Characterization of <Emphasis Type="Italic">Edwardsiella tarda rpoS</Emphasis>: effect on serum resistance,chondroitinase activity,biofilm formation,and autoinducer synthetases expression

In this study, rpoS gene was identified from Edwardsiella tarda EIB202 and its functional role was analyzed by using an in-frame deletion mutant ∆rpoS and the complemental strain rpoS ⁺. Compared with the wild type and rpoS ⁺, ∆rpoS was impaired in terms of the ability to survive under oxidative stress and nutrient starvation, as well as the resistance to 50% serum of Scophthalmus maximus in 3 h, demonstrating essential roles of RpoS in stress adaptation. The rpoS mutant also displayed markedly increased chondroitinase activity and biofilm formation. Real-time polymerase chain reaction revealed that the expression level of quorum sensing autoinducer synthetase genes luxS and edwI was increased by 3.7- and 2.5-fold in the rpoS mutant strain. Those results suggested that rpoS might be involved in the negative or positive regulation of chondroitinase and biofilm formation, or quorum sensing networks in E. tarda, respectively. Although there were no obvious differences between the wild-type and the rpoS mutant in adherence of epithelioma papulosum cyprini (EPC) cell and in the lethality on fish model, rpoS deletion leads to the drastically reduced capacity for E. tarda to internalize in EPC cells, indicating that RpoS was, while not the main, the factor required for the virulence network of E. tarda.     

Role of RpoS in stress survival,synthesis of extracellular autoinducer 2, and virulence in <Emphasis Type="Italic">Vibrio alginolyticus</Emphasis>

Vibrio alginolyticus, a marine bacterium, is an opportunistic pathogen capable of causing vibriosis with high mortality to fishes in the South China Sea. Stress resistance is very important for its survival in the natural environment and upon infection of the host. RpoS, an alternative sigma factor, is considered as an important regulator involved in stress response and virulence in many pathogens. In this study, the rpoS gene was cloned and characterized to evaluate the role of RpoS in V. alginolyticus. The predicted protein showed high identity with other reported rpoS gene products. The in-frame deleted mutation of rpoS in V. alginolyticus led to sensitivity of the strain to ethanol, hyperosmolarity, heat, and hydrogen peroxide challenges. Further studies showed that extracellular autoinducer 2 level, four of seven detected protease activities, and cytotoxicity of extracellular products were markedly decreased in the rpoS mutant compared with that in the wild-type strain. The results indicated that the global regulator RpoS was part of the regulatory networks of virulence and LuxS quorum sensing system.     

Role of RpoS in stress resistance,biofilm formation and quorum sensing of Shewanella baltica

Shewanella baltica is one of the most important bacterial species contributing to spoilage of seafood. Principally, RpoS has been recognized as the central regulator of stress resistance in many bacterial species. However, little is known about the role of RpoS in S. baltica. In this study, an rpoS mutant of S. baltica was constructed and analysed for its functions. The results showed that the survival rate of rpoS mutant decreased when treated with heat, ethanol and H₂O_2, while increased the resistance to NaCl. Moreover RpoS promoted the biofilm formation of S. baltica at 30°C, while declined at 4°C. Interestingly, the rpoS-deficient mutant showed increased swimming motility. Furthermore, the results revealed that the production of quorum-sensing (QS) signals such as cyclo-(l -Pro-l -Leu) and cyclo-(l -Pro-l -Phe) reduced in rpoS mutant. Mainly, rpoS positively regulated QS response regulators, as the expression of all luxR genes in rpoS mutant significantly decreased relative to wild type. This study reveals that RpoS is a major regulator involved in stress responses, biofilm formation and quorum sensing system in S. baltica. The present work provides significant information for the control of microbiological spoilage of seafood.     

