Michal Letek Polberg, PhD Centro de Investigacion del Cancer, Lab 18 Campus Miguel de Unamuno Universidad de Salamanca 37007-Salamanca SPAIN Phone: (34) (923) 294818 Fax: (34) (923) 294743
m.letek@usal.es
Short scientific biography:
My scientific background is rather broad. In general terms, I describe myself as a molecular biologist working on microbiology and cell biology.
During my doctorate, I was studying the cell division and cell growth of Corynebacteria to identify new targets for the development of novel antimicrobials. In addition, I was working on the potential use of these bacteria for arsenic bioremediation, developing new molecular tools for its genetic manipulation, and carrying out translational research in new molecular diagnostics for corynebacterial pathogens.
During my first postdoctoral years I was focused on the functional analysis of the genome of an intracellular pathogen, Rhodococcus equi. The aim was to identify and characterize the virulence pathways used by these bacteria to interact with their host cell, the lung macrophages. To this end, we reconstructed R. equi regulatory network and identified new virulence traits, which expression was coordinated with essential genes for the intracellular survival of this pathogen.
My research work is now focused on the molecular basis of autophagy activation in mammalian cells against different stimuli, such as intracellular proliferation of bacterial pathogens.
Abstract: Selective autophagy underlies many of the important physiological roles that autophagy plays in multicellular organisms, but the mechanisms involved in cargo selection are poorly understood. Here we describe a molecular mechanism that can target conventional endosomes for autophagic degradation. We show that the human transmembrane protein TMEM59 contains a minimal 19-amino-acid peptide in its intracellular domain that promotes LC3 labelling and lysosomal targeting of its own endosomal compartment. Interestingly, this peptide defines a novel protein motif that mediates interaction with the WD-repeat domain of ATG16L1, thus providing a mechanistic basis for the activity. The motif is represented with the same ATG16L1-binding ability in other molecules, suggesting a more general relevance. We propose that this motif may play an important role in targeting specific membranous compartments for autophagic degradation, and therefore it may facilitate the search for adaptor proteins that promote selective autophagy by engaging ATG16L1. Endogenous TMEM59 interacts with ATG16L1 and mediates autophagy in response to Staphylococcus aureus infection.
Abstract: Adaptation to endoplasmic reticulum (ER) stress depends on the activation of the unfolded protein response (UPR) stress sensor inositol-requiring enzyme 1α (IRE1α), which functions as an endoribonuclease that splices the mRNA of the transcription factor XBP-1 (X-box-binding protein-1). Through a global proteomic approach we identified the BCL-2 family member PUMA as a novel IRE1α interactor. Immun oprecipitation experiments confirmed this interaction and further detected the association of IRE1α with BIM, another BH3-only protein. BIM and PUMA double-knockout cells failed to maintain sustained XBP-1 mRNA splicing after prolonged ER stress, resulting in early inactivation. Mutation in the BH3 domain of BIM abrogated the physical interaction with IRE1α, inhibiting its effects on XBP-1 mRNA splicing. Unexpectedly, this regulation required BCL-2 and was antagonized by BAD or the BH3 domain mimetic ABT-737. The modulation of IRE1α RNAse activity by BH3-only proteins was recapitulated in a cell-free system suggesting a direct regulation. Moreover, BH3-only proteins controlled XBP-1 mRNA splicing in vivo and affected the ER stress-regulated secretion of antibodies by primary B cells. We conclude that a subset of BCL-2 family members participates in a new UPR-regulatory network, thus assuming apoptosis-unrelated functions.
Abstract: Although bacteria are considered the simplest life forms, we are now slowly unraveling their cellular complexity. Surprisingly, not only do bacterial cells have a cytoskeleton but also the building blocks are not very different from the cytoskeleton that our own cells use to grow and divide. Nonetheless, despite important advances in our understanding of the basic physiology of certain bacterial models, little is known about Actinobacteria, an ancient group of Eubacteria. Here we review current knowledge on the cytoskeletal elements required for bacterial cell growth and cell division, focusing on actinobacterial genera such as Mycobacterium, Corynebacterium, and Streptomyces. These include some of the deadliest pathogens on earth but also some of the most prolific producers of antibiotics and antitumorals.
