Stationary Phase Survival Research Articles

The VrrA sRNA controls a stationary phase survival factor Vrp of Vibrio cholerae

Small non-coding RNAs (sRNAs) are emerging regulatory elements in bacteria. The Vibrio cholerae sRNA VrrA has previously been shown to down-regulate outer membrane proteins (OmpA and OmpT) and biofilm matrix protein (RbmC) by base-pairing with the 5′ region of the corresponding mRNAs. In this study, we present an additional target of VrrA in V. cholerae, the mRNA coding for the ribosome binding protein Vrp. Vrp is homologous to ribosome-associated inhibitor A (RaiA) of Escherichia coli which facilitates stationary phase survival through ribosome hibernation. We show that VrrA down-regulates Vrp protein synthesis by base-pairing to the 5′ region of vrp mRNA and that the regulation requires the RNA chaperone protein, Hfq. We further demonstrate that Vrp is highly expressed during stationary phase growth and associates with the ribosome of V. cholerae. The effect of the Vrp protein in starvation survival is synergistic with that of the VC2530 protein, a homolog of the E. coli hibernation promoting factor HPF, suggesting a combined role for these proteins in ribosome hibernation in V. cholerae. Vrp and VC2530 are important for V. cholerae starvation survival under nutrient deficient conditions. While VC2530 is down-regulated in cells lacking vrrA, mutation of vrp results in VC2530 activation. This is the first report indicating a regulatory role for an sRNA, modulating stationary factors involved in bacterial ribosome hibernation.

Transcription level analysis of intracellular Burkholderia pseudomallei illustrates the role of BPSL1502 during bacterial interaction with human lung epithelial cells.

Melioidosis caused by Burkholderia pseudomallei is a globally important disease of increasing concern according to high case-fatality rate and epidemic spreading. The ability of B. pseudomallei to attach and invade host cells and subsequently survive intracellularly has stimulated many questions concerning the comprehension of bacterial pathogenesis progression. Transcription levels of intracellular B. pseudomallei genes in human lung epithelial cells were therefore analyzed using bioinformatic tools, RT-PCR and real time RT-PCR. Here, it is reported that the identification of bpsl1502, encoding B. pseudomallei SurE (stationary phase survival protein E) located in a global transcriptional regulation operon was accomplished. The up-regulation of B. pseudomallei SurE was demonstrated during intracellular survival of A549 cells at 12, 18, and 24 h post-infection. To investigate the role of this protein, a B. pseudomallei SurE defective mutant was constructed. The invasion and initial survival of the SurE mutants within the A549 cells were impaired. There was no difference, however, between the growth of B. pseudomallei SurE mutant as compared to the wild type in Luria-Bertani culture. These data suggest that SurE may assist B. pseudomallei host cells invade and facilitate early intracellular infection but is not crucial during the stationary growth phase. The identification of B. pseudomallei SurE provides more information of bacterial strategy during an early step of the pathogenesis process of melioidosis.

Oleoyl Coenzyme A Regulates Interaction of Transcriptional Regulator RaaS (Rv1219c) with DNA in Mycobacteria

We have recently shown that RaaS (regulator of antimicrobial-assisted survival), encoded by Rv1219c in Mycobacterium tuberculosis and by bcg_1279c in Mycobacterium bovis bacillus Calmette-Guérin, plays an important role in mycobacterial survival in prolonged stationary phase and during murine infection. Here, we demonstrate that long chain acyl-CoA derivatives (oleoyl-CoA and, to lesser extent, palmitoyl-CoA) modulate RaaS binding to DNA and expression of the downstream genes that encode ATP-dependent efflux pumps. Moreover, exogenously added oleic acid influences RaaS-mediated mycobacterial improvement of survival and expression of the RaaS regulon. Our data suggest that long chain acyl-CoA derivatives serve as biological indicators of the bacterial metabolic state. Dysregulation of efflux pumps can be used to eliminate non-growing mycobacteria.

The effect of the rpoSam allele on gene expression and stress resistance in Escherichia coli.