Low-Shear Modeled Microgravity Alters the Salmonella enterica Serovar Typhimurium Stress Response in an RpoS-Independent Manner

We have previously demonstrated that low-shear modeled microgravity (low-shear MMG) serves to enhance the virulence of a bacterial pathogen, Salmonella enterica serovar Typhimurium. The Salmonella response to low-shear MMG involves a signaling pathway that we have termed the low-shear MMG stimulon, though the identities of the low-shear MMG stimulon genes and regulatory factors are not known. RpoS is the primary sigma factor required for the expression of genes that are induced upon exposure to different environmental-stress signals and is essential for virulence in mice. Since low-shear MMG induces a Salmonella acid stress response and enhances Salmonella virulence, we reasoned that RpoS would be a likely regulator of the Salmonella low-shear MMG response. Our results demonstrate that low-shear MMG provides cross-resistance to several environmental stresses in both wild-type and isogenic rpoS mutant strains. Growth under low-shear MMG decreased the generation time of both strains in minimal medium and increased the ability of both strains to survive in J774 macrophages. Using DNA microarray analysis, we found no evidence of induction of the RpoS regulon by low-shear MMG but did find that other genes were altered in expression under these conditions in both the wild-type and rpoS mutant strains. Our results indicate that, under the conditions of these studies, RpoS is not required for transmission of the signal that induces the low-shear MMG stimulon. Moreover, our studies also indicate that low-shear MMG can be added to a short list of growth conditions that can serve to preadapt an rpoS mutant for resistance to multiple environmental stresses.     

Roles of DnaK and RpoS in Starvation-Induced Thermotolerance of Escherichia coli

DnaK is essential for starvation-induced resistance to heat, oxidation, and reductive division in Escherichia coli. Studies reported here indicate that DnaK is also required for starvation-induced osmotolerance, catalase activity, and the production of the RpoS-controlled Dps (PexB) protein. Because these dnaK mutant phenotypes closely resemble those of rpoS (ς³⁸) mutants, the relationship between DnaK and RpoS was evaluated directly during growth and starvation at 30°C in strains with genetically altered DnaK content. A starvation-specific effect of DnaK on RpoS abundance was observed. During carbon starvation, DnaK deficiency reduced RpoS levels threefold, while DnaK excess increased RpoS levels nearly twofold. Complementation of the dnaK mutation restored starvation-induced RpoS levels to normal. RpoS deficiency had no effect on the cellular concentration of DnaK, revealing an epistatic relationship between DnaK and RpoS. Protein half-life studies conducted at the onset of starvation indicate that DnaK deficiency significantly destabilized RpoS. RpoH (ς³²) suppressors of the dnaK mutant with restored levels of RpoS and dnaK rpoS double mutants were used to show that DnaK plays both an independent and an RpoS-dependent role in starvation-induced thermotolerance. The results suggest that DnaK coordinates sigma factor levels in glucose-starved E. coli.     

The variant rpoS allele of S. enteritidis strain 27655R does not affect virulence in a chick model nor constitutive curliation but does generate a cold-sensitive phenotype

Adherence of pathogenic Escherichia coli and Salmonella spp. to host cells is in part mediated by curli fimbriae which, along with other virulence determinants, are positively regulated by RpoS. Interested in the role and regulation of curli (SEF17) fimbriae of Salmonella enteritidis in poultry infection, we tested the virulence of naturally occurring S. enteritidis PT4 strains 27655R and 27655S which displayed constitutive and null expression of curli (SEF17) fimbriae, respectively, in a chick invasion assay and analysed their rpoS alleles. Both strains were shown to be equally invasive and as invasive as a wild-type phage type 4 strain and an isogenic derivative defective for the elaboration of curli. We showed that the rpoS allele of 27655S was intact even though this strain was non-curliated and we confirmed that a S. enteritidis rpoS::str^r null mutant was unable to express curli, as anticipated. Strain 27655R, constitutively curliated, possessed a frameshift mutation at position 697 of the rpoS coding sequence which resulted in a truncated product and remained curliated even when transduced to rpoS::str^r. Additionally, rpoS mutants are known to be cold-sensitive, a phenotype confirmed for strain 27655R. Collectively, these data indicated that curliation was not a significant factor for pathogenesis of S. enteritidis in this model and that curliation of strains 27655R and 27655S was independent of RpoS. Significantly, strain 27655R possessed a defective rpoS allele and remained virulent. Here was evidence that supported the concept that different naturally occurring rpoS alleles may generate varying virulence phenotypic traits.     

