Sigma Factor RpoS Research Articles

Residue L193P Mutant of RpoS Affects Its Activity During Biofilm Formation in Salmonella Pullorum

The role of alternative sigma factor RpoS in regulating biofilm formation may differ in various Salmonella Pullorum strains. In this study, the biofilm-forming ability of two Salmonella Pullorum strains S6702 and S11923-3 were compared. The biofilm forming ability of S11923-3 was much stronger than that of S6702. After knocking out the rpoS gene, S11923-3ΔrpoS had significantly reduced biofilm while S6702ΔrpoS demonstrated similar biofilm compared with each parent strain. The analysis of RpoS sequences indicated two amino acid substitutions (L193P and R293C) between S6702 and S11923-3 RpoS. A complementation study confirmed that the expression of S11923-3 RpoS rather than S6702 RpoS could restore the biofilm-forming ability of ΔrpoS strains and the L193P mutation contributed to the restoration of the biofilm-forming ability. Further study indicated that RpoS with the L193P mutant had significantly improved expression level and binding activity to RNAP and csgD gene promoter, which increased the efficacy of the csgD gene promoter and biofilm-forming ability. Therefore, the L193P mutation of RpoS is critical for stronger biofilm formation of Salmonella Pullorum.

Stringent starvation protein A and LuxI/LuxR-type quorum sensing system constitute a mutual positive regulation loop in Pseudoalteromonas

Bacteria commonly exhibit social activities through acyl-homoserine lactones (AHLs)-based quorum sensing (QS) systems to form their unique social network. The sigma factor RpoS is an important regulator that controls QS system in different bacteria. However, the upstream of RpoS involving regulation on QS system remains unclear. In Escherichia coli RpoS is regulated by stringent starvation protein A (SspA), which is dependent of histone-like nucleoid structuring protein (H-NS). To date, the connection between SspA and QS system is essentially unknown. Here, we characterized a typical LuxI/LuxR-type QS system in marine bacterium Pseudoalteromonas sp. T1lg65 which can produce four types of AHLs. The luxI encoding AHLs synthase and luxR encoding AHLs-responsive receptor are co-transcribed, providing advantages in rapidly amplifying QS signaling. Notably, SspA positively regulated luxI/luxR transcription by activating RpoS expression, which is mediated by H-NS. Interestingly, LuxR in turn positively regulated SspA expression. Therefore, SspA and QS system constitute a mutual positive regulation loop in T1lg65. In view of the crucial roles of SspA and QS system in environmental adaption, we believe that the improvement of bacterial tolerance to marine environments could be related to rapidly tuning SspA-involved QS programming.

Sublethal Paraquat Confers Multidrug Tolerance in Pseudomonas aeruginosa by Inducing Superoxide Dismutase Activity and Lowering Envelope Permeability.

Stressors and environmental cues shape the physiological state of bacteria, and thus how they subsequently respond to antibiotic toxicity. To understand how superoxide stress can modulate survival to bactericidal antibiotics, we examined the effect of intracellular superoxide generators, paraquat and menadione, on stationary-phase antibiotic tolerance of the opportunistic pathogen, Pseudomonas aeruginosa. We tested how pre-challenge with sublethal paraquat and menadione alters the tolerance to ofloxacin and meropenem in wild-type P. aeruginosa and mutants lacking superoxide dismutase (SOD) activity (sodAB), the paraquat responsive regulator soxR, (p)ppGpp signaling (relA spoT mutant), or the alternative sigma factor rpoS. We confirmed that loss of SOD activity impairs ofloxacin and meropenem tolerance in stationary phase cells, and found that sublethal superoxide generators induce drug tolerance by stimulating SOD activity. This response is rapid, requires de novo protein synthesis, and is RpoS-dependent but does not require (p)ppGpp signaling nor SoxR. We further showed that pre-challenge with sublethal paraquat induces a SOD-dependent reduction in cell-envelope permeability and ofloxacin penetration. Our results highlight a novel mechanism of hormetic protection by superoxide generators, which may have important implications for stress-induced antibiotic tolerance in P. aeruginosa cells.

The mode of action of the PSIR-3 photosensitizer in the photodynamic inactivation of Klebsiella pneumoniae is by the production of type II ROS which activate RpoE-regulated extracytoplasmic factors.