The RNA polymerase associated with RpoS transcribes many genes related to stationary phase and stress survival in Escherichia coli. The DNA sequence of rpoS exhibits a high degree of polymorphism. A C to T transition at position 99 of the rpoS ORF, which results in a premature amber stop codon often found in E. coli strains. The rpoSam mutant expresses a truncated and partially functional RpoS protein. Here, we present new evidence regarding rpoS polymorphism in common laboratory E. coli strains. One out of the six tested strains carries the rpoSam allele, but expressed a full-length RpoS protein owing to the presence of an amber supressor mutation. The rpoSam allele was transferred to a non-suppressor background and tested for RpoS level, stress resistance and for the expression of RpoS and sigma70-dependent genes. Overall, the rpoSam strain displayed an intermediate phenotype regarding stress resistance and the expression of σ(S)-dependent genes when compared to the wild-type rpoS(+) strain and to the rpoS null mutant. Surprisingly, overexpression of rpoSam had a differential effect on the expression of the σ(70)-dependent genes phoA and lacZ that, respectively, encode the enzymes alkaline phosphatase and β-galactosidase. The former was enhanced while the latter was inhibited by high levels of RpoSam.

Yeast DJ-1 superfamily members are required for diauxic-shift reprogramming and cell survival in stationary phase

The yeast Hsp31 minifamily proteins (Hsp31, Hsp32, Hsp33, Hsp34) belong to the highly conserved DJ-1 superfamily. The human DJ-1 protein is associated with cancer and neurodegenerative disorders, such as Parkinson disease. However, the precise function of human and yeast DJ-1 proteins is unclear. Here we show that the yeast DJ-1 homologs have a role in diauxic-shift (DS), characterized by metabolic reprogramming because of glucose limitation. We find that the Hsp31 genes are strongly induced in DS and in stationary phase (SP), and that deletion of these genes reduces chronological lifespan, impairs transcriptional reprogramming at DS, and impairs the acquisition of several typical characteristics of SP, including autophagy induction. In addition, under carbon starvation, the HSP31 family gene-deletion strains display impaired autophagy, disrupted target of rapamycin complex 1 (TORC1) localization to P-bodies, and caused abnormal TORC1-mediated Atg13 phosphorylation. Repression of TORC1 by rapamycin in the gene-deletion strains completely reversed their sensitivity to heat shock. Taken together, our data indicate that Hsp31 minifamily is required for DS reprogramming and cell survival in SP, and plays a role upstream of TORC1. The enhanced understanding of the cellular function of these genes sheds light into the biological role of other members of the superfamily, including DJ-1, which is an attractive target for therapeutic intervention in cancer and in Parkinson disease.

High-Resolution Profiling of Stationary-Phase Survival Reveals Yeast Longevity Factors and Their Genetic Interactions

Lifespan is influenced by a large number of conserved proteins and gene-regulatory pathways. Here, we introduce a strategy for systematically finding such longevity factors in Saccharomyces cerevisiae and scoring the genetic interactions (epistasis) among these factors. Specifically, we developed an automated competition-based assay for chronological lifespan, defined as stationary-phase survival of yeast populations, and used it to phenotype over 5,600 single- or double-gene knockouts at unprecedented quantitative resolution. We found that 14% of the viable yeast mutant strains were affected in their stationary-phase survival; the extent of true-positive chronological lifespan factors was estimated by accounting for the effects of culture aeration and adaptive regrowth. We show that lifespan extension by dietary restriction depends on the Swr1 histone-exchange complex and that a functional link between autophagy and the lipid-homeostasis factor Arv1 has an impact on cellular lifespan. Importantly, we describe the first genetic interaction network based on aging phenotypes, which successfully recapitulated the core-autophagy machinery and confirmed a role of the human tumor suppressor PTEN homologue in yeast lifespan and phosphatidylinositol phosphate metabolism. Our quantitative analysis of longevity factors and their genetic interactions provides insights into the gene-network interactions of aging cells.