Abstract: We report the genome of the facultative intracellular parasite Rhodococcus equi, the only animal pathogen within the biotechnologically important actinobacterial genus Rhodococcus. The 5.0-Mb R. equi 103S genome is significantly smaller than those of environmental rhodococci. This is due to genome expansion in nonpathogenic species, via a linear gain of paralogous genes and an accelerated genetic flux, rather than reductive evolution in R. equi. The 103S genome lacks the extensive catabolic and secondary metabolic complement of environmental rhodococci, and it displays unique adaptations for host colonization and competition in the short-chain fatty acid-rich intestine and manure of herbivores--two main R. equi reservoirs. Except for a few horizontally acquired (HGT) pathogenicity loci, including a cytoadhesive pilus determinant (rpl) and the virulence plasmid vap pathogenicity island (PAI) required for intramacrophage survival, most of the potential virulence-associated genes identified in R. equi are conserved in environmental rhodococci or have homologs in nonpathogenic Actinobacteria. This suggests a mechanism of virulence evolution based on the cooption of existing core actinobacterial traits, triggered by key host niche-adaptive HGT events. We tested this hypothesis by investigating R. equi virulence plasmid-chromosome crosstalk, by global transcription profiling and expression network analysis. Two chromosomal genes conserved in environmental rhodococci, encoding putative chorismate mutase and anthranilate synthase enzymes involved in aromatic amino acid biosynthesis, were strongly coregulated with vap PAI virulence genes and required for optimal proliferation in macrophages. The regulatory integration of chromosomal metabolic genes under the control of the HGT-acquired plasmid PAI is thus an important element in the cooptive virulence of R. equi.
Abstract: Corynebacteria grow by wall extension at the cell poles, with DivIVA being an essential protein orchestrating cell elongation and morphogenesis. DivIVA is considered a scaffolding protein able to recruit other proteins and enzymes involved in polar peptidoglycan biosynthesis. Partial depletion of DivIVA induced overexpression of cg3264, a previously uncharacterized gene that encodes a novel coiled coil-rich protein specific for corynebacteria and a few other actinomycetes. By partial depletion and overexpression of Cg3264, we demonstrated that this protein is an essential cytoskeletal element needed for maintenance of the rod-shaped morphology of Corynebacterium glutamicum, and it was therefore renamed RsmP (rod-shaped morphology protein). RsmP forms long polymers in vitro in the absence of any cofactors, thus resembling eukaryotic intermediate filaments. We also investigated whether RsmP could be regulated post-translationally by phosphorylation, like eukaryotic intermediate filaments. RsmP was phosphorylated in vitro by the PknA protein kinase and to a lesser extent by PknL. A mass spectrometric analysis indicated that phosphorylation exclusively occurred on a serine (Ser-6) and two threonine (Thr-168 and Thr-211) residues. We confirmed that mutagenesis to alanine (phosphoablative protein) totally abolished PknA-dependent phosphorylation of RsmP. Interestingly, when the three residues were converted to aspartic acid, the phosphomimetic protein accumulated at the cell poles instead of making filaments along the cell, as observed for the native or phosphoablative RsmP proteins, indicating that phosphorylation of RsmP is necessary for directing cell growth at the cell poles.
Abstract: The coiled-coil protein DivIVA is a determinant of apical growth and hyphal branching in Streptomyces coelicolor. We have investigated the properties of this protein and the involvement of different domains in its essential function and subcellular targeting. In S. coelicolor cell extracts, DivIVA was present as large oligomeric complexes that were not strongly membrane associated. The purified protein could self-assemble into extensive protein filaments in vitro. Two large and conspicuous segments in the amino acid sequence of streptomycete DivIVAs not present in other homologs, an internal PQG-rich segment and a carboxy-terminal extension, are shown to be dispensable for the essential function in S. coelicolor. Instead, the highly conserved amino-terminal of 22 amino acids was required and affected establishment of new DivIVA foci and hyphal branches, and an essential coiled-coil domain affected oligomerization of the protein.
Abstract: We identified the first enzymes that use mycothiol and mycoredoxin in a thiol/disulfide redox cascade. The enzymes are two arsenate reductases from Corynebacterium glutamicum (Cg_ArsC1 and Cg_ArsC2), which play a key role in the defense against arsenate. In vivo knockouts showed that the genes for Cg_ArsC1 and Cg_ArsC2 and those of the enzymes of the mycothiol biosynthesis pathway confer arsenate resistance. With steady-state kinetics, arsenite analysis, and theoretical reactivity analysis, we unraveled the catalytic mechanism for the reduction of arsenate to arsenite in C. glutamicum. The active site thiolate in Cg_ArsCs facilitates adduct formation between arsenate and mycothiol. Mycoredoxin, a redox enzyme for which the function was never shown before, reduces the thiol-arseno bond and forms arsenite and a mycothiol-mycoredoxin mixed disulfide. A second molecule of mycothiol recycles mycoredoxin and forms mycothione that, in its turn, is reduced by the NADPH-dependent mycothione reductase. Cg_ArsCs show a low specificity constant of approximately 5 m(-1) s(-1), typically for a thiol/disulfide cascade with nucleophiles on three different molecules. With the in vitro reconstitution of this novel electron transfer pathway, we have paved the way for the study of redox mechanisms in actinobacteria.