Due to increased bacterial multi-drug resistance (MDR), there is an antibiotic depletion to treat infectious diseases. Consequently, other promising options have emerged, such as the antimicrobial photodynamic inactivation therapy (aPDI) based on photosensitizer (PS) compounds to produce light-activated local oxidative stress (photooxidative stress). However, there are scarce studies regarding the mode of action of PS compounds to induce photooxidative stress on pathogenic γ-proteobacteria such as MDR-Klebsiella pneumoniae. The mode of action exerted by the cationic Ir(III)-based PS (PSIR-3) to inhibit the growth of K. pneumoniae was analyzed. RT-qPCR determined the transcriptional response induced by PSIR-3 on bacteria treated with aPDI. The expression levels of genes associated with a bacterial oxidative response, such as oxyR and sodA, and the extracytoplasmic, regulators rpoE and hfq were determined. Also, were determined the transcriptional response of the extracytoplasmic factors mrkD, acrB, magA, and rmpA. At 17 μW/cm2 photon flux and 4 μg/mL of the PSIR-3 compound, the K. pneumoniae growth was inhibited in 3 log10. Compared with untreated bacteria, the transcriptional response induced by PSIR-3 occurs via the extracytoplasmic sigma factor rpoE and hfq. In contrast, no participation in the oxyR pathway or induction of the sodA gene was observed. This response was accompanied by the upregulation of the extracytoplasmic virulence factors mrkD, magA, and rmpA. PDI aPDI produced by PSIR-3 kills K. pneumoniae and may induce damage to the bacterial envelope. The bacterium tries to avoid this injury by activation of extracytoplasmic factors mediated through the rpoE regulon.

Bacteria Modify Their Sensitivity to Chemerin-Derived Peptides by Hindering Peptide Association With the Cell Surface and Peptide Oxidation.

Chronic inflammatory skin diseases like psoriasis alter the local skin microbiome and lead to complications such as persistent infection with opportunistic/pathogenic bacteria. Disease-associated changes in microbiota may be due to downregulation of epidermal antimicrobial proteins/peptides, such as antimicrobial protein chemerin. Here, we show that chemerin and its bioactive derivatives have differential effects on the viability of different genera of cutaneous bacteria. The lethal effects of chemerin are enhanced by bacterial-derived ROS-induced chemerin peptide oxidation and suppressed by stationary phase sigma factor RpoS. Insight into the mechanisms underlying changes in the composition of cutaneous bacteria during autoreactive skin disease may provide novel ways to mobilize chemerin and its peptide derivatives for maximum antimicrobial efficacy.

Interactions between DksA and Stress-Responsive Alternative Sigma Factors Control Inorganic Polyphosphate Accumulation in Escherichia coli.

Bacteria synthesize inorganic polyphosphate (polyP) in response to a variety of different stress conditions. polyP protects bacteria by acting as a protein-stabilizing chaperone, metal chelator, or regulator of protein function, among other mechanisms. However, little is known about how stress signals are transmitted in the cell to lead to increased polyP accumulation. Previous work in the model enterobacterium Escherichia coli has indicated that the RNA polymerase-binding regulatory protein DksA is required for polyP synthesis in response to nutrient limitation stress. In this work, I set out to characterize the role of DksA in polyP regulation in more detail. I found that overexpression of DksA increases cellular polyP content (explaining the long-mysterious phenotype of dksA overexpression rescuing growth of a dnaK mutant at high temperatures) and characterized the roles of known functional residues of DksA in this process, finding that binding to RNA polymerase is required but that none of the other functions of DksA appear to be necessary. Transcriptomics revealed genome-wide transcriptional changes upon nutrient limitation, many of which were affected by DksA, and follow-up experiments identified complex interactions between DksA and the stress-sensing alternative sigma factors FliA, RpoN, and RpoE that impact polyP production, indicating that regulation of polyP synthesis is deeply entwined in the multifactorial stress response network of E. coliIMPORTANCE Inorganic polyphosphate (polyP) is an evolutionarily ancient, widely conserved biopolymer required for stress resistance and pathogenesis in diverse bacteria, but we do not understand how its synthesis is regulated. In this work, I gained new insights into this process by characterizing the role of the transcriptional regulator DksA in polyP regulation in Escherichia coli and identifying previously unknown links between polyP synthesis and the stress-responsive alternative sigma factors FliA, RpoN, and RpoE.