Clp chaperones and proteases are central in stress survival, virulence and antibiotic resistance of Staphylococcus aureus

Intracellular proteolysis carried out by energy-dependent proteases is one of the most conserved biological processes. In all cells proteolysis maintains and shapes the cellular proteome by ridding the cell of damaged proteins and by regulating abundance of functional proteins such as regulatory proteins. The ATP-dependent ClpP protease is highly conserved among eubacteria and in the chloroplasts and mitochondria of eukaryotic cells. In the serious human pathogen, Staphylococcus aureus inactivation of clpP rendered the bacterium avirulent emphasizing the central role of proteolysis in virulence. The contribution of the Clp proteins to virulence is likely to occur at multiple levels. First of all, both Clp ATPases and the Clp protease are central players in stress responses required to cope with the adverse conditions met in the host. The ClpP protease has a dual role herein, as it both eliminates stress-damaged proteins as well as ensures the timely degradation of major stress regulators such as Spx, LexA and CtsR. Additionally, as we will summarize in this review, Clp proteases and Clp chaperones impact on such central processes as virulence gene expression, cell wall metabolism, survival in stationary phase, and cell division. These observations together with recent findings that Clp proteins contribute to adaptation to antibiotics highlights the importance of this interesting proteolytic machinery both for understanding pathogenicity of the organism and for treating staphylococcal infections.

Integrity of the Saccharomyces cerevisiae Rpn11 Protein Is Critical for Formation of Proteasome Storage Granules (PSG) and Survival in Stationary Phase

Decline of proteasome activity has been reported in mammals, flies and yeasts during aging. In the yeast Saccharomyces cerevisiae, the reduction of proteolysis in stationary phase is correlated with disassembly of the 26S proteasomes into their 20S and 19S subcomplexes. However a recent report showed that upon entry into the stationary phase, proteasome subunits massively re-localize from the nucleus into mobile cytoplasmic structures called proteasome storage granules (PSGs). Whether proteasome subunits in PSG are assembled into active complexes remains an open question that we addressed in the present study. We showed that a particular mutant of the RPN11 gene (rpn11-m1), encoding a proteasome lid subunit already known to exhibit proteasome assembly/stability defect in vitro, is unable to form PSGs and displays a reduced viability in stationary phase. Full restoration of long-term survival and PSG formation in rpn11-m1 cells can be achieved by the expression in trans of the last 45 amino acids of the C-terminal domain of Rpn11, which was moreover found to co-localize with PSGs. In addition, another rpn11 mutant leading to seven amino acids change in the Rpn11 C-terminal domain, which exhibits assembled-26S proteasomes, is able to form PSGs but with a delay compared to the wild type situation. Altogether, our findings indicate that PSGs are formed of fully assembled 26S proteasomes and suggest a critical role for the Rpn11 protein in this process.

Pyruvate Oxidase Influences the Sugar Utilization Pattern and Capsule Production in Streptococcus pneumoniae

Pyruvate oxidase is a key function in the metabolism and lifestyle of many lactic acid bacteria and its activity depends on the presence of environmental oxygen. In Streptococcus pneumoniae the protein has been suggested to play a major role in metabolism and has been implicated in virulence, oxidative stress survival and death in stationary phase. Under semi-aerobic conditions, transcriptomic and metabolite profiling analysis of a spxB mutant grown on glucose showed minor changes compared to the wild type, apart from the significant induction of two operons involved in carbohydrate uptake and processing. This induction leads to a change in the sugar utilization capabilities of the bacterium, as indicated by the analysis of the growth profiles of the D39 parent and spxB mutant on alternative carbohydrates. Metabolic analysis and growth experiments showed that inactivation of SpxB has no effect on the glucose fermentation pattern, except under aerobic conditions. More importantly, we show that mutation of spxB results in the production of increased amounts of capsule, the major virulence factor of S. pneumoniae. Part of this increase can be attributed to induction of capsule operon (cps) transcription. Therefore, we propose that S. pneumoniae utilizes pyruvate oxidase as an indirect sensor of the oxygenation of the environment, resulting in the adaption of its nutritional capability and the amount of capsule to survive in the host.