Abstract: Corynebacterium glutamicum is a rod-shaped actinomycete with a distinct model of peptidoglycan synthesis during cell elongation, which takes place at the cell poles and is sustained by the essential protein DivIVA(CG) (C. glutamicum DivIVA). This protein contains a short conserved N-terminal domain and two coiled-coil regions: CC1 and CC2. Domain deletions and chimeric versions of DivIVA were used to functionally characterize the three domains, and all three were found to be essential for proper DivIVA(CG) function. However, in the presence of the N-terminal domain from DivIVA(CG), either of the two coiled-coil domains of DivIVA(CG) could be replaced by the equivalent coiled-coil domain of Bacillus subtilis DivIVA (DivIVA(BS)) without affecting the function of the original DivIVA(CG), and more than one domain had to be exchanged to lose function. Although no single domain was sufficient for subcellular localization or function, CC1 was mainly implicated in stimulating polar growth and CC2 in targeting to DivIVA(CG) assemblies at the cell poles in C. glutamicum.
Abstract: Time-lapse imaging of Streptomyces hyphae revealed foci of the essential protein DivIVA at sites where lateral branches will emerge. Overexpression experiments showed that DivIVA foci can trigger establishment of new zones of cell wall assembly, suggesting a key role of DivIVA in directing peptidoglycan synthesis and cell shape in Streptomyces.
Abstract: The pathogenic actinomycete Rhodococcus equi harbors different types of virulence plasmids associated with specific nonhuman hosts. We determined the complete DNA sequence of a vapB(+) plasmid, typically associated with pig isolates, and compared it with that of the horse-specific vapA(+) plasmid type. pVAPB1593, a circular 79,251-bp element, had the same housekeeping backbone as the vapA(+) plasmid but differed over an approximately 22-kb region. This variable region encompassed the vap pathogenicity island (PAI), was clearly subject to selective pressures different from those affecting the backbone, and showed major genetic rearrangements involving the vap genes. The pVAPB1593 PAI harbored five different vap genes (vapB and vapJ to -M, with vapK present in two copies), which encoded products differing by 24 to 84% in amino acid sequence from the six full-length vapA(+) plasmid-encoded Vap proteins, consistent with a role for the specific vap gene complement in R. equi host tropism. Sequence analyses, including interpolated variable-order motifs for detection of alien DNA and reconstruction of Vap family phylogenetic relationships, suggested that the vap PAI was acquired by an ancestor plasmid via lateral gene transfer, subsequently evolving by vap gene duplication and sequence diversification to give different (host-adapted) plasmids. The R. equi virulence plasmids belong to a new family of actinobacterial circular replicons characterized by an ancient conjugative backbone and a horizontally acquired niche-adaptive plasticity region.
Abstract: The Mur ligases play an essential role in the biosynthesis of bacterial cell-wall peptidoglycan and thus represent attractive targets for the design of novel antibacterials. These enzymes catalyze the stepwise formation of the peptide moiety of the peptidoglycan disaccharide peptide monomer unit. MurC is responsible of the addition of the first residue (L-alanine) onto the nucleotide precursor UDP-MurNAc. Phosphorylation of proteins by Ser/Thr protein kinases has recently emerged as a major physiological mechanism of regulation in prokaryotes. Herein, the hypothesis of a phosphorylation-dependent mechanism of regulation of the MurC activity was investigated in Corynebacterium glutamicum. We showed that MurC was phosphorylated in vitro by the PknA protein kinase. An analysis of the phosphoamino acid content indicated that phosphorylation exclusively occurred on threonine residues. Six phosphoacceptor residues were identified by mass spectrometry analysis, and we confirmed that mutagenesis to alanine residues totally abolished PknA-dependent phosphorylation of MurC. In vitro and in vivo ligase activity assays showed that the catalytic activity of MurC was impaired following mutation of these threonine residues. Further in vitro assays revealed that the activity of the MurC-phosphorylated isoform was severely decreased compared with the non-phosphorylated protein. To our knowledge, this is the first demonstration of a MurC ligase phosphorylation in vitro. The finding that phosphorylation is correlated with a decrease in MurC enzymatic activity could have significant consequences in the regulation of peptidoglycan biosynthesis.
Abstract: Bacterial cell growth and cell division are highly complicated and diversified biological processes. In most rod-shaped bacteria, actin-like MreB homologues produce helicoidal structures along the cell that support elongation of the lateral cell wall. An exception to this rule is peptidoglycan synthesis in the rod-shaped actinomycete Corynebacterium glutamicum, which is MreB-independent. Instead, during cell elongation this bacterium synthesizes new cell-wall material at the cell poles whereas the lateral wall remains inert. Thus, the strategy employed by C. glutamicum to acquire a rod-shaped morphology is completely different from that of Escherichia coli or Bacillus subtilis. Cell division in C. glutamicum also differs profoundly by the apparent absence in its genome of homologues of spatial or temporal regulators of cell division, and its cell division apparatus seems to be simpler than those of other bacteria. Here we review recent advances in our knowledge of the C. glutamicum cell cycle in order to further understand this very different model of rod-shape acquisition.