DksA plays an essential role in regulating the virulence of Borrelia burgdorferi.

The RNA polymerase-binding protein DksA, together with the alarmone nucleotides (p)ppGpp, mediates the stringent response to nutrient starvation in Borrelia burgdorferi. To date, the contribution of DksA to B. burgdorferi infection remains unknown. We report here that DksA is essential for B. burgdorferi to infect a mammalian host. dksA expression was highly induced during infection. Moreover, a dksA-deficient mutant was incapable of infecting mice. The mutant displayed growth defects when cultured in vitro and resistance to osmotic pressure was markedly reduced. These phenotypes were fully restored to those of the wild type when dksA mutation was complemented. We further showed that DksA controlled the expression of virulence-associated lipoprotein OspC, likely via the central alternative sigma factor RpoS. Synthesis of RpoS was abolished in the dksA mutant, but rpoS transcription remained unaffected. Additionally, we found that the expression of clpX, clpA, clpP, and clpP2 was significantly increased in the mutant, suggesting that DksA may post-transcriptionally regulate rpoS expression via its effect on ClpXP and/or ClpAP proteases. These combined data demonstrate that DksA regulates B. burgdorferi virulence at least partially through its influence on RpoS and OspC. This study thus elucidates that, in addition to function as a stringent response regulator, DksA promotes the transcription and/or translation of genes contributing to B. burgdorferi infectivity.

Identification of a Small Protein Involved in the Regulation of the Stationary Phase Sigma Factor RpoS

Upon encountering stressful environments, E coli induces the sigma factor RpoS to activate stationary phase and provide general protection against stress. To better understand the regulation of RpoS, we carried out an overexpression screen with an RpoS’‐‘LacZ fusion. This screen identified an intergenic region that increased RpoS expression and significantly slowed down growth when overexpressed. A short open reading frame within this region was sub cloned and shown to also induce RpoS expression and affect growth. Thus RpoS may be regulated by a novel small protein. This work highlights the importance of small proteins that were not originally annotated in the E coli genome.

Adaptation to Photooxidative Stress: Common and Special Strategies of the Alphaproteobacteria Rhodobacter sphaeroides and Rhodobacter capsulatus.

Photosynthetic bacteria have to deal with the risk of photooxidative stress that occurs in presence of light and oxygen due to the photosensitizing activity of (bacterio-) chlorophylls. Facultative phototrophs of the genus Rhodobacter adapt the formation of photosynthetic complexes to oxygen and light conditions, but cannot completely avoid this stress if environmental conditions suddenly change. R. capsulatus has a stronger pigmentation and faster switches to phototrophic growth than R. sphaeroides. However, its photooxidative stress response has not been investigated. Here, we compare both species by transcriptomics and proteomics, revealing that proteins involved in oxidation–reduction processes, DNA, and protein damage repair play pivotal roles. These functions are likely universal to many phototrophs. Furthermore, the alternative sigma factors RpoE and RpoHII are induced in both species, even though the genetic localization of the rpoE gene, the RpoE protein itself, and probably its regulon, are different. Despite sharing the same habitats, our findings also suggest individual strategies. The crtIB-tspO operon, encoding proteins for biosynthesis of carotenoid precursors and a regulator of photosynthesis, and cbiX, encoding a putative ferrochelatase, are induced in R. capsulatus. This specific response might support adaptation by maintaining high carotenoid-to-bacteriochlorophyll ratios and preventing the accumulation of porphyrin-derived photosensitizers.

Development and characterization of Escherichia coli triple reporter strains for investigation of population heterogeneity in bioprocesses

