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The diarrheal disease cholera is caused by the versatile and responsive bacterium Vibrio cholerae, which is capable of adapting to environmental changes. Among others, the alternative sigma factor RpoS activates response pathways, including regulation of motility‐ and chemotaxis‐related genes under nutrient‐poor conditions in V. cholerae. Although RpoS has been well characterised, links between RpoS and other regulatory networks remain unclear. In this study, we identified the ArcAB two‐component system to control rpoS transcription and RpoS protein stability in V. cholerae. In a manner similar to that seen in Escherichia coli, the ArcB kinase not only activates the response regulator ArcA but also RssB, the anti‐sigma factor of RpoS. Our results demonstrated that, in V. cholerae, RssB is phosphorylated by ArcB, which subsequently activates RpoS proteolysis. Furthermore, ArcA acts as a repressor of rpoS transcription. Additionally, we determined that the cysteine residue at position 180 of ArcB is crucial for signal recognition and activity. Thus, our findings provide evidence linking RpoS response to the anoxic redox control system ArcAB in V. cholerae. [ABSTRACT FROM AUTHOR]

Abstract Background The intrinsic concentration of RpoS, the second most abundant sigma factor, varies widely across the E. coli species. Bacterial isolates that express high levels of RpoS display high resistance to environmental stresses, such as temperature, pH and osmolarity shifts, but are less nutritional competent, making them less capable of utilising alternative nutrient sources. The role of RpoS in antibiotic resistance and persistence in standard laboratory domesticated strains has been examined in several studies, most demonstrating a positive role for RpoS. Results Using disk diffusion assays we examined bacterial resistance to 15 different antibiotics, including $$\upbeta$$ β -lactams (penicillins, monobactams, carbapenems and cephalosporins), aminoglycosides, quinolones and anti-folates, in a representative collection of 328 E. coli natural isolates displaying a continuum of different levels of RpoS. There was an overall trend that isolates with higher levels of RpoS were slightly more resistant to these antibiotics. In addition, the effect of RpoS on bacterial tolerance and persistence to 3 different antibiotics - ampicillin, ciprofloxacin and kanamycin was evaluated through time-kill curves. Again, there was a small beneficial effect of RpoS on tolerance and persistence to these antibiotics, but this difference was not statistically significant. Finally, a K-12 strain expressing high levels of RpoS was compared with its isogenic RpoS-null counterpart, and no significant effect of RpoS was found. Conclusion Based on a representative collection of the species E. coli, RpoS was found to have a very small impact on antibiotic resistance, tolerance, or persistence.

The general stress response (GSR) sigma factor RpoS from Escherichia coli has emerged as one of the key paradigms for study of how numerous signal inputs are accepted at multiple levels into a single pathway for regulation of gene expression output. While many studies have elucidated the key pathways controlling the production and activity of this sigma factor, recent discoveries have uncovered still more regulatory mechanisms which feed into the network. Moreover, while the regulon of this sigma factor comprises a large proportion of the E. coli genome, the downstream expression levels of all the RpoS target genes are not identically affected by RpoS upregulation but respond heterogeneously, both within and between cells. This minireview highlights the most recent developments in our understanding of RpoS regulation and expression, in particular those which influence the regulatory network at different levels from previously well-studied pathways. [ABSTRACT FROM AUTHOR]

Background: The intrinsic concentration of RpoS, the second most abundant sigma factor, varies widely across the E. coli species. Bacterial isolates that express high levels of RpoS display high resistance to environmental stresses, such as temperature, pH and osmolarity shifts, but are less nutritional competent, making them less capable of utilising alternative nutrient sources. The role of RpoS in antibiotic resistance and persistence in standard laboratory domesticated strains has been examined in several studies, most demonstrating a positive role for RpoS. Results: Using disk diffusion assays we examined bacterial resistance to 15 different antibiotics, including β -lactams (penicillins, monobactams, carbapenems and cephalosporins), aminoglycosides, quinolones and anti-folates, in a representative collection of 328 E. coli natural isolates displaying a continuum of different levels of RpoS. There was an overall trend that isolates with higher levels of RpoS were slightly more resistant to these antibiotics. In addition, the effect of RpoS on bacterial tolerance and persistence to 3 different antibiotics - ampicillin, ciprofloxacin and kanamycin was evaluated through time-kill curves. Again, there was a small beneficial effect of RpoS on tolerance and persistence to these antibiotics, but this difference was not statistically significant. Finally, a K-12 strain expressing high levels of RpoS was compared with its isogenic RpoS-null counterpart, and no significant effect of RpoS was found. Conclusion: Based on a representative collection of the species E. coli, RpoS was found to have a very small impact on antibiotic resistance, tolerance, or persistence. [ABSTRACT FROM AUTHOR]

Melioidosis, a human infectious disease with a high mortality rate in many tropical countries, is caused by the pathogen Burkholderia pseudomallei (B. pseudomallei). The function of the B. pseudomallei sigma S (RpoS) transcription factor in survival during the stationary growth phase and conditions of oxidative stress is well documented. Besides the rpoS, bioinformatics analysis of B. pseudomallei genome showed the existence of two rpoN genes, named rpoN1 and rpoN2. In this study, by using the mouse macrophage cell line RAW264.7 as a model of infection, the involvement of B. pseudomallei RpoS and RpoN2 in the invasion, intracellular survival leading to the reduction in multinucleated giant cell (MNGC) formation of RAW264.7 cell line were illustrated. We have demonstrated that the MNGC formation of RAW264.7 cell was dependent on a certain number of intracellular bacteria (at least 5 × 104). In addition, the same MNGC formation (15%) observed in RAW264.7 cells infected with either B. pseudomallei wild type with multiplicity of infection (MOI) 2 or RpoN2 mutant (∆rpoN2) with MOI 10 or RpoS mutant (∆rpoS) with MOI 100. The role of B. pseudomallei RpoS and RpoN2 in the regulation of type III secretion system on bipB-bipC gene expression was also illustrated in this study. [ABSTRACT FROM AUTHOR]

