Abstract: Two independent isolates of the gut commensal Lactobacillus johnsonii were sequenced. They belonged to the same clonal lineage and differed mainly by a 40.8 kb-long prophage LJ771 of the Sfi11 phage lineage. LJ771 shares close DNA sequence identity with Lactobacillus gasseri prophages. LJ771 coexists as integrated prophage and excised circular phage DNA, but phage DNA packaged into extracellular phage particles was not detected. Between the phage lysin gene and attR a likely mazE ("antitoxin")/pemK ("toxin") gene cassette was detected in LJ771, but not the L. gasseri prophages. Expressed pemK could only be cloned in E. coli together with the mazE gene. LJ771 was shown to be highly stable and could only be cured by the co-expression of mazE from a plasmid. The prophage was integrated into the methionine sulfoxide reductase gene (msrA) and complements the 5' end of the gene creating a protein with a slightly altered N terminal sequence. The two L. johnsonii strains showed identical in vitro growth and in vivo gut persistence phenotypes. Also on an isogenic background, prophage possession caused no growth disadvantage.
Abstract: Lactobacillus johnsonii strains NCC533 and ATCC 33200 (the type strain of this species) differed significantly in gut residence time (12 versus 5 days) after oral feeding to mice. Genes affecting the long gut residence time of the probiotic strain NCC533 were targeted for analysis. We hypothesized that genes specific for this strain, which are expressed during passage of the bacterium through the gut, affect the phenotype. When the DNA of the type strain was hybridized against a microarray of the sequenced NCC533 strain, we identified 233 genes that were specific for the long-gut-persistence isolate. Whole-genome transcription analysis of the NCC533 strain using the microarray format identified 174 genes that were strongly and consistently expressed in the jejunum of mice monocolonized with this strain. Fusion of the two microarray data sets identified three gene loci that were both expressed in vivo and specific to the long-gut-persistence isolate. The identified genes included LJ1027 and LJ1028, two glycosyltransferase genes in the exopolysaccharide synthesis operon; LJ1654 to LJ1656, encoding a sugar phosphotransferase system (PTS) transporter annotated as mannose PTS; and LJ1680, whose product shares 30% amino acid identity with immunoglobulin A proteases from pathogenic bacteria. Knockout mutants were tested in vivo. The experiments revealed that deletion of LJ1654 to LJ1656 and LJ1680 decreased the gut residence time, while a mutant with a deleted exopolysaccharide biosynthesis cluster had a slightly increased residence time.
Abstract: This study aimed to relate the acquisition of different antibiotic resistances and the corresponding physiological responses to cold stress of Lactobacillus delbrueckii subsp. bulgaricus strain CFL1. Six resistant mutants were spontaneously obtained and studied depending on the target of the antibiotic: (i) bacitracin and vancomycin (Bac(R), Van(R), wall synthesis), (ii) novobiocin (Nov(R), DNA replication), and (iii) kanamycin, spiramycin, streptomycin (Kan(R), Spi(R), Str(R), RNA translation). The mutations modified the growth and the cold stress response at three different physiological levels: (i) Van(R) and Spi(R) mutants showed significant lower growth rates compared to the wild type strain. (ii) Van(R) and Bac(R) mutants displayed a slightly higher resistance to a freezing-thawing challenge whereas Str(R) and Spi(R) mutants were more sensitive compared to the wild type. (iii) The recovery of acidification activity after freezing and during frozen storage was improved by considering the Nov(R) strain, but not with the Van(R) and Spi(R) mutants. Thus, acquisition of some antibiotic resistance by spontaneous mutation led to modification of the cold stress response. The hypothesis of a unique cellular thermostat is discussed regarding the diversity of the tested antibiotics.