BackgroundToday there is an increasing demand for high yielding robust and cost efficient biotechnological production processes. Although cells in these processes originate from isogenic cultures, heterogeneity induced by intrinsic and extrinsic influences is omnipresent. To increase understanding of this mechanistically poorly understood phenomenon, advanced tools that provide insights into single cell physiology are needed.ResultsTwo Escherichia coli triple reporter strains have been designed based on the industrially relevant production host E. coli BL21(DE3) and a modified version thereof, E. coli T7E2. The strains carry three different fluorescence proteins chromosomally integrated. Single cell growth is followed with EmeraldGFP (EmGFP)-expression together with the ribosomal promoter rrnB. General stress response of single cells is monitored by expression of sigma factor rpoS with mStrawberry, whereas expression of the nar-operon together with TagRFP657 gives information about oxygen limitation of single cells. First, the strains were characterized in batch operated stirred-tank bioreactors in comparison to wildtype E. coli BL21(DE3). Afterwards, applicability of the triple reporter strains for investigation of population heterogeneity in bioprocesses was demonstrated in continuous processes in stirred-tank bioreactors at different growth rates and in response to glucose and oxygen perturbation simulating gradients on industrial scale. Population and single cell level physiology was monitored evaluating general physiology and flow cytometry analysis of fluorescence distributions of the triple reporter strains. Although both triple reporter strains reflected physiological changes that were expected based on the expression characteristics of the marker proteins, the triple reporter strain based on E. coli T7E2 showed higher sensitivity in response to environmental changes. For both strains, noise in gene expression was observed during transition from phases of non-growth to growth. Apparently, under some process conditions, e.g. the stationary phase in batch cultures, the fluorescence response of EmGFP and mStrawberry is preserved, whereas TagRFP657 showed a distinct response.ConclusionsSingle cell growth, general stress response and oxygen limitation of single cells could be followed using the two triple reporter strains developed in this study. They represent valuable tools to study population heterogeneity in bioprocesses significantly increasing the level of information compared to the use of single reporter strains.

A DIVA vaccine strain lacking RpoS and the secondary messenger c-di-GMP for protection against salmonellosis in pigs

Salmonellosis is the second most common food-borne zoonosis in the European Union, with pigs being a major reservoir of this pathogen. Salmonella control in pig production requires multiple measures amongst which vaccination may be used to reduce subclinical carriage and shedding of prevalent serovars, such as Salmonella enterica serovar Typhimurium. Live attenuated vaccine strains offer advantages in terms of enhancing cell mediated immunity and allowing inoculation by the oral route. However, main failures of these vaccines are the limited cross-protection achieved against heterologous serovars and interference with serological monitoring for infection. We have recently shown that an attenuated S. Enteritidis strain (ΔXIII) is protective against S. Typhimurium in a murine infection model. ΔXIII strain harbours 13 chromosomal deletions that make it unable to produce the sigma factor RpoS and synthesize cyclic-di-GMP (c-di-GMP). In this study, our objectives were to test the protective effects of ΔXIII strain in swine and to investigate if the use of ΔXIII permits the discrimination of vaccinated from infected pigs. Results show that oral vaccination of pre-weaned piglets with ΔXIII cross-protected against a challenge with S. Typhimurium by reducing faecal shedding and ileocaecal lymph nodes colonization, both at the time of weaning and slaughter. Vaccinated pigs showed neither faecal shedding nor tissue persistence of the vaccine strain at weaning, ensuring the absence of ΔXIII strain by the time of slaughter. Moreover, lack of the SEN4316 protein in ΔXIII strain allowed the development of a serological test that enabled the differentiation of infected from vaccinated animals (DIVA).

Metagenomic Resolution of Functional Diversity in Copper Surface-Associated Marine Biofilms.

We used metagenomic sequencing combined with morphological and chemical analyses to investigate microbial taxa and functions related to copper-resistance and microbiologically influenced corrosion in mature copper-associated biofilms in coastal seawater for 44 months. Facultative anaerobic microbes such as Woeseia sp. were found to be the dominant groups on the copper surface. Genes related to stress response and possible heavy metal transport systems, especially RNA polymerase sigma factors (rpoE) and putative ATP-binding cassette (ABC) transport system permease protein (ABC.CD.P) were observed to be highly enriched in copper-associated biofilms, while genes encoding DNA-methyltransferase and RNA polymerase subunit were highly enriched in aluminum-associated biofilms and seawater planktonic cells, respectively. Moreover, copper-associated biofilms harbored abundant copper-resistance genes including cus, cop and pco, as well as abundant genes related to extracellular polymeric substances, indicating the presence of diverse copper-resistance patterns. The proportion of dsr in copper-associated biofilms, key genes related to sulfide production, was as low as that in aluminum biofilm and seawater, which ruled out the possibility of microbial sulfide-induced copper-corrosion under field conditions. These results may fill knowledge gaps about the in situ microbial functions of marine biofilms and their effects on toxic-metal corrosion.