Abstract: The actinomycete Corynebacterium glutamicum grows as rod-shaped cells by zonal peptidoglycan synthesis at the cell poles. In this bacterium, experimental depletion of the polar DivIVACG protein resulted in the inhibition of polar growth; consequently, these cells exhibited a coccoid morphology. This result demonstrated that DivIVA is required for cell elongation and the acquisition of a rod shape. DivIVA from Streptomyces or Mycobacterium localized to the cell poles of DivIVACG-depleted C. glutamicum and restored polar peptidoglycan synthesis, in contrast to DivIVAs from Bacillus subtilis or Streptococcus pneumoniae which localized at the septum of C. glutamicum. This confirmed that DivIVAs from actinomycetes are involved in polarized cell growth. DivIVACG localized at the septum after cell-wall synthesis had started and the nucleoids had already segregated, suggesting that in C. glutamicum DivIVA is not involved in cell division or chromosome segregation.
Notes: Original article. 2006 Impact factor: 3.993
Abstract: Of the five promoters detected for the ftsZ gene in Corynebacterium glutamicum, three were located within the coding region of the upstream ftsQ gene and two within the intergenic ftsQ-ftsZ region. The most distant ftsZ promoter showed activity in Escherichia coli and controlled high-level transcriptional expression of ftsZ in C. glutamicum. Quantitative Western blotting showed that all five promoters were active during the exponential growth phase and down-regulated during stationary phase. This tightly controlled expression of ftsZ in C. glutamicum indicated that small changes in the amount of FtsZ protein strongly affect bacterial cell viability. The controlled overexpression of ftsZ in C. glutamicum, using the promoter of the gntK gene (PgntK), resulted in approximately 5-fold overproduction of FtsZ, an increase in cell diameter, and a highly variable localization of the protein as spirals or tangles throughout the cell. These results suggest that the intracellular concentration of FtsZ is critical for productive septum formation in C. glutamicum. Our observations provide insight into the mechanisms used by the coryneform group, which lack actin homologues and many regulators of cell division, to control cell morphology.
Notes: Original article. 2006 Impact Factor: 2.455
Abstract: Analysis of the complete genome sequence of Corynebacterium glutamicum indicated that, in addition to ftsI, there are eight proteins with sequence motifs that are strongly conserved in penicillin binding proteins (PBPs): four genes that code for high-molecular-weight (HMW)-PBPs (PBP1a, PBP1b, PBP2a and PBP2b), two genes encoding low-molecular-weight PBPs (PBP4 and PBP4b) and two probable beta-lactamases (PBP5 and PBP6). Here, the function of the four HMW-PBPs in C. glutamicum was investigated using a combination of genetic knockouts, enhanced green fluorescent protein 2 (EGFP2) fusions and penicillin staining of membrane preparations. The four HMW-PBPs were expressed in a growing culture of C. glutamicum, but none of four pbp genes was individually essential for the growth of the bacterium, and only the simultaneous disruption of both pbp1b and pbp2b was lethal. The fused EGFP2-PBP proteins were functional in vivo, which allowed correct determination of their cellular localization. EGFP2 fusions to PBP1a, PBP1b and PBP2b localized at the poles and at the septum, whereas EGFP2-PBP2a was predominantly found at the septum. Cefsulodin treatment specifically delocalized PBP1a and PBP1b (class A HMW-PBPs), whereas mecillinam caused the specific delocalization of PBP2b and PBP2a (class B HMW-PBPs). The results provide new insight into the mechanisms involved in the synthesis of the cell wall in this bacterial species, which lacks a known actin-like cytoskeletal structure.
Notes: Original article. 2006 Impact Factor: 5.634 - N. Valbuena and M. Letek contributed equally to this work
Abstract: The natural resistance mechanisms of corynebacteria to respond to the environments containing high levels of arsenic were successfully adopted to develop inexpensive and selective extractants for submicrogram amounts of arsenic. Kinetic and equilibrium characteristics were evaluated, and a preliminary exploration of the capability of these strains to be used for arsenic speciation was also made in this work. Three kinetics models were used to fit the experimental data. It was found that the pseudo-first-order kinetics model was not quite adequate to describe the retention process, while the intraparticle diffusion and the pseudo-second-order kinetics models provide the best fits. The equilibrium isotherm showed that the retention of arsenic was consistent with the Langmuir equation and that the Freundlich and Dubinin-Radushkevich models provided poorer fits to the experimental data. The maximum effective retention capacity for arsenic was about 15.4 ng As/mg biomass. The amount of arsenic retained was directly measured in the biomass by forward planning a slurry electrothermal atomic absorption spectrometric procedure.