Abstract: Food industry tends towards the use of controlled microorganisms in order to improve its technologies including frozen starter production. The fungus Geotrichum candidum, which is currently found in various environments, is widely used as ripening agent in some specific cheese making process. In order to optimize the cryopreservation of this microorganism, freezing experiments were carried out using a Peltier cooler-heater incubator, which permits to control the temperature downshift from +20 to -10 degrees C in time period ranges from 20 to 40min depending on the experiments. Concomitantly, study of the effect of an industrial ice nucleator protein derived from Pseudomonas syringae (SNOMAX) on the dynamic of freezing of G. candidum was carried out. Our results showed that the addition of this protein in the microbiological suspension has different complementary effects: (i) the synchronization of the different samples nucleation, leading to an homogeneous and earlier freezing, (ii) the increase of the freezing point temperature from -8.6 to -2.6 degrees C, (iii) a significant decrease of the lethality of G. candidum cells subjected to a freezing-thawing cycles challenge.
Abstract: Work with pathogens like Vibrio cholerae has shown major differences between genes expressed in bacteria grown in vitro and in vivo. To explore this subject for commensals, we investigated the transcription of the Lactobacillus johnsonii NCC533 genome during in vitro and in vivo growth using the microarray technology. During broth growth, 537, 626, and 277 of the 1,756 tested genes were expressed during exponential phase, "adaptation" (early stationary phase), and stationary phase, respectively. One hundred one, 150, and 33 genes, respectively, were specifically transcribed in these three phases. To explore the in vivo transcription program, we fed L. johnsonii containing a resistance plasmid to antibiotic-treated mice. After a 2-day washout phase, we determined the viable-cell counts of lactobacilli that were in the lumina and associated with the mucosae of different gut segments. While the cell counts showed a rather uniform distribution along the gut, we observed marked differences with respect to the expression of the Lactobacillus genome. The largest number of transcribed genes was in the stomach (n = 786); the next-largest numbers occurred in the cecum (n = 391) and the jejunum (n = 296), while only 26 Lactobacillus genes were transcribed in the colon. In vitro and in vivo transcription programs overlapped only partially. One hundred ninety-one of the transcripts from the lactobacilli in the stomach were not detected during in vitro growth; 202 and 213 genes, respectively, were transcribed under all in vitro and in vivo conditions; but the core transcriptome for all growth conditions comprised only 103 genes. Forty-four percent of the NCC533 genes were not detectably transcribed under any of the investigated conditions. Nontranscribed genes were clustered on the genome and enriched in the variable-genome part. Our data revealed not only major differences between in vitro- and in vivo-expressed genes in a Lactobacillus gut commensal organism but also marked changes in the expression of genes along the digestive tract.
Abstract: Freezing of prokaryotic and eukaryotic microorganisms is the main interest in the study of cold stress responses of living organisms. In parallel, applications which arise from this approach are of two types: (i) optimization of the frozen starters used in food processing; and (ii) improvement of the ex situ preservation of microorganisms in collections. Currently, cryopreservation of microorganisms in collections is carried out in cryotubes, and bibliographical references related to freezing microorganisms packaged in straws are scarce. In this context, a preliminary study was completed to evaluate the technological potential of ionomeric resin straws compared to polycarbonate cryo-tubes. Survival under freezing stress was tested on three microorganisms selected for their biotechnological interest: two lactic acid bacteria, Lactococcus lactis subsp. cremoris and Lactobacillus delbrueckii subsp. bulgaricus and a deuteromycete fungus, Geotrichum candidum. The stress was carried out by repeated freezing-thawing cycles to artificially accelerate the lethal effect of freezing on the microorganisms. Two main results were obtained: (i) the survival rate values (per freezing-thawing cycle) seems to depend on the thermal type of the studied microorganism, and (ii) there was no, under our experimental conditions, significant difference between straws and tubes. However, conservation in the resin straws lead to a slight increase in the survival of L. cremoris and G. candidum compared to microtubes. In those conditions, straws seems an alternative system to securely store frozen microorganisms with three main characteristics: (i) a high resistance to thermal stress, (ii) a safe closing by hermetic weld, and (iii) a system for inviolable identification.