Characterization of the RpoS Status of Clinical Isolates of Salmonella enterica

The stationary-phase-inducible sigma factor, σ^S (RpoS), is the master regulator of the general stress response in Salmonella and is required for virulence in mice. rpoS mutants can frequently be isolated from highly passaged laboratory strains of Salmonella. We examined the rpoS status of 116 human clinical isolates of Salmonella, including 41 Salmonella enterica serotype Typhi strains isolated from blood, 38 S. enterica serotype Typhimurium strains isolated from blood, and 37 Salmonella serotype Typhimurium strains isolated from feces. We examined the abilities of these strains to produce the σ^S protein, to express RpoS-dependent catalase activity, and to resist to oxidative stress in the stationary phase of growth. We also carried out complementation experiments with a cloned wild-type rpoS gene. Our results showed that 15 of the 41 Salmonella serotype Typhi isolates were defective in RpoS. We sequenced the rpoS allele of 12 strains. This led to identification of small insertions, deletions, and point mutations resulting in premature stop codons or affecting regions 1 and 2 of σ^S, showing that the rpoS mutations are not clonal. Thus, mutant rpoS alleles can be found in freshly isolated clinical strains of Salmonella serotype Typhi, and they may affect virulence properties. Interestingly however, no rpoS mutants were found among the 75 Salmonella serotype Typhimurium isolates. Strains that differed in catalase activity and resistance to hydrogen peroxide were found, but the differences were not linked to the rpoS status. This suggests that Salmonella serotype Typhimurium rpoS mutants are counterselected because rpoS plays a role in the pathogenesis of Salmonella serotype Typhimurium in humans or in the transmission cycle of the disease.     

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 σ⁷⁰-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.     

Polyamines Stimulate the Level of the σ38 Subunit (RpoS) of Escherichia coli RNA Polymerase,Resulting in the Induction of the Glutamate Decarboxylase-dependent Acid Response System via the gadE Regulon

To study the physiological roles of polyamines, we carried out a global microarray analysis on the effect of adding polyamines to an Escherichia coli mutant that lacks polyamines because of deletions in the genes in the polyamine biosynthetic pathway. Previously, we have reported that the earliest response to polyamine addition is the increased expression of the genes for the glutamate-dependent acid resistance system (GDAR). We also presented preliminary evidence for the involvement of rpoS and gadE regulators. In the current study, further confirmation of the regulatory roles of rpoS and gadE is shown by a comparison of genome-wide expression profiling data from a series of microarrays comparing the genes induced by polyamine addition to polyamine-free rpoS⁺/gadE⁺ cells with genes induced by polyamine addition to polyamine-free ΔrpoS/gadE⁺ and rpoS⁺/ΔgadE cells. The results indicate that most of the genes in the E. coli GDAR system that are induced by polyamines require rpoS and gadE. Our data also show that gadE is the main regulator of GDAR and other acid fitness island genes. Both polyamines and rpoS are necessary for the expression of gadE gene from the three promoters of gadE (P1, P2, and P3). The most important effect of polyamine addition is the very rapid increase in the level of RpoS sigma factor. Our current hypothesis is that polyamines increase the level of RpoS protein and that this increased RpoS level is responsible for the stimulation of gadE expression, which in turn induces the GDAR system in E. coli.     

Monitoring of Vibrio harveyi quorum sensing activity in real time during infection of brine shrimp larvae

Quorum sensing, bacterial cell-to-cell communication, has been linked to the virulence of pathogenic bacteria. Indeed, in vitro experiments have shown that many bacterial pathogens regulate the expression of virulence genes by this cell-to-cell communication process. Moreover, signal molecules have been detected in samples retrieved from infected hosts and quorum sensing disruption has been reported to result in reduced virulence in different host–pathogen systems. However, data on in vivo quorum sensing activity of pathogens during infection of a host are currently lacking. We previously reported that quorum sensing regulates the virulence of Vibrio harveyi in a standardised model system with gnotobiotic brine shrimp (Artemia franciscana) larvae. Here, we monitored quorum sensing activity in Vibrio harveyi during infection of the shrimp, using bioluminescence as a read-out. We found that wild-type Vibrio harveyi shows a strong increase in quorum sensing activity early during infection. In this respect, the bacteria behave remarkably similar in different larvae, despite the fact that only half of them survive the infection. Interestingly, when expressed per bacterial cell, Vibrio harveyi showed around 200-fold higher maximal quorum sensing-regulated bioluminescence when associated with larvae than in the culture water. Finally, the in vivo quorum sensing activity of mutants defective in the production of one of the three signal molecules is consistent with their virulence, with no detectable in vivo quorum sensing activity in AI-2- and CAI-1-deficient mutants. These results indicate that AI-2 and CAI-1 are the dominant signals during infection of brine shrimp.     

