstract: Cortial alpha-crystallin was size-fractionated by gel filtration on Ultrogel AcA22 and charge-fractionated by anion-exchange chromatography on DE-52 DEAE-cellulose using gradient elution. Electron microscopy demonstrates that both native and reassociated alpha-crystallin are heterogeneous populations of spherical or slightly ellipsoidal molecules with diameters of 13.5--16.0 nm (maximum at 14.0--15.0 nm) for native alpha-crystallin and 8.5--12.5 nm (maximum at 10.0--10.5 nm) for reassociated alpha-crystallin. An enormous charge heterogeneity of native alpha-crystallin was detected, which is shown to arise from variations in the stoichiometry of the 5 main types of subunits. The molar ratio of acidic chains (A2, A1 and A1/2-151) to basic chains (B2 and B1) varies from 70/30--80/20 (averaging about 3/1) and the amount of deamidated chains (A1 and B1) varies from 7--37%. Recombination of the subunits, after dissociation in 6 M urea, leads to a charge heterogeneity of reassociated alpha-crystallin very similar to that of native alpha-crystallin. Therefore, specific formation of pure A or B chain aggregates is not preferred. Instead, random combination of subunits is theoretically shown to be sufficient to describe the observed charge microheterogeneity of both reassociated and native alpha-crystallin. No obvious relationship exists between size and charge heterogeneity. Within these ranges of molecular weight and subunit composition there are more than 1000 different combinations of A2, A1, A1/2-151, B2 and B1 conceivable.
Abstract: Fifty amino acids are analyzed as their N-heptafluorobutyryl isobutyl esters by gas chromatography on a column of 3% OV-101 on Gas-Chrom Q. The identification and quantification of unusual amino acids, specifically those occurring in the marine environment, in the presence of common protein amino acids is reported. Regular relationships between elution temperatures and amino acid structures are found, thus allowing the estimation of elution temperatures of other amino acids.