Carbon Starvation Induces the Expression of PprB-Regulated Genes in Pseudomonas aeruginosa.

Pseudomonas aeruginosa can cause severe infections in humans. This bacterium often adopts a biofilm lifestyle that is hard to treat. In several previous studies, the PprA-PprB two-component system (TCS), which controls the expression of type IVb pili, BapA adhesin, and CupE fimbriae, was shown to be involved in biofilm formation (M. Romero, H. Silistre, L. Lovelock, V. J. Wright, K.-G. Chan, et al., Nucleic Acids Res 46:6823-6840, 2018, https://doi.org/10.1093/nar/gky324; S. de Bentzmann, C. Giraud, C. S. Bernard, V. Calderon, F. Ewald F, et al., PLoS Pathog 8:e1003052, 2012, https://doi.org/10.1371/journal.ppat.1003052). However, signals or environmental conditions that can trigger the PprA-PprB TCS are still unknown, and the molecular mechanisms of PprB-mediated biofilm formation are poorly characterized. Here, we report that carbon starvation stress (CSS) can induce the expression of pprB and genes in the PprB regulon. CSS-induced pprB transcription is mediated by the stress response sigma factor RpoS rather than the two-component sensor PprA. We also observed a strong negative regulation of PprB on the transcription of itself. Further experiments showed that PprB overexpression greatly enhanced cell-cell adhesion (CCA) and cell-surface adhesion (CSA) in P. aeruginosa Specifically, under the background of PprB overexpression, both the BapA adhesin and CupE fimbriae displayed positive effects on CCA and CSA, while the type IVb pili showed an unexpected negative effect on CCA and no effect on CSA. In addition, expression of the PprB regulon genes were significantly increased in 3-day colony biofilms, indicating a possible carbon limitation state. The CSS-RpoS-PprB-Bap/Flp/CupE pathway identified in this study provides a new perspective on the process of biofilm formation in carbon-limited environments.IMPORTANCE Typically, the determination of the external signals that can trigger a regulatory system is crucial to understand the regulatory logic and inward function of that system. The PprA-PprB two-component system was reported to be involved in biofilm formation in Pseudomonas aeruginosa, but the signals triggering this system are unknown. In this study, we found that carbon starvation stress (CSS) induces transcription of pprB and genes in the PprB regulon through an RpoS-dependent pathway. Increased PprB expression leads to enhanced cell-cell adhesion (CCA) and cell-surface adhesion (CSA) in P. aeruginosa Both CCA and CSA are largely dependent on the Bap secretion system and are moderately dependent on the CupE fimbriae. Our findings suggest that PprB reinforces the structure of biofilms under carbon-limited conditions, and the Bap secretion system and CupE fimbriae are two potential targets for biofilm treatment.

Increased mistranslation protects E.coli from protein misfolding stress due to activation of a RpoS-dependent heat shock response.

The misincorporation of an incorrect amino acid into a polypeptide during protein synthesis is considered a detrimental phenomenon. A mistranslated protein is often misfolded and degraded or nonfunctional and results in an increased cost to quality control machinery. Despite these costs, errors during protein synthesis are common in bacteria. Here, we report that mistranslation in Escherichia coli increase the protein level of the heat shock sigma factor RpoH and protect cells against heat stress. Surprisingly, this increase in RpoH due to mistranslation is dependent on the presence of the general stress response sigma factor RpoS. This report provides evidence for a protective function of mistranslation and suggests a novel regulatory role of RpoS in the heat shock response.

Death Rate of E.coli during Starvation Is Set by Maintenance Cost and Biomass Recycling.

To break down organismal fitness into molecular contributions, costs and benefits of cellular components must be analyzed in all phases of the organism's life cycle. Here, we establish the required quantitative approach for the death phase of the model bacterium Escherichia coli. We show that in carbon starvation, an exponential decay of viability emerges as a collective phenomenon, with viable cells recycling nutrients from cell carcasses to maintain viability. The observed collective death rate is determined by the maintenance rate of viable cells and the amount of nutrients recovered from dead cells. Using this relation, we study the cost of a wasteful enzyme during starvation and the benefit of the stress response sigma factor RpoS. While the enzyme increases maintenance and thereby the death rate, RpoS improves biomass recycling, decreasing the death rate. Our approach thus enables quantitative analyses of how cellular components affect the survival of non-growing cells.