Bacterial Hypoxic Responses Revealed as Critical Determinants of the Host-Pathogen Outcome by TnSeq Analysis of Staphylococcus aureus Invasive Infection

Staphylococcus aureus is capable of infecting nearly every organ in the human body. In order to infiltrate and thrive in such diverse host tissues, staphylococci must possess remarkable flexibility in both metabolic and virulence programs. To investigate the genetic requirements for bacterial survival during invasive infection, we performed a transposon sequencing (TnSeq) analysis of S. aureus during experimental osteomyelitis. TnSeq identified 65 genes essential for staphylococcal survival in infected bone and an additional 148 mutants with compromised fitness in vivo. Among the loci essential for in vivo survival was SrrAB, a staphylococcal two-component system previously reported to coordinate hypoxic and nitrosative stress responses in vitro. Healthy bone is intrinsically hypoxic, and intravital oxygen monitoring revealed further decreases in skeletal oxygen concentrations upon S. aureus infection. The fitness of an srrAB mutant during osteomyelitis was significantly increased by depletion of neutrophils, suggesting that neutrophils impose hypoxic and/or nitrosative stresses on invading bacteria. To more globally evaluate staphylococcal responses to changing oxygenation, we examined quorum sensing and virulence factor production in staphylococci grown under aerobic or hypoxic conditions. Hypoxic growth resulted in a profound increase in quorum sensing-dependent toxin production, and a concomitant increase in cytotoxicity toward mammalian cells. Moreover, aerobic growth limited quorum sensing and cytotoxicity in an SrrAB-dependent manner, suggesting a mechanism by which S. aureus modulates quorum sensing and toxin production in response to environmental oxygenation. Collectively, our results demonstrate that bacterial hypoxic responses are key determinants of the staphylococcal-host interaction.     

Engineering Synthetic Multistress Tolerance in Escherichia coli by Using a Deinococcal Response Regulator,DR1558

Cellular robustness is an important trait for industrial microbes, because the microbial strains are exposed to a multitude of different stresses during industrial processes, such as fermentation. Thus, engineering robustness in an organism in order to push the strains toward maximizing yield has become a significant topic of research. We introduced the deinococcal response regulator DR1558 into Escherichia coli (strain Ec-1558), thereby conferring tolerance to hydrogen peroxide (H₂O₂). The reactive oxygen species (ROS) level in strain Ec-1558 was reduced due to the increased KatE catalase activity. Among four regulators of the oxidative-stress response, OxyR, RpoS, SoxS, and Fur, we found that the expression of rpoS increased in Ec-1558, and we confirmed this increase by Western blot analysis. Electrophoretic mobility shift assays showed that DR1558 bound to the rpoS promoter. Because the alternative sigma factor RpoS regulates various stress resistance-related genes, we performed stress survival analysis using an rpoS mutant strain. Ec-1558 was able to tolerate a low pH, a high temperature, and high NaCl concentrations in addition to H₂O₂, and the multistress tolerance phenotype disappeared in the absence of rpoS. Microarray analysis clearly showed that a variety of stress-responsive genes that are directly or indirectly controlled by RpoS were upregulated in strain Ec-1558. These findings, taken together, indicate that the multistress tolerance conferred by DR1558 is likely routed through RpoS. In the present study, we propose a novel strategy of employing an exogenous response regulator from polyextremophiles for strain improvement.     

ClpXP affects the cell metabolism of <Emphasis Type="Italic">Salmonella typhimurium</Emphasis> partially in an RpoS-dependent manner

Introduction

ClpXP protease is an important proteolytic system in Salmonella enterica serovar typhimurium (S. typhimurium). Inactivation of ClpXP by deletion of clpP resulted in overproduction of RpoS and a growth defect phenotype. Only one report has indicated that deleting rpoS can restore the growth of a S. typhimurium clpP mutant to the wild-type level. Whether overproduction of RpoS is responsible for the growth deficiency resulting from clpP disruption and how ClpXP affects the cell metabolism of S. typhimurium remain to be elucidated.

Objectives

The aim of this study is to investigate the effect of ClpXP on cell metabolism of S. typhimurium and explore the possible co-effect of RpoS associated with ClpXP in cell metabolism.