Pseudomonas aeruginosa is an important opportunistic pathogen. Several of its virulence‐related processes, including the synthesis of pyocyanin (PYO) and biofilm formation, are controlled by quorum sensing (QS). It has been shown that the alternative sigma factor RpoS regulates QS through the reduction of lasR and rhlR transcription (encoding QS regulators). However, paradoxically, the absence of RpoS increases PYO production and biofilm development (that are RhlR dependent) by unknown mechanisms. Here, we show that RpoS represses pqsE transcription, which impacts the stability and activity of RhlR. In the absence of RpoS, rhlR transcript levels are reduced but not the RhlR protein concentration, presumably by its stabilization by PqsE, whose expression is increased. We also report that PYO synthesis and the expression of pqsE and phzA1B1C1D1E1F1G1 operon exhibit the same pattern at different RpoS concentrations, suggesting that the RpoS‐dependent PYO production is due to its ability to modify PqsE concentration, which in turn modulates the activation of the phzA1 promoter by RhlR. Finally, we demonstrate that RpoS favors the expression of Vfr, which activates the transcription of lasR and rhlR. Our study contributes to the understanding of how RpoS modulates the QS response in P. aeruginosa, exerting both negative and positive regulation. [ABSTRACT FROM AUTHOR]

Introduction: An extended-spectrum beta-lactamase (ESBL)-hypervirulent Klebsiella pneumoniae (HvKP) strain HKE9 was isolated from the blood in an outpatient. Methods: The effect of the global regulatory factor RpoS on antimicrobial resistance, pathogenicity, and environmental adaptability was elucidated. Results: HKE9 is a novel ST3355 (K20/O2a) hypervirulent strain with a positive string test and resistant to cephems except cefotetan. It has a genome size of 5.6M, including two plasmids. CTX-M-15 was found in plasmid 2, and only ompk37 was found in the chromosome. HKE9 could produce bacterial siderophores, and genes of enterobactin, yersiniabactin, aerobactin, and salmochelin have been retrieved in the genome. As a global regulatory factor, knockout of rpoS did not change antimicrobial resistance or hemolytic phenotype while increasing the virulence to Galleria mellonella larvae and showing higher viscosity. Moreover, rpoS knockout can increase bacterial competitiveness and cell adhesion ability. Interestingly, HKE9-M-rpoS decreased resistance to acidic pH, high osmotic pressure, heat shock, and ultraviolet and became sensitive to disinfectants (H2O2, alcohol, and sodium hypochlorite). Although there were 13 Type 6 secretion system (T6SS) core genes divided into two segments with tle1 between segments in the chromosome, transcriptomic analysis showed that rpoS negatively regulated T4SS located on plasmid 2, type 1, and type 3 fimbriae and positively regulate genes responsible for acidic response, hyperosmotic pressure, heat shock, oxidative stress, alcohol and hypochlorous acid metabolism, and quorum sensing. Discussion: Here, this novel ST3355 ESBL-HvKP strain HKE9 may spread via various clonal types. The important regulation effect of rpoS is the enhanced tolerance and resistance to environmental stress and disinfectants, which may be at the cost of reducing virulence and regulated by T4SS. [ABSTRACT FROM AUTHOR]

This review addresses the involvement of DNA supercoiling in the development of virulence and antibiotic profiles for uropathogenic Escherichia coli and the emergence of new pathotypes such as strain ST131 (serotype O25:H4). The mechanism suggests a role for topoisomerase enzymes and associated mutations in altering the chromosomal supercoiling state and introducing the required DNA twists for expression of intrinsic β-lactamase by ampC and certain virulence factors. In Escherichia coli, constitutive hyperexpression of intrinsic ampC is associated with specific mutations in the promoter and attenuator regions. However, many reports have documented the involvement of slow growth interventions in the expression of intrinsic resistance determinants. There is evidence that a stationary phase transcriptional switch protein, “BolA,” is involved in the expression of the intrinsic ampC gene under starvation conditions. The process involves changes in the activity of the enzyme “gyrase,” which leads to a change in the chromosomal DNA topology. Consequently, the DNA is relaxed, and the expression of the bolA gene is upregulated. The evolution of the extraintestinal pathogenic E. coli strain ST131 has demonstrated successful adaptability to various stress conditions and conferred compensatory mutations that endowed the microbe with resistance to fluoroquinolones and β-lactams. The results of this study provided new insights into the evidence for the influence of DNA topology in the expression of virulence genes and various determinants of antibiotic resistance (e.g., the intrinsic ampC gene) in Escherichia coli pathotypes. [ABSTRACT FROM AUTHOR]

The general stress response (GSR) sigma factor RpoS from Escherichia coli has emerged as one of the key paradigms for study of how numerous signal inputs are accepted at multiple levels into a single pathway for regulation of gene expression output. While many studies have elucidated the key pathways controlling the production and activity of this sigma factor, recent discoveries have uncovered still more regulatory mechanisms which feed into the network. Moreover, while the regulon of this sigma factor comprises a large proportion of the E. coli genome, the downstream expression levels of all the RpoS target genes are not identically affected by RpoS upregulation but respond heterogeneously, both within and between cells. This minireview highlights the most recent developments in our understanding of RpoS regulation and expression, in particular those which influence the regulatory network at different levels from previously well-studied pathways.