Abstract: AIMS: Assessment of genetic diversity within the species Geotrichum candidum and development of tools to trace the strains that play an important role in the agro food industry. METHODS AND RESULTS: RAM-PCR and RAPD-PCR techniques were assessed for their ability to discriminate 57 strains of various morphotypes, substrates and geographical origin. The techniques were complementary and, when combined, allowed us to discriminate isolates. Moreover, we established a link between a taxon and its occupation of an ecological niche, which should not be confused with the substrate of isolation. CONCLUSIONS: We observed a high degree of diversity, which could be linked to the variety of the ecological niches chosen and to the high degree of morphological polymorphism encountered within the species. SIGNIFICANCE AND IMPACT OF THE STUDY: Used in combination, RAM-PCR and RAPD-PCR permit traceability and monitoring systems for G. candidum strains during food processing.
Abstract: Geotrichum candidum is an ascomycetous anamorph yeast-like fungus found in various habitats. It is a component of the natural flora of milk and is used as a maturing agent for both soft and hard cheeses. This microorganism displays phenotypic variability and may act as an opportunist pathogen, causing geotrichosis. Cytological analysis of G. candidum strain ATCC 204307 showed this strain to have eight chromosomes. We prepared chromosomal DNA from 13 strains of G. candidum differing in habitat and morphotype. We used pulsed field gel electrophoresis (PFGE) in two sets of conditions to determine the size of the chromosomal DNA molecules. The strains investigated had five to eight chromosomes, 0.6 to 4.5 Mb in size. We estimated genome size in these 13 strains to be between 11 and 19 Mb. Pulsed-field gel electrophoresis profiles showed a high degree of polymorphism, indicating considerable variability between strains. Genome size and the presence of large chromosomes appeared to be correlated with morphotype. Strains with a mold-like or intermediate morphotype tended to have larger genomes than strains with a yeast-like morphotype did.
Abstract: Geotrichum candidum is a yeast-like fungus used as ripening starter in cheese making. The present study focused on chemical stress pretreatments affecting survival of G. candidum ATCC 204307 to freeze-thaw stress. Cryotolerance of G. candidum cells was induced by pretreatment with NaCl, CaCl2, or MgCl2, indicating heterologous phenotypic adaptation to freeze-thaw stress (- 20 to 25 degrees C) by osmotic stress. Furthermore, the nystatin, an antifungal compound, was shown to be a cryotolerance inducer.
Abstract: This study was conducted to investigate the ability of cryoprotective chemicals to induce phenotypic cryoadaptation in Lactobacillus delbrueckii ssp. bulgaricus CIP 101027T. Tolerance to negative temperature stress (freezing at -20 degrees C and thawing at 37 degrees C) was induced by pretreatment with Me(2)SO, glycerol, lactose, sucrose, and trehalose. Interestingly, Me(2)SO has a significantly greater cryoprotective effect than glycerol. Furthermore, lactose, sucrose, and trehalose, often referred to as osmotica, were shown to have greater cryoadaptive than cryoprotective properties. These results suggest that bacteria such as L. delbrueckii ssp. bulgaricus could be phenotypically adapted to freezing and thawing by an osmotic stress applied prior to freeze-thaw stress.
Abstract: The effect of cold stress on Geotrichum candidum was investigated at chill and freezing temperatures. Specific growth rates were determined at various temperatures and plotted according to the Ratkowsky and Arrhenius equations. The obtained profiles led to the determination of characteristics including the activation energy and notional minimum temperatures. At temperatures below the optimum single linear slopes were observed. At freezing temperatures, the loss of viability of cell populations was proportional to the number of freezing-thawing cycles. Nevertheless, the ability of G. candidum to survive this challenge depended on the physiological conditions prior to the freezing stress. The loss of viability was growth phase specific. Cells harvested in stationary phase showed a higher resistance compared to those obtained with cells in exponential phase. Furthermore, the cells of G. candidum could be adapted to the freeze-thaw challenge by pre-treatment at chill temperatures. This phenomenon known as cryotolerance was a function of the duration of the preincubation exposure.
