CtsR, the Master Regulator of Stress-Response in Oenococcus oeni, Is a Heat Sensor Interacting With ClpL1

Oenococcus oeni is a lactic acid bacterium responsible for malolactic fermentation of wine. While many stress response mechanisms implemented by O. oeni during wine adaptation have been described, little is known about their regulation. CtsR is the only regulator of stress response genes identified to date in O. oeni. Extensively characterized in Bacillus subtilis, the CtsR repressor is active as a dimer at 37°C and degraded at higher temperatures by a proteolytic mechanism involving two adapter proteins, McsA and McsB, together with the ClpCP complex. The O. oeni genome does not encode orthologs of these adapter proteins and the regulation of CtsR activity remains unknown. In this study, we investigate CtsR function in O. oeni by using antisense RNA silencing in vivo to modulate ctsR gene expression. Inhibition of ctsR gene expression by asRNA leads to a significant loss in cultivability after heat shock (58%) and acid shock (59%) highlighting the key role of CtsR in the O. oeni stress response. Regulation of CtsR activity was studied using a heterologous expression system to demonstrate that O. oeni CtsR controls expression and stress induction of the O. oeni hsp18 gene when produced in a ctsR-deficient B. subtilis strain. Under heat stress conditions, O. oeni CtsR acts as a temperature sensor and is inactivated at growth temperatures above 33°C. Finally, using an E. coli bacterial two-hybrid system, we showed that CtsR and ClpL1 interact, suggesting a key role for ClpL1 in controlling CtsR activity in O. oeni

Nonselective Bottlenecks Control the Divergence and Diversification of Phase-Variable Bacterial Populations

Phase variation occurs in many pathogenic and commensal bacteria and is a major generator of genetic variability. A putative advantage of phase variation is to counter reductions in variability imposed by nonselective bottlenecks during transmission. Genomes of Campylobacter jejuni, a widespread food-borne pathogen, contain multiple phase-variable loci whose rapid, stochastic variation is generated by hypermutable simple sequence repeat tracts. These loci can occupy a vast number of combinatorial expression states (phasotypes) enabling populations to rapidly access phenotypic diversity. The imposition of nonselective bottlenecks can perturb the relative frequencies of phasotypes, changing both within-population diversity and divergence from the initial population. Using both in vitro testing of C. jejuni populations and a simple stochastic simulation of phasotype change, we observed that single-cell bottlenecks produce output populations of low diversity but with bimodal patterns of either high or low divergence. Conversely, large bottlenecks allow divergence only by accumulation of diversity, while interpolation between these extremes is observed in intermediary bottlenecks. These patterns are sensitive to the genetic diversity of initial populations but stable over a range of mutation rates and number of loci. The qualitative similarities of experimental and in silico modeling indicate that the observed patterns are robust and applicable to other systems where localized hypermutation is a defining feature. We conclude that while phase variation will maintain bacterial population diversity in the face of intermediate bottlenecks, narrow transmission-associated bottlenecks could produce host-to-host variation in bacterial phenotypes and hence stochastic variation in colonization and disease outcomes

<i>Staphylococcus aureus </i>Transcriptome Architecture:From Laboratory to Infection-Mimicking Conditions

Staphylococcus aureus is a major pathogen that colonizes about 20% of the human population. Intriguingly, this Gram-positive bacterium can survive and thrive under a wide range of different conditions, both inside and outside the human body. Here, we investigated the transcriptional adaptation of S. aureus HG001, a derivative of strain NCTC 8325, across experimental conditions ranging from optimal growth in vitro to intracellular growth in host cells. These data establish an extensive repertoire of transcription units and non-coding RNAs, a classification of 1412 promoters according to their dependence on the RNA polymerase sigma factors SigA or SigB, and allow identification of new potential targets for several known transcription factors. In particular, this study revealed a relatively low abundance of antisense RNAs in S. aureus, where they overlap only 6% of the coding genes, and only 19 antisense RNAs not co-transcribed with other genes were found. Promoter analysis and comparison with Bacillus subtilis links the small number of antisense RNAs to a less profound impact of alternative sigma factors in S. aureus. Furthermore, we revealed that Rho-dependent transcription termination suppresses pervasive antisense transcription, presumably originating from abundant spurious transcription initiation in this A+T-rich genome, which would otherwise affect expression of the overlapped genes. In summary, our study provides genome-wide information on transcriptional regulation and non-coding RNAs in S. aureus as well as new insights into the biological function of Rho and the implications of spurious transcription in bacteria