Bacterial behavior has been studied under microgravity conditions, but very little is known about it under lunar and Martian gravitational regimes. An Earth-based approach was designed and implemented using inclined clinostats and an in-house-developed code to determine the optimal clinorotation angular speed for bacterial liquid cultures of 5 RPM. With this setup, growth dynamics, phenotypic changes, and sensitivity to antibiotics (minimum inhibitory concentration (MIC) of two different classes of antibiotics) for three Escherichia coli strains (including uropathogenic) were examined under simulated micro-, lunar, and Martian gravities. The results included increased growth under simulated micro- and lunar gravities for some strains, and higher concentrations of antibiotics needed under simulated lunar gravity with respect to simulated micro- and Martian gravities. Clinostat-produced results can be considered suggestive but not determinative of what might be expected in altered gravity, as there is still a need to systematically verify these simulation devices' ability to accurately replicate phenomena observed in space. Nevertheless, this approach serves as a baseline to start interrogating key cellular and molecular aspects relevant to microbial processes on the lunar and Martian surfaces.

ABSTRACT The alternative sigma factor RpoS regulates transcription of over 1,000 genes in Escherichia coli in response to many different stresses. RpoS levels rise continuously after exposure to stress, and the consequences of changing levels of RpoS for the temporal patterns of expression of RpoS-regulated genes have not been described. We measured RpoS levels at various times during the entry to stationary phase, or in response to high osmolarity or low temperature, and found that the time required to reach maximum levels varied by several hours. We quantified the transcriptome across these stresses using RNA-seq. The number of differentially expressed genes differed among stresses, with 1,379 DE genes identified in stationary phase, 633 in high osmolarity, and 302 in cold shock. To quantify the timing of gene expression, we fit sigmoid or double sigmoid models to differentially expressed genes in each stress. During the entry into stationary phase, genes whose expression rose earlier tended to be those that had been found to respond most strongly to low levels of RpoS. The timing of individual gene’s expression was not correlated across stresses. Taken together, our results demonstrate E. coli activates RpoS with different timing in response to different stresses, which in turn generates a unique pattern of timing of the transcription response to each stress. IMPORTANCE Bacteria adapt to changing environments by altering the transcription of their genes. Specific proteins can regulate these changes. This study explored how a single protein called RpoS controls how many genes change expression during adaptation to three stresses. We found that: (i) RpoS is responsible for activating different genes in different stresses; (ii) that during a stress, the timing of gene activation depends on the what stress it is; and (iii) that how much RpoS a gene needs in order to be activated can predict when that gene will be activated during the stress of stationary phase.

In several Gram‐negative bacteria, the general stress response is mediated by the alternative sigma factor RpoS, a subunit of RNA polymerase that confers promoter specificity. In Escherichia coli, regulation of protein levels of RpoS involves the adaptor protein RssB, which binds RpoS for presenting it to the ClpXP protease for its degradation. However, in species from the Pseudomonadaceae family, RpoS is also degraded by ClpXP, but an adaptor has not been experimentally demonstrated. Here, we investigated the role of an E. coli RssB‐like protein in two representative Pseudomonadaceae species such as Azotobacter vinelandii and Pseudomonas aeruginosa. In these bacteria, inactivation of the rssB gene increased the levels and stability of RpoS during exponential growth. Downstream of rssB lies a gene that encodes a protein annotated as an anti‐sigma factor antagonist (rssC). However, inactivation of rssC in both A. vinelandii and P. aeruginosa also increased the RpoS protein levels, suggesting that RssB and RssC work together to control RpoS degradation. Furthermore, we identified an in vivo interaction between RssB and RpoS only in the presence of RssC using a bacterial three‐hybrid system. We propose that both RssB and RssC are necessary for the ClpXP‐dependent RpoS degradation during exponential growth in two species of the Pseudomonadaceae family. [ABSTRACT FROM AUTHOR]

Understanding the functional role of bacterial genes in the persistence of Salmonella in plant organs can facilitate the development of agricultural practices to mitigate food safety risks associated with the consumption of fresh produce contaminated with Salmonella spp. Our study showed that Salmonella enterica subsp. enterica serotype Typhimurium (strain MDD14) persisted less in inoculated tomato plants than other Salmonella Typhimurium strains tested (JSG210, JSG626, JSG634, JSG637, JSG3444, and EV030415; P < 0.01). In-vitro assays performed in limited-nutrient conditions (growth rate, biofilm production, and motility) were inconclusive in explaining the in-planta phenotype observed with MDD14. Whole-genome sequencing combined with non-synonymous single nucleotide variations analysis was performed to identify genomic differences between MDD14 and the other Salmonella Typhimurium strains. The genome of MDD14 contained a truncated version (123 bp N-terminal) of yicC and a mutated version of rpoS (two non-synonymous substitutions, i.e., G66E and R82C), which are two stress-induced proteins involved in iron acquisition, environmental sensing, and cell envelope integrity. The rpoS and yicC genes were deleted in Salmonella Typhimurium JSG210 with the Lambda Red recombining system. Both mutants had limited persistence in tomato plant organs, similar to that of MDD14. In conclusion, we demonstrated that YicC and RpoS are involved in the persistence of Salmonella in tomato plants in greenhouse conditions and, thus, could represent potential targets to mitigate persistence of Salmonella spp. in planta. [Graphic: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Both alternative sigma factor RpoS and ubiquitous secondary messenger c-di-GMP participate in the biofilm forming of Salmonella Pullorum; however, the relationship between RpoS and c-di-GMP-regulated genes during biofilm forming remains unclear. In this study, nine genes related with c-di-GMP regulation were found to be differentially expressed (P<0.01) by RNA-seq analysis when compared with S. Pullorum strain S9 and its rpoS deletion strain S9S. Specifically, the rpoS deletion strain S9S had higher transcription level of gene STM1703 and lower c-di-GMP concentration and biofilm-forming ability than S9 (P<0.01). The STM1703 gene deletion in strains S9 and S9S significantly enhanced the c-di-GMP concentration and the biofilm-forming ability (P<0.01). qRT–PCR analysis showed that rpoS deletion or P193L substitution in RpoS increased the transcription level of the STM1703 gene by decreasing the transcription levels of the csrA and STM1344 genes (P<0.01). Overall, RpoS-mediated STM1703 negatively regulates the biofilm formation of S. Pullorum by degrading c-di-GMP. [ABSTRACT FROM AUTHOR]