Alternative Sigma Factor RpoX Is a Part of the RpoE Regulon and Plays Distinct Roles in Stress Responses, Motility, Biofilm Formation, and Hemolytic Activities in the Marine Pathogen Vibrio alginolyticus.

Vibrio alginolyticus is one of the most abundant microorganisms in marine environments and is also an opportunistic pathogen mediating high-mortality vibriosis in marine animals. Alternative sigma factors play essential roles in bacterial pathogens in the adaptation to environmental changes during infection and the adaptation to various niches, but little is known about them for V. alginolyticus Our previous investigation indicated that the transcript level of the gene rpoX significantly decreased in an RpoE mutant. Here, we found that rpoX was highly expressed in response to high temperature and low osmotic stress and was under the direct control of the alternative sigma factor RpoE and its own product RpoX. Moreover, transcriptome sequencing (RNA-seq) results showed that RpoE and RpoX had different regulons, although they coregulated 105 genes at high temperature (42°C), including genes associated with biofilm formation, motility, virulence, regulatory factors, and the stress response. RNA-seq and chromatin immunoprecipitation sequencing (ChIP-seq) analyses as well as electrophoretic mobility shift assays (EMSAs) revealed the distinct binding motifs of RpoE and RpoX proteins. Furthermore, quantitative real-time reverse transcription-PCR (qRT-PCR) analysis also confirmed that RpoX can upregulate genes associated with flagella, biofilm formation, and hemolytic activities at higher temperatures. rpoX abrogation does not appear to attenuate virulence toward model fish at normal temperature. Collectively, data from this study demonstrated the regulatory cascades of RpoE and an alternative sigma factor, RpoX, in response to heat and osmotic stresses and their distinct and overlapping roles in pathogenesis and stress responses in the marine bacterium V. alginolyticusIMPORTANCE The alternative sigma factor RpoE is essential for the virulence of Vibrio alginolyticus toward marine fish, coral, and other animals in response to sea surface temperature increases. In this study, we characterized another alternative sigma factor, RpoX, which is induced at high temperatures and under low-osmotic-stress conditions. The expression of rpoX is under the tight control of RpoE and RpoX. Although RpoE and RpoX coregulate 105 genes, they are programming different regulatory functions in stress responses and virulence in V. alginolyticus These findings illuminated the RpoE-RpoX-centered regulatory cascades and their distinct and overlapping regulatory roles in V. alginolyticus, which facilitates unraveling of the mechanisms by which the bacterium causes diseases in various sea animals in response to temperature fluctuations as well as the development of appropriate strategies to tackle infections by this bacterium.

Coxiella burnetii RpoS Regulates Genes Involved in Morphological Differentiation and Intracellular Growth.

Coxiella burnetii, the etiological agent of Q fever, undergoes a unique biphasic developmental cycle where bacteria transition from a replicating (exponential-phase) large cell variant (LCV) form to a nonreplicating (stationary-phase) small cell variant (SCV) form. The alternative sigma factor RpoS is an essential regulator of stress responses and stationary-phase physiology in several bacterial species, including Legionella pneumophila, which has a developmental cycle superficially similar to that of C. burnetii Here, we used a C. burnetii ΔrpoS mutant to define the role of RpoS in intracellular growth and SCV development. Growth yields following infection of Vero epithelial cells or THP-1 macrophage-like cells with the rpoS mutant in the SCV form, but not the LCV form, were significantly lower than that of wild-type bacteria. RNA sequencing and whole-cell mass spectrometry of the C. burnetii ΔrpoS mutant revealed that a substantial portion of the C. burnetii genome is regulated by RpoS during SCV development. Regulated genes include those involved in stress responses, arginine transport, peptidoglycan remodeling, and synthesis of the SCV-specific protein ScvA. Genes comprising the dot/icm locus, responsible for production of the Dot/Icm type 4B secretion system, were also dysregulated in the rpoS mutant. These data were corroborated with independent assays demonstrating that the C. burnetii ΔrpoS strain has increased sensitivity to hydrogen peroxide and carbenicillin and a thinner cell wall/outer membrane complex. Collectively, these results demonstrate that RpoS is an important regulator of genes involved in C. burnetii SCV development and intracellular growth.IMPORTANCE The Q fever bacterium Coxiella burnetii has spore-like environmental stability, a characteristic that contributes to its designation as a potential bioweapon. Stability is likely conferred by a highly resistant, small cell variant (SCV) stationary-phase form that arises during a biphasic developmental cycle. Here, we define the role of the alternative sigma factor RpoS in regulating genes associated with SCV development. Genes involved in stress responses, amino acid transport, cell wall remodeling, and type 4B effector secretion were dysregulated in the rpoS mutant. Cellular impairments included defects in intracellular growth, cell wall structure, and resistance to oxidants. These results support RpoS as a central regulator of the Coxiella developmental cycle and identify developmentally regulated genes involved in morphological differentiation.