Accumulation of inorganic polyphosphate enables stress endurance and catalytic vigour in Pseudomonas putida KT2440

BackgroundAccumulation of inorganic polyphosphate (polyP), a persistent trait throughout the whole Tree of Life, is claimed to play a fundamental role in enduring environmental insults in a large variety of microorganisms. The share of polyP in the tolerance of the soil bacterium Pseudomonas putida KT2440 to a suite of physicochemical stresses has been studied on the background of its capacity as a host of oxidative biotransformations.ResultsCells lacking polyphosphate kinase (Ppk), which expectedly presented a low intracellular polyP level, were more sensitive to a number of harsh external conditions such as ultraviolet irradiation, addition of β-lactam antibiotics and heavy metals (Cd2+ and Cu2+). Other phenotypes related to a high-energy phosphate load (e.g., swimming) were substantially weakened as well. Furthermore, the ppk mutant was consistently less tolerant to solvents and its survival in stationary phase was significantly affected. In contrast, the major metabolic routes were not significantly influenced by the loss of Ppk as diagnosed from respiration patterns of the mutant in phenotypic microarrays. However, the catalytic vigour of the mutant decreased to about 50% of that in the wild-type strain as estimated from the specific growth rate of cells carrying the catabolic TOL plasmid pWW0 for m-xylene biodegradation. The catalytic phenotype of the mutant was restored by over-expressing ppk in trans. Some of these deficits could be explained by the effect of the ppk mutation on the expression profile of the rpoS gene, the stationary phase sigma factor, which was revealed by the analysis of a PrpoS → rpoS‘-’lacZ translational fusion. Still, every stress-related effect of lacking Ppk in P. putida was relatively moderate as compared to some of the conspicuous phenotypes reported for other bacteria.ConclusionsWhile polyP can be involved in a myriad of cellular functions, the polymer seems to play a relatively secondary role in the genetic and biochemical networks that ultimately enable P. putida to endure environmental stresses. Instead, the main value of polyP could be ensuring a reservoire of energy during prolonged starvation. This is perhaps one of the reasons for polyP persistence in live systems despite its apparent lack of essentiality.

Dramatic Structural Changes Resulting from the Loss of a Crucial Hydrogen Bond in the Hinge Region Involved in C-Terminal Helix Swapping in SurE: A Survival Protein from Salmonella typhimurium

Domain swapping is an interesting feature of some oligomeric proteins in which each protomer of the oligomer provides an identical surface for exclusive interaction with a segment or domain belonging to another protomer. Here we report results of mutagenesis experiments on the structure of C-terminal helix swapped dimer of a stationary phase survival protein from Salmonella typhimurium (StSurE). Wild type StSurE is a dimer in which a large helical segment at the C-terminus and a tetramerization loop comprising two β strands are swapped between the protomers. Key residues in StSurE that might promote C-terminal helix swapping were identified by sequence and structural comparisons. Three mutants in which the helix swapping is likely to be avoided were constructed and expressed in E. coli. Three-dimensional X-ray crystal structures of the mutants H234A and D230A/H234A could be determined at 2.1 Å and 2.35 Å resolutions, respectively. Contrary to expectations, helix swapping was mostly retained in both the mutants. The loss of the crucial D230 OD2– H234 NE2 hydrogen bond (2.89 Å in the wild type structure) in the hinge region was compensated by new inter and intra-chain interactions. However, the two fold molecular symmetry was lost and there were large conformational changes throughout the polypeptide. In spite of these changes, the dimeric structure and an approximate tetrameric organization were retained, probably due to the interactions involving the tetramerization loop. Mutants were mostly functionally inactive, highlighting the importance of precise inter-subunit interactions for the symmetry and function of StSurE.