Pyocyanin is a phenazine with redox activity produced by Pseudomonas aeruginosa that is harmful to other bacteria and eukaryotic organisms by generating reactive oxygen species. Gene regulation of pyocyanin synthesis has been addressed in the PAO1 and PA14 strains and involves the three‐quorum sensing systems Las, Rhl, and Pqs; the regulators RsaL, MvaU, and RpoS, and the posttranscriptional Rsm system, among others. Here, we determined how RsmA regulates pyocyanin synthesis in P. aeruginosa ID4365, an overproducer strain. We found that, in the protease peptone glucose ammonium salts medium, rsmA inactivation increases pyocyanin production compared with the wild‐type strains ID4365, PAO, and PA14. We showed that RsmA regulates inversely the expression of both phz operons involved in pyocyanin synthesis; particularly the phz2 operon is positively regulated at the transcriptional level indirectly through MvaU. In addition, we found that the phz1 operon contributes mainly to pyocyanin synthesis and that RsmA negatively regulates phzM and phzS expression. Finally, we showed that translation of the sigma factor RpoS is positively regulated by RsmA, and the expression of rpoS under an independent promoter decreases pyocyanin production in the IDrsmA strain. These results indicate that RsmA regulates not only the genes for pyocyanin production but also their regulators. [ABSTRACT FROM AUTHOR]

The rhizosphere microbe Pseudomonas protegens FD6 possesses beneficial traits such as the production of antibiotics like pyoluteorin (Plt) and 2,4-diacetylphloroglucinol (2,4-DAPG). The alternative RpoS (σ38 factor), as a master regulator, activates or inhibits the transcription of stationary phase genes in several biocontrol organisms. Here, we investigated the complicated function and regulatory mechanism of RpoS in the biosynthesis of 2,4-DAPG and Plt in strain FD6. Phenotypic assays suggested that ΔrpoS was impaired in biofilm formation, swimming motility, swarming motility, and resistance to stress, such as heat, H2O2 and 12% ethanol. The RpoS mutation significantly increased both 2,4-DAPG and Plt production and altered the transcription and translation of the biosynthetic genes phlA and pltL, indicating that RpoS inhibited antibiotic production by FD6 at both the transcriptional and translational levels. RpoS negatively controlled 2,4-DAPG biosynthesis and transcription of the 2,4-DAPG operon phlACBD by directly interacting with the promoter sequences of phlG and phlA. In addition, RpoS significantly inhibited Plt production and the expression of its operon pltLABCDEFG by directly binding to the promoter regions of pltR, pltL and pltF. Further analyzes demonstrated that a putative R147 mutation in the RpoS binding domain abolished its inhibitory activity on the expression of pltL and phlA. Overall, our results reveal the pleiotropic regulatory function of RpoS in P. protegens FD6 and provide the basis for improving antibiotic biosynthesis by genetic engineering in biocontrol organisms. [ABSTRACT FROM AUTHOR]

Enterobacter cloacae is widely distributed in the aquatic environment, and has been determined as a novel pathogen of various aquatic animals recently. Our previous studies have indicated E. cloacae caused repeated infections in Macrobrachium rosenbergii, suggesting a high survival ability of the bacteria, and rpoS gene has been known to regulate stress response and virulence of many bacteria. In this study, the E. cloacae-rpoS RNAi strain was constructed by RNAi technology, and the regulation role of rpoS in stress resistance and virulence of E. cloacae was explored by transcriptomic and phenotype analysis. The transcriptome analysis showed a total of 488 differentially expressed genes (DEGs) were identified between rpoS-RNAi and wild-type strains, including 30 up-regulated genes and 458 down-regulated genes, and these down-regulated DEGs were mainly related to environmental response, biofilm formation, bacterial type II secretory system, flagellin, fimbrillin, and chemotactic protein which associated with bacterial survival and virulence. The phenotype changes also showed the E. cloacae-rpoS RNAi strain exhibited significantly decreasing abilities of survival in environmental stresses (starvation, salinity, low pH, and oxidative stress), biofilm production, movement, adhesion to cells, pathogenicity, and colonization to M. rosenbergii. These results reveal that rpoS plays an important regulatory role in environmental stress adaptation and virulence of E. cloacae. [ABSTRACT FROM AUTHOR]