Abstract: The diversity of the prokaryotes that have been studied, combined with the many different effects of low temperature, has led to an extensive literature concerning cold stress responses in mesophilic bacteria. The aim of this review is to discuss the effects of cold on the behavior of bacteria. The following three responses will be described: (i) biochemical modifications consisting first of membrane fatty acid desaturation and second of the synthesis of cold stress proteins, (ii) physiological responses of the cells to permit growth at low temperatures above 0 degrees C and cryotolerance at lower temperatures, and (iii) control of the cold shock response at a transcriptional and/or translational level. This paper reviews knowledge, most of which has been acquired in the last 10 years, in the field of cold stress responses. It is hoped that these data will help to focus attention on the metabolic responses associated with environmental disturbance.
Abstract: Salt-induced genes in the cyanobacterium Anabaena sp. strain PCC 7120 were identified by use of a Tn5-based transposon bearing luxAB as a reporter. The genomic sequence adjacent to one site of insertion of the transposon was identical in part to the sequence of the lti2 gene, which was previously identified in a differential screen for cold-induced transcripts in Anabaena variabilis. The lti2-like gene was induced by sucrose and other osmotica and by low temperature, in addition to salt. Regulatory components necessary for the induction of this gene by osmotica were sought by a further round of transposon mutagenesis. One mutant that displayed reduced transcriptional activity of the lti2-like gene in response to exposure to osmotica had an insertion in an open reading frame, which was denoted orrA, whose predicted product showed sequence similarity to response regulators from two-component regulatory systems. The corresponding mutation was reconstructed and was shown, like the second-site transposon mutation, to result in reduced response to osmotic stress. Induction of the lux reporter gene by osmotica was restored by complementation with a genomic fragment containing the entire open reading frame for the presumptive response regulator, whereas a fragment containing a truncated copy of the open reading frame for the response regulator did not complement the mutation.
Abstract: Transfer of Enterococcus faecalis to a cold temperature (8 degrees C for 4 to 30 h) led to increased expression of 11 cold shock proteins (CSPs). Furthermore, this mesophilic prokaryote synthesized 10 cold acclimation proteins, five of them distinct from CSPs, during continuous growth (4 days) at the same temperature (8 degrees C).
Abstract: Growth at low positive temperatures and induced phenotypic resistance to extreme cold temperature (freezing/thawing cycles) of Enterococcus faecalis were investigated. The effect of low temperatures on the specific growth rates was studied; use of Arrhenius profile and Ratkovsky 'square-root' model allowed determination of the 'temperature characteristic' (mu approximately equal to 13,800 cal mol-1), the critical temperature (Tcrit approximately equal to 17.9 degrees C) and the notional minimum growth temperature (T0 approximately equal to 3.6 degrees C). Preincubation of Ent. faecalis cells at low temperatures (8-16 degrees C) during periods corresponding to their generation time resulted in an increased ability of the bacterial cells to withstand short periods of freezing/thawing (-20 degrees C/+37 degrees C) challenge. Moreover, the increase of the incubation period at low positive temperature led to a higher degree of adaptation.
Abstract: Like in other organisms tested to date, adapted cells of Lactococcus lactis subsp. lactis IL1403 pretreated at 42 degrees C for 30 min develop a thermotolerant state, i.e. an increased ability to survive subsequent exposure to a lethal challenge temperature (52 degrees C for 15 or 30 min). In different cellular systems, chemicals as diverse as divalent metal salts, natural or synthetic compounds trigger the development of thermotolerance. Yet, in L. lactis subsp. lactis IL1403, among the 17 chemicals tested, only four induced this transient increased tolerance to heat: cadmium chloride, mercury chloride, sodium azide and beta-mercaptoethanol. Intriguingly, none of these four compounds induced the synthesis of three major heat shock proteins (DnaK, GroEL and hsp104-analogue), which are believed to be responsible for thermotolerance in most organisms. It is suggested that: (i) the lesions produced by these various 'proteotoxic' agents are fundamentally different from those produced by heat; (ii) heat shock protein synthesis and transient induced tolerance to heat are not tightly correlated phenomena in L. lactis subsp. lactis as they are in Escherichia coli and some other organisms.