Iron, glucose and intrinsic factors alter sphingolipid composition as yeast cells enter stationary phase

Survival of Saccharomyces cerevisiae cells, like most microorganisms, requires switching from a rapidly dividing to a non-dividing or stationary state. To further understand how cells navigate this switch, we examined sphingolipids since they are key structural elements of membranes and also regulate signaling pathways vital for survival. During and after the switch to a non-dividing state there is a large increase in total free and sphingolipid-bound long chain-bases and an even larger increase in free and bound C20-long-chain bases, which are nearly undetectable in dividing cells. These changes are due to intrinsic factors including Orm1 and Orm2, ceramide synthase, Lcb4 kinase and the Tsc3 subunit of serine palmitoyltransferase as well as extrinsic factors including glucose and iron. Lowering the concentration of glucose, a form of calorie restriction, decreases the level of LCBs, which is consistent with the idea that reducing the level of some sphingolipids enhances lifespan. In contrast, iron deprivation increases LCB levels and decreases long term survival; however, these phenomena may not be related because iron deprivation disrupts many metabolic pathways. The correlation between increased LCBs and shorter lifespan is unsupported at this time. The physiological rise in LCBs that we observe may serve to modulate nutrient transporters and possibly other membrane phenomena that contribute to enhanced stress resistance and survival in stationary phase.

A prototrophic deletion mutant collection for yeast metabolomics and systems biology

A prototrophic deletion mutant collection for yeast metabolomics and systems biology

Polyphosphate degradation in stationary phase triggers biofilm formation via LuxS quorum sensing system in Escherichia coli.

In most natural environments, association with a surface in a structure known as biofilm is the prevailing microbial life-style of bacteria. Polyphosphate (polyP), an ubiquitous linear polymer of hundreds of orthophosphate residues, has a crucial role in stress responses, stationary-phase survival, and it was associated to bacterial biofilm formation and production of virulence factors. In previous work, we have shown that Escherichia coli cells grown in media containing a critical phosphate concentration >37 mM maintained an unusual high polyP level in stationary phase. The aim of the present work was to analyze if fluctuations in polyP levels in stationary phase affect biofilm formation capacity in E. coli. Polymer levels were modulated by the media phosphate concentration or using mutant strains in polyP metabolism. Cells grown in media containing phosphate concentrations higher than 25 mM were defective in biofilm formation. Besides, there was a disassembly of 24 h preformed biofilm by the addition of high phosphate concentration to the medium. These phenotypes were related to the maintenance or re-synthesis of polyP in stationary phase in static conditions. No biofilm formation was observed in ppk−ppx− or ppk−ppx−/ppk+ strains, deficient in polyP synthesis and hydrolysis, respectively. luxS and lsrK mutants, impaired in autoinducer-2 quorum sensing signal metabolism, were unable to form biofilm unless conditioned media from stationary phase wild type cells grown in low phosphate were used. We conclude that polyP degradation is required for biofilm formation in sufficient phosphate media, activating or triggering the production of autoinducer-2. According to our results, phosphate concentration of the culture media should be carefully considered in bacterial adhesion and virulence studies.

Investigation into the Physiologies of Aeromonas veronii in vitro and Inside the Digestive Tract of the Medicinal Leech Using RNA-seq

Host-associated microbial communities are widespread in nature and vital to the health and fitness of the host. Deciphering the physiology of the microbiome in vivo is critical to understanding the molecular basis of the symbiosis. Recently, the development and application of high-throughput sequencing techniques, particularly RNA-seq, for studying microbial communities has enabled researchers to address not only which microbes are present in a given community but also how the community functions. For microbes that can also be cultivated in the laboratory, RNA-seq provides the opportunity to identify genes that are differentially expressed during symbiosis by comparing in vitro to in vivo transcriptomes. In the current study, we used RNA-seq to identify genes expressed by the digestive-tract microbiome of the medicinal leech, Hirudo verbana, and by one of the two dominant symbionts, Aeromonas veronii, in a rich medium. We used a comparative approach to identify genes differentially expressed during symbiosis and gain insight into the symbiont's physiology in vivo. Notable findings include evidence for the symbionts experiencing environmental stress, performing arginine catabolism, and expressing noncoding RNAs that are implicated in stationary phase survival, a state in which A. veronii persists for months within the host.