Notes: Original article. 2006 Impact Factor: 2.459
Abstract: Arsenic is an extremely toxic metalloid that, when present in high concentrations, severely threatens the biota and human health. Arsenic contamination of soil, water, and air is a global growing environmental problem due to leaching from geological formations, the burning of fossil fuels, wastes generated by the gold mining industry present in uncontrolled landfills, and improper agriculture or medical uses. Unlike organic contaminants, which are degraded into harmless chemical species, metals and metalloids cannot be destroyed, but they can be immobilized or transformed into less toxic forms. The ubiquity of arsenic in the environment has led to the evolution in microbes of arsenic defense mechanisms. The most common of these mechanisms is based on the presence of the arsenic resistance operon (ars), which codes for: (i) a regulatory protein, ArsR; (ii) an arsenite permease, ArsB; and (iii) an enzyme involved in arsenate reduction, ArsC. Corynebacterium glutamicum, which is used for the industrial production of amino acids and nucleotides, is one of the most arsenic-resistant microorganisms described to date (up to 12 mM arsenite and >400 mM arseniate). Analysis of the C. glutamicum genome revealed the presence of two complete ars operons (ars1 and ars2) comprising the typical three-gene structure arsRBC, with an extra arsC1 located downstream from arsC1 (ars1 operon), and two orphan genes (arsB3 and arsC4). The involvement of both ars operons in arsenic resistance in C. glutamicum was confirmed by disruption and amplification of those genes. The strains obtained were resistant to up to 60 mM arsenite, one of the highest levels of bacterial resistance to arsenite so far described. Using tools for the genetic manipulation of C. glutamicum that were developed in our laboratory, we are attempting to obtain C. glutamicum mutant strains able to remove arsenic from contaminated water.
Abstract: The actinomycete Corynebacterium amycolatum is a saprophytic bacterium usually associated with the human skin, but it is at present considered an emergent pathogen as it is isolated from nosocomial settings from samples of immunosuppressed patients. The conventional method to distinguish C. amycolatum from closely related species is mainly based on phenotypic or chemotaxonomic studies. We developed a molecular method to identify rapidly C. amycolatum based on the use of different primers for amplification of the cell division divIVA gene using conventional or real-time PCR. This technique was used for the first time to distinguish C. amycolatum from the closely related Corynebacterium striatum, Corynebacterium minutissimum and Corynebacterium xerosis, without the requirement of further molecular analysis. The suitability of the identification method was tested on 51 clinical isolates belonging to the nonlipophilic fermentative group of corynebacteria (cluster C. striatum/C. amycolatum), which were accurately characterized by sequencing a 0.8 kb fragment of the 16S rRNA gene.
Notes: Original article. 2006 Impact Factor: 2.068
Abstract: The genes involved in gluconate catabolism (gntP and gntK) in Corynebacterium glutamicum are scattered in the chromosome, and no regulatory genes are apparently associated with them, in contrast with the organization of the gnt operon in Escherichia coli and Bacillus subtilis. In C. glutamicum, gntP and gntK are essential genes when gluconate is the only carbon and energy source. Both genes contain upstream regulatory regions consisting of a typical promoter and a hypothetical cyclic AMP (cAMP) receptor protein (CRP) binding region but lack the expected consensus operator region for binding of the GntR repressor protein. Expression analysis by Northern blotting showed monocistronic transcripts for both genes. The expression of gntP and gntK is not induced by gluconate, and the gnt genes are subject to catabolite repression by sugars, such as glucose, fructose, and sucrose, as was detected by quantitative reverse transcription-PCR (qRT-PCR). Specific analysis of the DNA promoter sequences (PgntK and PgntP) was performed using bifunctional promoter probe vectors containing mel (involved in melanin production) or egfp2 (encoding a green fluorescent protein derivative) as the reporter gene. Using this approach, we obtained results parallel to those from qRT-PCR. An applied example of in vivo gene expression modulation of the divIVA gene in C. glutamicum is shown, corroborating the possible use of the gnt promoters to control gene expression. glxR (which encodes GlxR, the hypothetical CRP protein) was subcloned from the C. glutamicum chromosomal DNA and overexpressed in corynebacteria; we found that the level of gnt expression was slightly decreased compared to that of the control strains. The purified GlxR protein was used in gel shift mobility assays, and a specific interaction of GlxR with sequences present on PgntP and PgntK fragments was detected only in the presence of cAMP.
Notes: Original article. 2006 Impact Factor: 3.993
Abstract: In Corynebacterium glutamicum, as in many Gram-positive bacteria, the cell division gene ftsI is located at the beginning of the dcw cluster, which comprises cell division- and cell wall-related genes. Transcriptional analysis of the cluster revealed that ftsI is transcribed as part of a polycistronic mRNA, which includes at least mraZ, mraW, ftsL, ftsI and murE, from a promoter that is located upstream of mraZ. ftsI appears also to be expressed from a minor promoter that is located in the intergenic ftsL-ftsI region. It is an essential gene in C. glutamicum, and a reduced expression of ftsI leads to the formation of larger and filamentous cells. A translational GFP-FtsI fusion protein was found to be functional and localized to the mid-cell of a growing bacterium, providing evidence of its role in cell division in C. glutamicum. This study involving proteomic analysis (using 2D SDS-PAGE) of a C. glutamicum strain that has partially depleted levels of FtsI reveals that at least 20 different proteins were overexpressed in the organism. Eight of these overexpressed proteins, which include DivIVA, were identified by MALDI-TOF. Overexpression of DivIVA was confirmed by Western blotting using anti-DivIVA antibodies, and also by fluorescence microscopy analysis of a C. glutamicum RESF1 strain expressing a chromosomal copy of a divIVA-gfp transcriptional fusion. Overexpression of DivIVA was not observed when FtsI was inhibited by cephalexin treatment or by partial depletion of FtsZ.