Contaminated fresh produce has been routinely linked to outbreaks of Salmonellosis. Multiple studies have identified Salmonella enterica factors associated with successful colonization of diverse plant niches and tissues. It has also been well documented that S. enterica can benefit from the conditions generated during plant disease by host-compatible plant pathogens. In this study, we compared the capacity of two common S. enterica research strains, 14028s and LT2 (strain DM10000) to opportunistically colonize the leaf apoplast of two model plant hosts Arabidopsis thaliana and Nicotiana benthamiana during disease. While S. enterica 14028s benefited from co-colonization with plant-pathogenic Pseudomonas syringae in both plant hosts, S. enterica LT2 was unable to benefit from Pto co-colonization in N. benthamiana. Counterintuitively, LT2 grew more rapidly in ex planta N. benthamiana apoplastic wash fluid with a distinctly pronounced biphasic growth curve in comparison with 14028s. Using allelic exchange, we demonstrated that both the N. benthamiana infection-depedent colonization and apoplastic wash fluid growth phenotypes of LT2 were associated with mutations in the S. enterica rpoS stress-response sigma factor gene. Mutations of S. enterica rpoS have been previously shown to decrease tolerance to oxidative stress and alter metabolic regulation. We identified rpoS-dependent alterations in the utilization of L-malic acid, an abundant carbon source in N. benthamiana apoplastic wash fluid. We also present data consistent with higher relative basal reactive oxygen species (ROS) in N. benthamiana leaves than in A. thaliana leaves. The differences in basal ROS may explain the host-dependent disease co-colonization defect of the rpoS-mutated LT2 strain. Our results indicate that the conducive environment generated by pathogen modulation of the apoplast niche can vary from hosts to host even with a common disease-compatible pathogen. [ABSTRACT FROM AUTHOR]

Although RNA structures play important roles in regulating gene expression, the mechanism and function of mRNA folding in plant bacterial pathogens remain elusive. Therefore, we perform dimethyl sulfate sequencing (DMS-seq) on the Pseudomonas syringae under nutrition-rich and -deficient conditions, revealing that the mRNA structure changes substantially in the minimal medium (MM) that tunes global translation efficiency (TE), thereby inducing virulence. This process is led by the increased expression of hfq , which is directly activated by transcription regulators RpoS and CysB. The co-occurrence of Hfq and RpoS in diverse bacteria and the deep conservation of Hfq Y25 is critical for RNA-mediated regulation and implicates the wider biological importance of mRNA structure and feedback loops in the control of global gene expression. [Display omitted] • Under virulence conditions, RNA structure changes in Pseudomonas savastanoi • Hfq plays a vital role in regulating RNA structure following MM stimulation • The Y25 site of Hfq is crucial for modulating RNA structure • RpoS and CysB regulate hfq expression under virulence conditions Hua et al. demonstrate that RNA structure changes after virulence condition stimulation. The Y25 site of Hfq is crucial for regulating RNA structure. The expression of hfq is regulated by RpoS and CysB under MM conditions. [ABSTRACT FROM AUTHOR]

Food preservatives are crucial in controlling microbial growth in processed foods to maintain food safety. Bacterial biofilms pose a threat in the food chain by facilitating persistence on a range of surfaces and food products. Cells in a biofilm are often highly tolerant of antimicrobials and can evolve in response to antimicrobial exposure. Little is known about the efficacy of preservatives against biofilms and their potential impact on the evolution of antimicrobial resistance. In this study we investigated how Salmonella enterica serovar Typhimurium responded to subinhibitory concentrations of four food preservatives (sodium chloride, potassium chloride, sodium nitrite or sodium lactate) when grown planktonically and in biofilms. We found that each preservative exerted a unique selective pressure on S. Typhimurium populations. There was a trade-off between biofilm formation and growth in the presence of three of the four preservatives, where prolonged preservative exposure resulted in reduced biofilm biomass and matrix production over time. All three preservatives selected for mutations in global stress response regulators rpoS and crp. There was no evidence for any selection of cross-resistance to antibiotics after preservative exposure. In conclusion, we showed that preservatives affect biofilm formation and bacterial growth in a compound specific manner. We showed trade-offs between biofilm formation and preservative tolerance, but no antibiotic cross-tolerance. This indicates that bacterial adaptation to continuous preservative exposure, is unlikely to affect food safety or contribute to antibiotic resistance. • Preservative-specific evolutionary adaptation of Salmonella was shown over time. • A trade-off between adaptation and biofilm formation was observed. • No cross-resistance to antibiotics was seen after preservative exposure. • Mutations were found to be preservative-specific, with some common ones like rpoS and crp. [ABSTRACT FROM AUTHOR]