Resistance to oxidative stress by inner membrane protein ElaB is regulated by OxyR and RpoS.

SummaryC‐tail anchored inner membrane proteins are a family of proteins that contain a C‐terminal transmembrane domain but lack an N‐terminal signal sequence for membrane targeting. They are widespread in eukaryotes and prokaryotes and play critical roles in membrane traffic, apoptosis and protein translocation in eukaryotes. Recently, we identified and characterized in Escherichia coli a new C‐tail anchored inner membrane, ElaB, which is regulated by the stationary phase sigma factor RpoS. ElaB is important for resistance to oxidative stress but the exact mechanism is unclear. Here, we show that ElaB functions as part of the adaptive oxidative stress response by maintaining membrane integrity. Production of ElaB is induced by oxidative stress at the transcriptional level. Moreover, elaB expression is also regulated by the key regulator OxyR via an OxyR binding site in the promoter of elaB. OxyR induces the expression of elaB in the exponential growth phase, while excess OxyR reduces elaB expression in an RpoS‐dependent way in the stationary phase. In addition, deletion of elaB reduced fitness compared to wild‐type cells after prolonged incubation. Therefore, we determined how ElaB is regulated under oxidative stress: RpoS and OxyR coordinately control the expression of inner membrane protein ElaB.

Polyphosphate kinase 1 of Burkholderia pseudomallei controls quorum sensing, RpoS and host cell invasion

Burkholderia pseudomallei is a Gram negative bacterium and the causative agent of melioidosis. Nonetheless, how virulence factors and pathogenic mechanisms are regulated have been elusive. In this study, we determined a role of polyphosphate kinase 1 (Ppk1) in regulation of quorum sensing (QS) and the sigma factor RpoS, and identified genes co-regulated by Ppk1, QS and RpoS. We find that Ppk1 positively controls autoinducer production and expression of rpoS transcript. Proteomic analysis identified 70 protein spots that are differentially expressed between B. pseudomallei wildtype and its ppk1-deficient strain. Within Ppk1regulated proteins, expression of 31 proteins are co-regulated by both RpoS and QS, whose functions of the majority of these proteins are associated with energy production and stress response. Moreover, expression of proteins involved in type III secretion system (T3SS) is also controlled by Ppk1. Quantitative PCR analysis confirmed that the T3SS genes bipB, bsaR and hrpK are down-regulated in ppk1 mutant. In addition, the ppk1-deficient strain exhibits defects in adhesion and invasion into human lung epithelial cells. Our work therefore reveals regulation of virulence factors and a regulatory mechanism of RpoS and QS by Ppk1, which altogether participate in gene expression control, and might be crucial for pathogenicity of B. pseudomallei. SignificancePolyphosphate kinase1 (Ppk1), which is a key enzyme in polyphosphate biosynthesis, is pivotal for virulence of the melioidosis pathogen B. pseudomallei. This enzyme is not present in human. Therefore, it has been proposed to be a key target for anti-bacterial drugs. An important step toward development of novel antibiotics and therapeutic strategies is an analysis of proteins that are controlled by Ppk1. By using proteomics, we find that Ppk1 co-regulates virulence-associated genes together with quorum sensing (QS) and the sigma factor RpoS. Moreover, we reveal that Ppk1 is critical for bacterial adhesion and host cell invasion, supporting the finding from our proteome analysis.

Molecular genetics of salmonela survival and resistance

Molecular genetics of salmonela survival and resistance