Notes: Original article. 2006 Impact Factor: 3.139
Abstract: Corynebacterium glutamicum is a Gram-positive bacterium that lacks the cell division FtsA protein and actin-like MreB proteins responsible for determining cylindrical cell shape. When the cell division ftsZ gene from C. glutamicum (ftsZ(Cg)) was cloned in different multicopy plasmids, the resulting constructions could not be introduced into C. glutamicum; it was assumed that elevated levels of FtsZ(Cg) result in lethality. The presence of a truncated ftsZ(Cg) and a complete ftsZ(Cg) under the control of Plac led to a fourfold reduction in the intracellular levels of FtsZ, generating aberrant cells displaying buds, branches and knots, but no filaments. A 20-fold reduction of the FtsZ level by transformation with a plasmid carrying the Escherichia coli lacI gene dramatically reduced the growth rate of C. glutamicum, and the cells were larger and club-shaped. Immunofluorescence microscopy of FtsZ(Cg) or visualization of FtsZ(Cg)-GFP in C. glutamicum revealed that most cells showed one fluorescent band, most likely a ring, at the mid-cell, and some cells showed two fluorescent bands (septa of future daughter cells). When FtsZ(Cg)-GFP was expressed from Plac, FtsZ rings at mid-cell, or spirals, were also clearly visible in the aberrant cells; however, this morphology was not entirely due to GFP but also to the reduced levels of FtsZ expressed from Plac. Localization of FtsZ at the septum is not negatively regulated by the nucleoid, and therefore the well-known occlusion mechanism seems not to operate in C. glutamicum.
Notes: Original article. 2005 Impact Factor: 3.173
Abstract: Corynebacterium glutamicum is able to grow in media containing up to 12 mM arsenite and 500 mM arsenate and is one of the most arsenic-resistant microorganisms described to date. Two operons (ars1 and ars2) involved in arsenate and arsenite resistance have been identified in the complete genome sequence of Corynebacterium glutamicum. The operons ars1 and ars2 are located some distance from each other in the bacterial chromosome, but they are both composed of genes encoding a regulatory protein (arsR), an arsenite permease (arsB), and an arsenate reductase (arsC); operon ars1 contains an additional arsenate reductase gene (arsC1') located immediately downstream from arsC1. Additional arsenite permease and arsenate reductase genes (arsB3 and arsC4) scattered on the chromosome were also identified. The involvement of ars operons in arsenic resistance in C. glutamicum was confirmed by gene disruption experiments of the three arsenite permease genes present in its genome. Wild-type and arsB3 insertional mutant C. glutamicum strains were able to grow with up to 12 mM arsenite, whereas arsB1 and arsB2 C. glutamicum insertional mutants were resistant to 4 mM and 9 mM arsenite, respectively. The double arsB1-arsB2 insertional mutant was resistant to only 0.4 mM arsenite and 10 mM arsenate. Gene amplification assays of operons ars1 and ars2 in C. glutamicum revealed that the recombinant strains containing the ars1 operon were resistant to up to 60 mM arsenite, this being one of the highest levels of bacterial resistance to arsenite so far described, whereas recombinant strains containing operon ars2 were resistant to only 20 mM arsenite. Northern blot and reverse transcription-PCR analysis confirmed the presence of transcripts for all the ars genes, the expression of arsB3 and arsC4 being constitutive, and the expression of arsR1, arsB1, arsC1, arsC1', arsR2, arsB2, and arsC2 being inducible by arsenite.
Notes: Original article. 2005 Impact Factor: 3.818
Abstract: Bacterial cell size and morphology are enormously diverse. The molecular factors of morphogenesis are well understood in certain bacterial models and fairly conserved throughout a broad spectrum of bacterial species, as follows. In most bacteria, the tubulin-like FtsZ protein polymerizes at the mid cell , thereby generating the scaffold of the bacterial cell division septum. Actin-like MreB homologues are required for cell elongation at the lateral walls of Escherichia coli or Bacillus subtilis. Whereas FtsZ is conserved in Corynebacterium glutamicum, mreB homologues are absent in the corynebacterial genomes sequenced to date. Furthermore, in these bacteria, cell elongation occurs at the polar ends in a mycelial fashion. This process is structurally maintained from the inside of the cell by oligomers created through self-interaction of DivIVA, a coiled-coil-rich cytoskeletal protein that interacts with the molecular machinery of the polar cell wall synthesis. In addition, the molecular factors involved in the spatio-temporal regulation of bacterial cell division are also missing in C. glutamicum. However, certain serine/threonine kinases have been reported recently in this organism that could be implicated in a tight regulation of cytokinesis through protein phosphorylation. Since numerous antibiotics target bacterial cell division or cell elongation genes, a detailed understanding of these processes could enable the development of novel antibiotics for treating bacterial infections caused by pathogenic Corynebacteria or by the closely related Mycobacteria, Nocardia, or Rhodococcus.