Discovery, taxonomic distribution, and phenotypic characterization of a gene required for 3-methylhopanoid production

Hopanoids methylated at the C-3 position are a subset of bacterial triterpenoids that are readily preserved in modern and ancient sediments and in petroleum. The production of 3-methylhopanoids by extant aerobic methanotrophs and their common occurrence in modern and fossil methane seep communities, in conjunction with carbon isotope analysis, has led to their use as biomarker proxies for aerobic methanotrophy. In addition, these lipids are also produced by aerobic acetic acid bacteria and, lacking carbon isotope analysis, are more generally used as indicators for aerobiosis in ancient ecosystems. However, recent genetic studies have brought into question our current understanding of the taxonomic diversity of methylhopanoid-producing bacteria and have highlighted that a proper interpretation of methylhopanes in the rock record requires a deeper understanding of their cellular function. In this study, we identified and deleted a gene, hpnR, required for methylation of hopanoids at the C-3 position in the obligate methanotroph Methylococcus capsulatus strain Bath. Bioinformatics analysis revealed that the taxonomic distribution of HpnR extends beyond methanotrophic and acetic acid bacteria. Phenotypic analysis of the M. capsulatus hpnR deletion mutant demonstrated a potential physiological role for 3-methylhopanoids; they appear to be required for the maintenance of intracytoplasmic membranes and cell survival in late stationary phase. Therefore, 3-methylhopanoids may prove more useful as proxies for specific environmental conditions encountered during stationary phase rather than a particular bacterial group.

Role of RelA and SpoT in Burkholderia pseudomallei virulence and immunity.

Burkholderia pseudomallei is a Gram-negative soil bacterium and the causative agent of melioidosis, a disease of humans and animals. It is also listed as a category B bioterrorism threat agent by the U.S. Centers for Disease Control and Prevention, and there is currently no melioidosis vaccine available. Small modified nucleotides such as the hyperphosphorylated guanosine molecules ppGpp and pppGpp play an important role as signaling molecules in prokaryotes. They mediate a global stress response under starvation conditions and have been implicated in the regulation of virulence and survival factors in many bacterial species. In this study, we created a relA spoT double mutant in B. pseudomallei strain K96243, which lacks (p)ppGpp-synthesizing enzymes, and investigated its phenotype in vitro and in vivo. The B. pseudomallei ΔrelA ΔspoT mutant displayed a defect in stationary-phase survival and intracellular replication in murine macrophages. Moreover, the mutant was attenuated in the Galleria mellonella insect model and in both acute and chronic mouse models of melioidosis. Vaccination of mice with the ΔrelA ΔspoT mutant resulted in partial protection against infection with wild-type B. pseudomallei. In summary, (p)ppGpp signaling appears to represent an essential component of the regulatory network governing virulence gene expression and stress adaptation in B. pseudomallei, and the ΔrelA ΔspoT mutant may be a promising live-attenuated vaccine candidate.

A histone-like protein of Helicobacter pylori protects DNA from stress damage and aids host colonization

Genomic DNA in a bacterial cell is folded into a compact structure called a nucleoid, and nucleoid-associated proteins are responsible for proper assembly of active higher-order genome structures. The human gastric pathogen Helicobacter pylori express a nucleoid-associated protein encoded by the hup gene, which is the homolog to the Escherichia coli histone-like protein HU. An H. pylori hup mutant strain (X47 hup:cat) showed a defect in stationary phase survival. The X47 hup:cat mutant was more sensitive to the DNA damaging agent mitomycin C, and displayed a decreased frequency of DNA recombination, indicating Hup plays a significant role in facilitating DNA recombinational repair. The X47 hup:cat mutant was also sensitive to both oxidative and acid stress, conditions that H. pylori commonly encounters in the host. The hup mutant cells survived significantly (7-fold) less upon exposure to macrophages than the wild type strain. In a mouse infection model, the hup mutant strain displayed a greatly reduced ability to colonize host stomachs. The geometric means of colonization number for the wild type and hup mutant were 6×105 and 1.5×104CFU/g stomachs, respectively. Complementation of the hup strain by chromosomal insertion of a functional hup gene restored oxidative stress resistance, DNA transformation frequency, and mouse colonization ability to the wild type level. We directly demonstrated that the purified His-tagged H. pylori Hup protein can protect (in vitro) an H. pylori-derived DNA fragment from oxidative damage.