Method

We constructed a clpP rpoS double deletion mutant TT-19 (ΔclpP ΔrpoS TT-1) using a two-step phage transduction technique. We then compared the metabolite fingerprints of Salmonella rpoS deletion mutant TT-14 (ΔrpoS TT-1), clpP deletion mutant TT-16 (ΔclpP TT-1), and clpP rpoS double deletion mutant TT-19 (ΔclpP ΔrpoS TT-1) with those of the wild-type strain TT-1 by using gas chromatography coupled with mass spectrometry (GC–MS).

Results

Deletion of rpoS recovered only a part of the growth of Salmonella clpP mutant. Further metabolome analysis indicated that clpP disruption changed the levels of 16 extra- and 19 intracellular substances, while the extracellular concentrations of 4 compounds (serine, l-5-oxoproline, l-glutamic acid, and l-tryptophan) and intracellular concentrations of 10 compounds (l-isoleucine, glycine, serine, l-methionine, l-phenylalanine, malic acid, citric acid, urea, putrescine, and 6-hydroxypurine) returned to their wild-type levels when rpoS was also deleted.

Conclusion

ClpXP affects the cell metabolism of S. typhimurium partially in an RpoS-dependent manner.

     

RpoS-Dependent Stress Response and Exoenzyme Production in Vibrio vulnificus

Vibrio vulnificus is an estuarine bacterium capable of causing rapidly fatal infections through both ingestion and wound infection. Like other opportunistic pathogens, V. vulnificus must adapt to potentially stressful environmental changes while living freely in seawater, upon colonization of the oyster gut, and upon infection of such diverse hosts as humans and eels. In order to begin to understand the ability of V. vulnificus to respond to such stresses, we examined the role of the alternate sigma factor RpoS, which is important in stress response and virulence in many pathogens. An rpoS mutant of V. vulnificus strain C7184o was constructed by homologous recombination. The mutant strain exhibited a decreased ability to survive diverse environmental stresses, including exposure to hydrogen peroxide, hyperosmolarity, and acidic conditions. The most striking difference was a high sensitivity of the mutant to hydrogen peroxide. Albuminase, caseinase, and elastase activity were detected in the wild type but not in the mutant strain, and an additional two hydrolytic activities (collagenase and gelatinase) were reduced in the mutant strain compared to the wild type. Additionally, the motility of the rpoS mutant was severely diminished. Overall, these studies suggest that rpoS in V. vulnificus is important for adaptation to environmental changes and may have a role in virulence.     

RpoS Regulates Essential Virulence Factors Remaining to Be Identified in Borrelia burgdorferi

Background

Since the RpoN-RpoS regulatory network was revealed in the Lyme disease spirochete Borrelia burgdorferi a decade ago, both upstream and downstream of the pathway have been intensively investigated. While significant progress has been made into understanding of how the network is regulated, most notably, discovering a relationship of the network with Rrp2 and BosR, only three crucial virulence factors, including outer surface protein C (OspC) and decorin-binding proteins (Dbps) A and B, are associated with the pathway. Moreover, for more than 10 years no single RpoS-controlled gene has been found to be critical for infection, raising a question about whether additional RpoS-dependent virulence factors remain to be identified.

Methodology/Principal Findings

The rpoS gene was deleted in B. burgdorferi; resulting mutants were modified to constitutively express all the known virulence factors, OspC, DbpA and DbpB. This genetic modification was unable to restore the rpoS mutant with infectivity.

Conclusions/Significance

The inability to restore the rpoS mutant with infectivity by simultaneously over-expressing all the three virulence factors allows us to conclude RpoS also regulates essential genes that remain to be identified in B. burgdorferi.     

Quorum Sensing Controls Swarming Motility of Burkholderia glumae through Regulation of Rhamnolipids

Burkholderia glumae is a plant pathogenic bacterium that uses an acyl-homoserine lactone-mediated quorum sensing system to regulate protein secretion, oxalate production and major virulence determinants such as toxoflavin and flagella. B. glumae also releases surface-active rhamnolipids. In Pseudomonas aeruginosa and Burkholderia thailandensis, rhamnolipids, along with flagella, are required for the social behavior called swarming motility. In the present study, we demonstrate that quorum sensing positively regulates the production of rhamnolipids in B. glumae and that rhamnolipids are necessary for swarming motility also in this species. We show that a rhlA- mutant, which is unable to produce rhamnolipids, loses its ability to swarm, and that this can be complemented by providing exogenous rhamnolipids. Impaired rhamnolipid production in a quorum sensing-deficient B. glumae mutant is the main factor responsible for its defective swarming motility behaviour.