Abstract: Rhodococcus equi is the only animal pathogen among the rhodococci. A soil inhabitant, R. equi is prevalent in the farm environment where it uses herbivore manure as growth substrate. In addition to its saprophytic lifestyle, R. equi has the ability to colonize animal host tissues, causing pyogranulomatous infections in a variety of mammals. Although clearly a multihost pathogen, R. equi is best known as the etiologic agent of a severe contagious bronchopneumonic disease in horses. It also causes deadly opportunistic infections in immunosuppressed individuals, particularly tuberculosis-like cavitary pneumonia in HIV-infected patients. R. equi is an intracellular parasite that replicates within a modified phagocytic vacuole in macrophages. Its pathogenicity depends on a horizontally-acquired genomic island carried on a virulence plasmid. This plasmid virulence locus encodes a family of surface-associated antigens, the Vap proteins. The products of the vap island confer the ability to survive within macrophages and recent evidence suggests they are also involved in infectious tropism towards specific animal host species (horses, pigs, and cattle). The complete DNA sequence of the R. equi genome has been recently determined and a detailed analysis will soon be published. This chapter reviews the current, âpregenomicâ state of knowledge about the biology and virulence of this fascinating pathogenic actinomycete.
Abstract: Unlike organic contaminants, which are degraded
into harmless chemical species, metals and metalloids
can not be destroyed but they can be immobilized or
transformed into less toxic forms. Arsenic health
problems are occurring today, mainly due to the high
arsenic levels present in drinking water, or associated
to specific professional activities. Arsenic enters the
biosphere by leaching from geological formations or
by human action which include gold mining, medical
(chemotherapeutics agents), agricultural (arsenical-containing fungicides,
pesticides and herbicides), and industrial uses. The toxicity of arsenic is
associated to the inorganic forms of arsenic, arsenate [As(V)] and arsenite
[As(III)], which react with cellular enzymes and proteins, modifying its
functional behaviour. The most common microbial arsenic resistance is based
on the presence of an arsenic resistance operon (ars) which basically encodes
for (i) a regulatory protein, (ii) an arsenite permease and (iii) an enzyme
involved in arsenate reduction. Corynebacterium glutamicum is one of the
most arsenic-resistant microorganisms described up to date and many of the
molecular biology techniques developed during the last two decades are being
used in our laboratory to develop efficient strains for arsenic remediation
(biosorption or biocontention) of contaminated environments.
Abstract: The expression of genes coding for heterologous extracellular enzymes or proteins in corynebacteria has provided new capacities to these industrially important microorganisms, such as the use of the culture media as sources of essential amino acids and hydrolytic enzymes that can be used as complements in animal food or for the production of enzymes with industrial, clinical, or pharmaceutical applications. Using genetic manipulation techniques, several corynebacteria strains expressing genes coding for hydrolytic enzymes or proteins have been constructed in different laboratories. Such strains carry antibiotic resistance genes and consequently they cannot be used in the food industry due to the stringent regulations on genetically manipulated microorganisms. To solve this problem, here we describe a general method for the construction of engineered corynebacteria bearing a single copy of a gene coding for a hydrolytic enzyme or a desired protein in its chromosome where it is stably maintained with no selective pressure and lacking any antibiotic resistance gene.
Abstract: The gnt promoters from Corynebacterium glutamicum were used to control the expression of
essential genes in this microorganism. Using these promoters we obtained partially depleted
DivIVA strains, which showed coccoid morphology as a consequence of the total lack of polar
growth. The partial depletion of DivIVA in C. glutamicum was complemented by DivIVAs from
actinomycetes, but not by DivIVAs from Bacillus subtilis or Streptococcus pneumoniae. The
partially depleted Corynebacterium strains were also complemented by quimeric versions of the
DivIVA protein, in which a conserved region was exchanged with the corresponding domain of
DivIVA from B. subtilis, despite the great difference in size of the respective coiled-coil regions.
Changes of the divIVA level of expression lead to an aberrant chromosome segregation, but
DivIVA seems to be indirectly involved in this process because it localizes at the midcell only
when the peptidoglycan synthesis has started and nucleoids are completely segregated. In
addition, in C. glutamicum the localization of FtsZ at the septum is not negatively regulated by
nucleoid occlusion or other temporal and spatial known regulators. Finally, as a practical
application of these results, the divIVA gene was used as a target for PCR amplification in order
to identify pathogenic corynebacteria.