Experimental evolution of Escherichia coli K-12 W3110 by serial dilutions for 2,200 generations at high pH extended the range of sustained growth from pH 9.0 to pH 9.3. pH 9.3-adapted isolates showed mutations in DNA-binding regulators and envelope proteins. One population showed an IS1 knockout of phoB (encoding the positive regulator of the phosphate regulon). A phoB::kanR knockout increased growth at high pH. phoB mutants are known to increase production of fermentation acids, which could enhance fitness at high pH. Mutations in pcnB [poly(A) polymerase] also increased growth at high pH. Three out of four populations showed deletions of torI, an inhibitor of TorR, which activates expression of torCAD (trimethylamine N-oxide respiration) at high pH. All populations showed point mutations affecting the stationary-phase sigma factor RpoS, either in the coding gene or in genes for regulators of RpoS expression. RpoS is required for survival at extremely high pH. In our microplate assay, rpoS deletion slightly decreased growth at pH 9.1. RpoS protein accumulated faster at pH 9 than at pH 7. The RpoS accumulation at high pH required the presence of one or more antiadaptors that block degradation (IraM, IraD, and IraP). Other genes with mutations after high-pH evolution encode regulators, such as those encoded by yobG (mgrB) (PhoPQ regulator), rpoN (nitrogen starvation sigma factor), malI, and purR, as well as envelope proteins, such as those encoded by ompT and yahO Overall, E. coli evolution at high pH selects for mutations in key transcriptional regulators, including phoB and the stationary-phase sigma factor RpoS.IMPORTANCEEscherichia coli in its native habitat encounters high-pH stress such as that of pancreatic secretions. Experimental evolution over 2,000 generations showed selection for mutations in regulatory factors, such as deletion of the phosphate regulator PhoB and mutations that alter the function of the global stress regulator RpoS. RpoS is induced at high pH via multiple mechanisms.

Contaminated fresh produce has been routinely linked to outbreaks of Salmonellosis. Multiple studies have identified Salmonella enterica factors associated with successful colonization of diverse plant niches and tissues. It has also been well documented that S. enterica can benefit from the conditions generated during plant disease by host-compatible plant pathogens. In this study, we compared the capacity of two common S. enterica research strains, 14028s and LT2 (strain DM10000) to opportunistically colonize the leaf apoplast of two model plant hosts Arabidopsis thaliana and Nicotiana benthamiana during disease. While S. enterica 14028s benefited from co-colonization with plant-pathogenic Pseudomonas syringae in both plant hosts, S. enterica LT2 was unable to benefit from Pto co-colonization in N. benthamiana. Counterintuitively, LT2 grew more rapidly in ex planta N. benthamiana apoplastic wash fluid with a distinctly pronounced biphasic growth curve in comparison with 14028s. Using allelic exchange, we demonstrated that both the N. benthamiana infection-depedent colonization and apoplastic wash fluid growth phenotypes of LT2 were associated with mutations in the S. enterica rpoS stress-response sigma factor gene. Mutations of S. enterica rpoS have been previously shown to decrease tolerance to oxidative stress and alter metabolic regulation. We identified rpoS-dependent alterations in the utilization of L-malic acid, an abundant carbon source in N. benthamiana apoplastic wash fluid. We also present data consistent with higher relative basal reactive oxygen species (ROS) in N. benthamiana leaves than in A. thaliana leaves. The differences in basal ROS may explain the host-dependent disease co-colonization defect of the rpoS-mutated LT2 strain. Our results indicate that the conducive environment generated by pathogen modulation of the apoplast niche can vary from hosts to host even with a common disease-compatible pathogen.

Shewanella baltica is the dominant bacterium that causes spoilage of seafood. RpoS is an alternative sigma factor regulating stress adaptation in many bacteria. However, the detailed regulatory mechanism of RpoS in S. baltica remains unclear. This study aims to investigate the regulatory function of RpoS on spoilage activity and adhesion ability in S. baltica. Results revealed that RpoS had no effect on the growth of S. baltica, but positively regulated the spoilage potential of S. baltica accompanied by a slower decline of total volatile basic nitrogen, lightness, and the sensory score of fish fillets inoculated with rpoS mutant. RpoS negatively regulated the adhesion ability, which was manifested in that the bacterial number of rpoS mutant adhered to stainless steel coupon was higher than that of the S. baltica in the early stage, and the biofilm formed on glass slide by rpoS mutant was thicker and tighter compared with S. baltica. Transcriptomic analysis showed that a total of 397 differentially expressed genes were regulated by RpoS. These genes were mainly enrichment in flagellar assembly, fatty acid metabolism/degradation, and RNA degradation pathways, which were associated with motility, biofilm formation and cold adaptation. This study demonstrated that RpoS is a primary regulator involved in flagellar assembly mediated biofilm formation and cold adaptation-related spoilage activity of S. baltica. Our research will provide significant insights into the control of microbiological spoilage in seafood. [ABSTRACT FROM AUTHOR]

Background: Salmonella entericaserovar Choleraesuis (S.C) is a swine enteric pathogen causing paratyphoid fever, enterocolitis, and septicemia in piglets. S. C is mainly transmitted through the fecal-oral route. Vaccination is an effective strategy for preventing and controlling Salmonella infection., Results: Herein, we used CRISPR-Cas9 technology to knockout the virulence regulatory genes, rpoS, and slyA of S. C and constructed the ∆rpoS, ∆slyA, and ∆rpoS ∆slyA strains. The phenotypic characteristics of the mutant strains remained unchanged compared with the parental wild-type strain. In vivo study, unlike the wild-type strain, the ∆slyA and ∆rpoS ∆slyA strains alleviated splenomegaly, colon atrophy, and lower bacterial loads in the spleen, liver, ileum, and colon. These mutant strains survived in Peyer's patches (PPs) and mesenteric lymph nodes (MLN) for up to 15 days post-infection. Furthermore, the immunization of the ∆rpoS ∆slyA strain induced robust humoral and cellular immune responses., Conclusions: Consequently, vaccination with ∆rpoS ∆slyA conferred a high percentage of protection against lethal invasive Salmonella, specifically S. C, in mice. This study provided novel insights into the development of live-attenuated vaccines against the infection of S. C., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