Genotypic diversity of stress response in Lactobacillus plantarum, Lactobacillus paraplantarum and Lactobacillus pentosus

Lactobacillus plantarum, Lactobacillus pentosus and Lactobacillus paraplantarum are three closely related species which are widespread in food and non-food environments, and are important as starter bacteria or probiotics. In order to evaluate the phenotypic diversity of stress tolerance in the L. plantarum group and the ability to mount an adaptive heat shock response, the survival of exponential and stationary phase and of heat adapted exponential phase cells of six L. plantarum subsp. plantarum, one L. plantarum subsp. argentoratensis, one L. pentosus and two L. paraplantarum strains selected in a previous work upon exposure to oxidative, heat, detergent, starvation and acid stresses was compared to that of the L. plantarum WCFS1 strain. Furthermore, to evaluate the genotypic diversity in stress response genes, ten genes (encoding for chaperones DnaK, GroES and GroEL, regulators CtsR, HrcA and CcpA, ATPases/proteases ClpL, ClpP, ClpX and protease FtsH) were amplified using primers derived from the WCFS1 genome sequence and submitted to restriction with one or two endonucleases. The results were compared by univariate and multivariate statistical methods. In addition, the amplicons for hrcA and ctsR were sequenced and compared by multiple sequence alignment and polymorphism analysis.Although there was evidence of a generalized stress response in the stationary phase, with increase of oxidative, heat, and, to a lesser extent, starvation stress tolerance, and for adaptive heat stress response, with increased tolerance to heat, acid and detergent, different growth phases and adaptation patterns were found. Principal component analysis showed that while heat, acid and detergent stresses respond similarly to growth phase and adaptation, tolerance to oxidative and starvation stresses implies completely unrelated mechanisms. A dendrogram obtained using the data from multilocus restriction typing (MLRT) of stress response genes clearly separated two groups of L. plantarum strains from the other species but there was no correlation between genotypic grouping and grouping obtained on the basis of the stress response pattern, nor with the phylograms obtained from hrcA and ctsR sequences. Differences in sequence in L. plantarum strains were mostly due to single nucleotide polymorphisms with a high frequency of synonymous nucleotide changes and, while hrcA was characterized by an excess of low frequency polymorphism, very low diversity was found in ctsR sequences. Sequence alignment of hrcA allowed a correct discrimination of the strains at the species level, thus confirming the relevance of stress response genes for taxonomy.

Construction and characterization of a null allele of the hfq gene of the dissimilatory metal reducing bacterium Shewanella oneidensis

Shewanella oneidensis is a dissimilatory metal reducing bacterium that can utilize a range of terminal electron acceptors when grown under anaerobic conditions. Our primary goal is to identify and characterize genes that encode small regulatory non‐coding RNAs (sRNAs) in S. oneidensis. sRNA genes are regulated by changes in environmental conditions and can themselves regulate other genes by inexact base pairing to their mRNA targets. We are particularly interested in sRNAs that may regulate the process of metal reduction.The highly conserved RNA chaperone protein Hfq has been widely implicated in bacterial sRNA function. We have constructed a null allele of the S. oneidensis hfq gene. The S. oneidensis hfq mutant has a modest delayed growth phenotype. In addition, our mutant displays reduced survival in stationary phase. Our hfq mutant also exhibits a decreased rate of Cr(VI) reduction when grown under anaerobic conditions compared to the rate of Cr(VI) reduction by wild type S. oneidensis. We are currently evaluating the growth and survival of the Shewanella hfq null mutant. We are also investigating the mutant's sensitivity to a variety of physical stresses, evaluating the capacity of the hfq mutant to reduce metals other than Cr(VI), and determining the role of Shewanella hfq in sRNA function.This research is funded by a grant from the RI‐INBRE program, award # P20RR016457‐10.