High-Resolution Melting Genotyping of Enterococcus faecium Based on Multilocus Sequence Typing Derived Single Nucleotide Polymorphisms

We have developed a single nucleotide polymorphism (SNP) nucleated high-resolution melting (HRM) technique to genotype Enterococcus faecium. Eight SNPs were derived from the E. faecium multilocus sequence typing (MLST) database and amplified fragments containing these SNPs were interrogated by HRM. We tested the HRM genotyping scheme on 85 E. faecium bloodstream isolates and compared the results with MLST, pulsed-field gel electrophoresis (PFGE) and an allele specific real-time PCR (AS kinetic PCR) SNP typing method. In silico analysis based on predicted HRM curves according to the G+C content of each fragment for all 567 sequence types (STs) in the MLST database together with empiric data from the 85 isolates demonstrated that HRM analysis resolves E. faecium into 231 “melting types” (MelTs) and provides a Simpson's Index of Diversity (D) of 0.991 with respect to MLST. This is a significant improvement on the AS kinetic PCR SNP typing scheme that resolves 61 SNP types with D of 0.95. The MelTs were concordant with the known ST of the isolates. For the 85 isolates, there were 13 PFGE patterns, 17 STs, 14 MelTs and eight SNP types. There was excellent concordance between PFGE, MLST and MelTs with Adjusted Rand Indices of PFGE to MelT 0.936 and ST to MelT 0.973. In conclusion, this HRM based method appears rapid and reproducible. The results are concordant with MLST and the MLST based population structure

Differential Actions of Chlorhexidine on the Cell Wall of Bacillus subtilis and Escherichia coli

Chlorhexidine is a chlorinated phenolic disinfectant used commonly in mouthwash for its action against bacteria. However, a comparative study of the action of chlorhexidine on the cell morphology of Gram-positive and Gram-negative bacteria is lacking. In this study, the actions of chlorhexidine on the cell morphology were identified with the aids of electron microscopy. After exposure to chlorhexidine, numerous spots of indentation on the cell wall were found in both Bacillus subtilis and Escherichia coli. The number of indentation spots increased with time of incubation and increasing chlorhexidine concentration. Interestingly, the dented spots found in B. subtilis appeared mainly at the hemispherical caps of the cells, while in E. coli the dented spots were found all over the cells. After being exposed to chlorhexidine for a prolonged period, leakage of cellular contents and subsequent ghost cells were observed, especially from B subtilis. By using 2-D gel/MS-MS analysis, five proteins related to purine nucleoside interconversion and metabolism were preferentially induced in the cell wall of E. coli, while three proteins related to stress response and four others in amino acid biosynthesis were up-regulated in the cell wall materials of B. subtilis. The localized morphological damages together with the biochemical and protein analysis of the chlorhexidine-treated cells suggest that chlorhexidine may act on the differentially distributed lipids in the cell membranes/wall of B. subtilis and E. coli

BaSysBio: Towards an understanding of dynamic transcriptional regulation at global scale in bacteria: a systems biology approach

9th International Conference on Systems Biology, ICSB 2008, 22-28 August, Goteborg, SwedenBaSysBio adopts a systems biology approach in which quantitative experimental data will be generated for each step of the information flow and will fuel computational modelling. High throughput technologies (living cell arrays, tiling DNA microarrays, MDLC proteomics and quantitative metabolomics) will be developed in conjunction with new computational modelling concepts to facilitate the understanding of biological complexity. Models will simulate the cellular transcriptional responses to environmental changes and their impact on metabolism and proteome dynamics. The iterative process of simulations and model-driven targeted experiments will generate novel hypotheses about the mechanistic nature of dynamic cellular responses, unravel emerging systems properties and ultimately provide an efficient roadmap to tackle novel, pathogenic organismsN

Feasibility of Modified Surviving Sepsis Campaign Guidelines in a Resource-Restricted Setting Based on a Cohort Study of Severe S. Aureus Sepsis

[This corrects the article on p. e29858 in vol. 7.]