ABSTRACT The discovery that biomechanical forces regulate microbial virulence was established with the finding that physiological low fluid shear (LFS) forces altered gene expression, stress responses, and virulence of the enteric pathogen Salmonella enterica serovar Typhimurium during the log phase. These log phase LFS-induced phenotypes were independent of the master stress response regulator, RpoS (σS). Given the central importance of RpoS in regulating stationary-phase stress responses of S. Typhimurium cultured under conventional shake flask and static conditions, we examined its role in stationary-phase cultures grown under physiological LFS. We constructed an isogenic rpoS mutant derivative of wild-type S. Typhimurium and compared the ability of these strains to survive in vitro pathogenesis-related stresses that mimic those encountered in the infected host and environment. We also compared the ability of these strains to colonize (adhere, invade, and survive within) human intestinal epithelial cell cultures. Unexpectedly, LFS-induced resistance of stationary-phase S. Typhimurium cultures to acid and bile salts stresses did not rely on RpoS. Likewise, RpoS was dispensable for stationary-phase LFS cultures to adhere to and survive within intestinal epithelial cells. In contrast, the resistance of these cultures to challenges of oxidative and thermal stresses, and their invasion into intestinal epithelial cells was influenced by RpoS. These findings expand our mechanistic understanding of how physiological fluid shear forces modulate stationary-phase S. Typhimurium physiology in unexpected ways and provide clues into microbial mechanobiology and nuances of Salmonella responses to microenvironmental niches in the infected host. IMPORTANCE Bacterial pathogens respond dynamically to a variety of stresses in the infected host, including physical forces of fluid flow (fluid shear) across their surfaces. While pathogens experience wide fluctuations in fluid shear during infection, little is known about how these forces regulate microbial pathogenesis. This is especially important for stationary-phase bacterial growth, which is a critical period to understand microbial resistance, survival, and infection potential, and is regulated in many bacteria by the general stationary-phase stress response protein RpoS. Here, we showed that, unlike conventional culture conditions, several stationary-phase Salmonella pathogenic stress responses were not impacted by RpoS when bacteria were cultured under fluid shear conditions relevant to those encountered in the intestine of the infected host. These findings offer new insight into how physiological fluid shear forces encountered by Salmonella during infection might impact pathogenic responses in unexpected ways that are relevant to their disease-causing ability.

The transcription factor RpoS of Escherichia coli controls many genes important for tolerance of a variety of stress conditions. IraD promotes the post-translation stability of RpoS by inhibition of RssB, an adaptor protein for ClpXP degradation. We have previously documented DNA damage induction of iraD expression, independent of the SOS response. Both iraD and rpoS are required for tolerance to DNA damaging treatments such as H2O2 and the replication inhibitor azidothymidine in the log phase of growth. Using luciferase gene fusions to the 672 bp iraD upstream region, we show here that both promoters of iraD are induced by azidothymidine. Genetic analysis suggests that both promoters are repressed by DnaA-ATP, partially dependent on a putative DnaA box at -81bp and are regulated by regulatory inactivation of DnaA, dependent on the DnaN processivity clamp. By electrophoretic mobility shift assays, we show that purified DnaA protein binds to the iraD upstream region, so DnaA regulation of IraD is likely to be direct. DNA damage induction of iraD during log phase growth is abolished in the dnaA-T174P mutant, suggesting that DNA damage, in some way, relieves DnaA repression, possibly through the accumulation of replication clamps and enhanced regulatory inactivation of DnaA. We also demonstrate that the RNA-polymerase associated factor, stringent starvation protein A, induced by the accumulation of ppGpp, also affects iraD expression, with a positive effect on constitutive expression and a negative effect on azidothymidine-induced expression. [ABSTRACT FROM AUTHOR]

Cholera is a life-threatening diarrheal disease caused by the human pathogenic bacterium Vibrio cholerae. Regulatory elements are essential for bacterial transition between the natural aquatic environment and the human host. One of them is the alternative sigma factor RpoS and its anti-sigma factor RssB. Regulation principles seem to be conserved among RpoS/RssB interaction modes between V. cholerae and Enterobacteriaceae species, however the associated input and output pathways seem different. In Escherichia coli, RpoS/RssB is important for the activation of an emergency program to increase persistence and survival. Whereas, it activates motility and chemotaxis in V. cholerae, used strategically to escape from starvation conditions. We characterised a starvation-induced interaction model showing a negative feedback loop between RpoS and RssB expression. We showed by genotypic and phenotypic analysis that rssB influences motility, growth behaviour, colonization fitness, and post-infectious survival. Furthermore, we found that RssB itself is a substrate for proteolysis and a critical Asp mutation was identified and characterised to influence rssB phenotypes and their interaction with RpoS. In summary, we present novel information about the regulatory interaction between RpoS and RssB being active under in vivo colonization conditions and mark an extension to the feedback regulation circuit, showing that RssB is a substrate for proteolysis.