Notes: Directors: Dr. Jose A Gil and Dr. Luis M Mateos;
European Doctorate - Cum Laude
Abstract: We identified the first enzymes, which use mycothiol and mycoredoxin in a thiol/disulfide redox cascade. The enzymes are two arsenate reductases from Corynebacterium glutamicum (Cg_ArsC1 and Cg_ArsC2), which play a key role in the defence against arsenate. In vivo knockouts showed that the genes for Cg_ArsC1 and Cg_ArsC2 and those of the enzymes of the mycothiol biosynthesis pathway confer arsenate resistance. With steady-state kinetics, arsenite analysis, and theoretical reactivity analysis, we unravelled the catalytic mechanism for the reduction of arsenate to arsenite in C. glutamicum. The active site thiolate in Cg_ArsCs facilitates adduct formation between arsenate and mycothiol. Mycoredoxin - a redox enzyme for which the function was never shown before - reduces the thiol- arseno bond, forms arsenite and a mycothiol-mycoredoxin mixed disulfide. A second molecule of mycothiol recycles mycoredoxin and forms mycothione that on its turn is reduced by the NADPH-dependent mycothione reductase. Cg_ArsCs show a low specificity constant of ~5 M -1 s -1, typically for a thiol/disulfide cascade with nucleophiles on three different molecules. With the in vitro reconstitution of this novel electron transfer pathway, we have paved the way for the study of redox mechanisms in actinobacteria.
Abstract: In Escherichia coli and Bacillus subtilis elongation occurs by the insertion of new cell wall material in the cylindrical portion of the cell supported by an actin-like cytoskeleton (MreB/Mbl), while the tubulin-like cytoskeleton (FtsZ) directs cell wall synthesis at the division apparatus (Daniel and Errington, 2003). However, no homologues of the bacterial actin-like proteins MreB/Mbl have been identified in corynebacteria (Ramos et al., 2005), suggesting that cell wall synthesis is organized differently. It is also evident that there are no apparent homologues for positive or negative regulators of cell division, e.g., ftsA, zipA, ezrA, noc, or the min system (Ramos et al., 2005). Interestingly, divIVA, a component of the min system in B. subtilis, is also present in corynebacteria, although the DivIVA protein seems to be involved in the regulation of polar growth (Ramos et al., 2003), as is the case in Streptomyces coelicolor (Flardh, 2003).
Abstract: The amino acid producer Corynebacterium glutamicum was used as a model for the identification of new targets for antimicrobial agents against pathogenic corynebacteria. We are studying the C. glutamicum essential divIVA gene, firstly described in Mycobacterium tuberculosis as wag31 and coding for the highly immunogenic Antigen 84. In Bacillus subtillis DivIVA is involved in the correct localization of Z-ring (the scaffold of the division septum), and also in chromosome segregation.
DivIVA is involved in the apical growth of the rod-shaped C. glutamicum and localizes mainly at the cell poles but also at the septum. A partial depletion of DivIVA in C. glutamicum leads to a coccoid morphology which was reverted when transformed with divIVA from C. glutamicum, Mycobacterium tuberculosis or Streptomyces coelicolor. On the other hand, divIVAs of B. subtillis and S. pneumoniae did not complement the lack of this gene in C. glutamicum, suggesting that the localization of Z-ring might be regulated in a different way in actinomycetes. Immunofluorescence microscopy of both FtsZ and DivIVA revealed that DivIVA is probably involved in the final steps of cell division since it localizes at mid-cell after localization of FtsZ and remains during septation.
Previous results in our laboratory indicated that a ratio between DivIVA and FtsI (a Penicillin Binding Protein -PBP- involved in the synthesis of peptidoglycan at the septum) is needed for the proper morphology of C. glutamicum. Two-Hybrid System (2HS) analysis reveals a protein-protein interaction between: (i) DivIVA and FtsI, (ii) FtsW and FtsZ (as it was suggested in M. tuberculosis), and (iii) DivIVA with itself, confirming its oligomerization. Proteomic studies of the DivIVA partially depleted mutant and further 2HS analysis will be needed to elucidate the functions of DivIVA.
Abstract: Genes gntK and gntP are involved in gluconic acid metabolism in different bacteria. Corynebacterium glutamicum, a Gram-positive microorganism traditionally used for aminoacids and nucleotide production, is able to metabolize gluconic acid as unique energy source. We cloned and sequenced gntK and gntP genes which were located far away in the chromosome.
Internal fragments from genes gntK and gntP were cloned into the E. coli mobilizable vector pK18mob obtaining respectively constructions pKMM3 and pKP1, these âsuicideâ plasmids (in corynebacteria) were used for gene disruption assays in C. glutamicum. Mutants obtained from these experiments were unable (in both cases) to grow in defined media with gluconate. Gene disruption of these genes were corroborated by Southern hybridization analysis.
These results confirm the functional metabolic pathway of gluconic acid in Corynebacterium glutamicum ATCC13032. The genes involved in this pathway in the most of the analyzed bacteria conform the gnt operon (gntKP) in the chromosome; in contrast, these genes in C. glutamicum are disposed far away in the chromosome although they are functional and apparently regulated by catabolic repression.