Abstract: Mutants of Anabaena sp. strain PCC 7120 that are incapable of sustained growth with air as the sole source of nitrogen were generated by using Tn5-derived transposons. Nitrogenase was expressed only in mutants that showed obvious morphological signs of heterocyst differentiation. Even under rigorously anaerobic conditions, nitrogenase was not synthesized in filaments that were unable to develop heterocysts. These results suggest that competence to synthesize nitrogenase requires a process that leads to an early stage of visible heterocyst development and are consistent with the idea that synthesis of nitrogenase is under developmental control (J. Elhai and C. P. Wolk, EMBO J. 9:3379-3388, 1990). We isolated mutants in which differentiation was arrested at an intermediate stage of heterocyst formation, suggesting that differentiation proceeds in stages; those mutants, as well as mutants with aberrant heterocyst envelopes and a mutant with defective respiration, expressed active nitrogenase under anaerobic conditions only. These results support the idea that the heterocyst envelope and heterocyst respiration are required for protection of nitrogenase from inactivation by oxygen. In the presence of air, such mutants contained less nitrogenase than under anaerobic conditions, and the Fe-protein was present in a posttranslationally modified inactive form. We conclude that internal partial oxygen pressure sufficient to inactivate nitrogenase is insufficient to repress synthesis of the enzyme completely. Among mutants with an apparently intact heterocyst envelope and normal respiration, three had virtually undetectable levels of dinitrogenase reductase under all conditions employed. However, three others expressed oxygen-sensitive nitrogenase activity, suggesting that respiration and barrier to diffusion of gases may not suffice for oxygen protection of nitrogenase in these mutants; two of these mutants reduced acetylene to ethylene and ethane.
Abstract: Anabaena, a filamentous cyanobacterium, is of developmental interest because, when deprived of fixed nitrogen, it shows patterned differentiation of N2-fixing cells called heterocysts. To help elucidate its early responses to a decrease in nitrogen, we used a derivative of transposon Tn5 to generate transcriptional fusions of promoterless bacterial luciferase genes, luxAB, to the Anabaena genome. Genes that responded to removal of fixed nitrogen or to other environmental shifts by increased or decreased transcription were identified by monitoring the luminescence of colonies from transposon-generated libraries. The Tn5 derivative transposed in Anabaena at ca. 1-4 x 10(-5) per cell and permitted high-resolution mapping of its position and orientation in the genome and facile cloning of contiguous genomic DNA.
Abstract: The degree of retention of whole cells of Synechocystis strain PCC 6803 on DEAE-cellulose columns was shown to depend on their content of exopolysaccharides, which are at least in part responsible for the external negative charge of the cells. This feature was used for the isolation of mutants modified in the apparent viscosity caused by these macromolecular constituents. When a wild-type suspension was loaded onto a DE52 column, the cells eluting in the two extreme fractions of a 0 to 5 M NaCl step gradient represented 10 to 10 of the total eluted population. The accuracy of the procedure was established through the analysis of four clones: Suc(0)32 and Suc(0)65 (0 M) and Suc(5)64A and Suc(5)61 (5 M). The decreased viscosity of the exopolymers of the two 0 M clones, which appeared identical, could be related to the production of molecules less charged in uronic acids and more readily liberated from the cells. The two 5 M clones exhibited a lower sedimentation velocity, correlating with either a 60% increase in uronic acid and a doubling of the specific viscosity of the exopolysaccharides [clone Suc(5)64A] or a doubling of the per-cell production of polymers otherwise identical to those from wild-type cells [clone Suc(5)61].