Photodynamic and Antibiotic Therapy Impair the Pathogenesis of Enterococcus faecium in a Whole Animal Insect Model

Enterococcus faecium has emerged as one of the most important pathogens in healthcare-associated infections worldwide due to its intrinsic and acquired resistance to many antibiotics, including vancomycin. Antimicrobial photodynamic therapy (aPDT) is an alternative therapeutic platform that is currently under investigation for the control and treatment of infections. PDT is based on the use of photoactive dye molecules, widely known as photosensitizer (PS). PS, upon irradiation with visible light, produces reactive oxygen species that can destroy lipids and proteins causing cell death. We employed Galleria mellonella (the greater wax moth) caterpillar fatally infected with E. faecium to develop an invertebrate host model system that can be used to study the antimicrobial PDT (alone or combined with antibiotics). In the establishment of infection by E. faecium in G. mellonella, we found that the G. mellonella death rate was dependent on the number of bacterial cells injected into the insect hemocoel and all E. faecium strains tested were capable of infecting and killing G. mellonella. Antibiotic treatment with ampicillin, gentamicin or the combination of ampicillin and gentamicin prolonged caterpillar survival infected by E. faecium (P = 0.0003, P = 0.0001 and P = 0.0001, respectively). In the study of antimicrobial PDT, we verified that methylene blue (MB) injected into the insect followed by whole body illumination prolonged the caterpillar survival (P = 0.0192). Interestingly, combination therapy of larvae infected with vancomycin-resistant E. faecium, with antimicrobial PDT followed by vancomycin, significantly prolonged the survival of the caterpillars when compared to either antimicrobial PDT (P = 0.0095) or vancomycin treatment alone (P = 0.0025), suggesting that the aPDT made the vancomycin resistant E. faecium strain more susceptible to vancomycin action. In summary, G. mellonella provides an invertebrate model host to study the antimicrobial PDT and to explore combinatorial aPDT-based treatments

The Pleiotropic CymR Regulator of Staphylococcus aureus Plays an Important Role in Virulence and Stress Response

We have characterized a novel pleiotropic role for CymR, the master regulator of cysteine metabolism. We show here that CymR plays an important role both in stress response and virulence of Staphylococcus aureus. Genes involved in detoxification processes, including oxidative stress response and metal ion homeostasis, were differentially expressed in a ΔcymR mutant. Deletion of cymR resulted in increased sensitivity to hydrogen peroxide-, disulfide-, tellurite- and copper-induced stresses. Estimation of metabolite pools suggests that this heightened sensitivity could be the result of profound metabolic changes in the ΔcymR mutant, with an increase in the intracellular cysteine pool and hydrogen sulfide formation. Since resistance to oxidative stress within the host organism is important for pathogen survival, we investigated the role of CymR during the infectious process. Our results indicate that the deletion of cymR promotes survival of S. aureus inside macrophages, whereas virulence of the ΔcymR mutant is highly impaired in mice. These data indicate that CymR plays a major role in virulence and adaptation of S. aureus for survival within the host

Thermoregulation of Capsule Production by Streptococcus pyogenes

The capsule of Streptococcus pyogenes serves as an adhesin as well as an anti-phagocytic factor by binding to CD44 on keratinocytes of the pharyngeal mucosa and the skin, the main entry sites of the pathogen. We discovered that S. pyogenes HSC5 and MGAS315 strains are further thermoregulated for capsule production at a post-transcriptional level in addition to the transcriptional regulation by the CovRS two-component regulatory system. When the transcription of the hasABC capsular biosynthetic locus was de-repressed through mutation of the covRS system, the two strains, which have been used for pathogenesis studies in the laboratory, exhibited markedly increased capsule production at sub-body temperature. Employing transposon mutagenesis, we found that CvfA, a previously identified membrane-associated endoribonuclease, is required for the thermoregulation of capsule synthesis. The mutation of the cvfA gene conferred increased capsule production regardless of temperature. However, the amount of the capsule transcript was not changed by the mutation, indicating that a post-transcriptional regulator mediates between CvfA and thermoregulated capsule production. When we tested naturally occurring invasive mucoid strains, a high percentage (11/53, 21%) of the strains exhibited thermoregulated capsule production. As expected, the mucoid phenotype of these strains at sub-body temperature was due to mutations within the chromosomal covRS genes. Capsule thermoregulation that exhibits high capsule production at lower temperatures that occur on the skin or mucosal surface potentially confers better capability of adhesion and invasion when S. pyogenes penetrates the epithelial surface