Vibrio mimicus is a pathogenic bacterium that cause red body disease in Macrobrachium nipponense , leading to high mortality and financial loss. Based on previous studies, rpoS gene contribute to bacterial pathogenicity during infection, but the role of RpoS involved in the immune response of M. nipponense under V. mimicus infection remains unclear. In this study, the pathogen load and the RNA-seq of M. nipponense under wild-type and Δ rpoS strain V. mimicus infection were investigated. Over the entire infection period, the Δ rpoS strain pathogen load was always lower than that of the wild-type strain in the M. nipponense hemolymph, hepatopancreas, gill and muscle. Furthermore, the expression level of rpoS gene in the hepatopancreas was the highest at 24 hours post infection (hpi), then the samples of hepatopancreas tissue infected with the wild type and Δ rpoS strain at 24 hpi were selected for RNA-seq sequencing. The results revealed a significant change in the transcriptomes of the hepatopancreases infected with Δ rpoS strain. In contrast to the wild-type infected group, the Δ rpoS strain infected group exhibited differentially expressed genes (DEGs) enriched in 181 KEGG pathways at 24 hpi. Among these pathways, 8 immune system-related pathways were enriched, including ECM-receptor interaction, PI3K-Akt signaling pathway, Rap1 signaling pathway, Gap junction, and Focal adhesion, etc. Among these pathways, up-regulated genes related to Kazal-type serine protease inhibitors, S-antigen protein, copper zinc superoxide dismutase, tight junction protein, etc. were enriched. This study elucidates that rpoS can affect tissue bacterial load and immune-related pathways, thereby impacting the survival rate of M. nipponense under V. mimicus infection. These findings validate the potential of rpoS as a promising target for the development of a live attenuated vaccine against V. mimicus. • The mutant of rpoS gene can reduce the pathogen load in M. nipponense tissues.. • RpoS is crucial in M. nipponense immune response to V. mimicus.. • Targeting rpoS gene can enhance M. nipponense survival against V. mimicus infection.. [ABSTRACT FROM AUTHOR]

A trade-off between reproduction and survival is a characteristic of many organisms. In bacteria, growth is constrained when cellular resources are channelled towards environmental stress protection. At the core of this trade-off in Escherichia coli is RpoS, a sigma factor that diverts transcriptional resources towards general stress resistance. The constancy of RpoS levels in natural isolates is unknown. A uniform RpoS content in E. coli would impart a narrow range of resistance properties to the species, whereas a diverse set of RpoS levels in nature should result in a diverse range of stress susceptibilities. We explore the diversity of trade-off settings and phenotypes by measuring the level of RpoS protein in strains of E. coli cohabiting in a natural environment. Strains from a stream polluted with domestic waste were investigated in monthly samples. Analyses included E. coli phylogroup classification, RpoS protein level, RpoS-dependent stress phenotypes and the sequencing of rpoS mutations. The most striking finding was the continuum of RpoS levels, with a 100-fold range of RpoS amounts consistently found in individuals in the stream. Approximately 1.8% of the sampled strains carried null or non-synonymous mutations in rpoS. The natural isolates also exhibited a broad (>100-fold) range of stress resistance responses. Our results are consistent with the view that a multiplicity of survival-multiplication trade-off settings is a feature of the species E. coli. The phenotypic diversity resulting from the trade-off permits bet-hedging and the adaptation of E. coli strains to a very broad range of environments. [ABSTRACT FROM AUTHOR]

The ppk1 gene encodes polyphosphate kinase (PPK1), which is the major catalytic enzyme that Escherichia coli utilizes to synthesize inorganic polyphosphate (polyP). The aim of this study was to explore the role of PPK1 in the pathogenesis of Enterohemorrhagic E. coli O157:H7 (EHEC O157:H7). An isogenic in-frame ppk1 deletion mutant (Δ ppk1) and ppk1 complemented mutant (Cppk1) were constructed and characterized in comparison to wild-type (WT) EHEC O157:H7 strain EDL933w by microscope observation and growth curve analysis. Survival rates under heat stress and acid tolerance, both of which the bacteria would face during pathogenesis, were compared among the three strains. LoVo cells and a murine model of intestinal colitis were used as the in vitro and in vivo models, respectively, to evaluate the effect of PPK1 on adhesion and invasion during the process of pathogenesis. Real-time reverse-transcription PCR of regulatory gene rpoS , adhesion gene eae , and toxin genes stx1 and stx2 was carried out to corroborate the results from the in vitro and in vivo models. The ppk1 deletion mutant exhibited disrupted polyP levels, but not morphology and growth characteristics. The survival rate of the Δ ppk1 strain under stringent environmental conditions was lower as compared with WT and Cppk1. The in vitro assays showed that deletion of the ppk1 gene reduced the adhesion, formation of attaching and effacing (A/E) lesions, and invasive ability of EHEC O157:H7. Moreover, the virulence of the Δ ppk1 in BALB/c mice was weaker as compared with the other two strains. Additionally, mRNA expression of rpoS , eae , stx1 and stx2 were consistent with the in vitro and in vivo results. In conclusion: EHEC O157:H7 requires PPK1 for both survival under harsh environmental conditions and virulence in vivo. [ABSTRACT FROM AUTHOR]

Biofilms are a widespread multicellular form of bacterial life. The spatial structure and emergent properties of these communities depend on a polymeric extracellular matrix architecture that is orders of magnitude larger than the cells that build it. Using as a model the wrinkly macrocolony biofilms of Escherichia coli, which contain amyloid curli fibers and phosphoethanolamine (pEtN)-modified cellulose as matrix components, we summarize here the structure, building, and function of this large-scale matrix architecture. Based on different sigma and other transcription factors as well as second messengers, the underlying regulatory network reflects the fundamental trade-off between growth and survival. It controls matrix production spatially in response to long-range chemical gradients, but it also generates distinct patterns of short-range matrix heterogeneity that are crucial for tissue-like elasticity and macroscopic morphogenesis. Overall, these biofilms confer protection and a potential for homeostasis, thereby reducing maintenance energy, which makes multicellularity an emergent property of life itself. [ABSTRACT FROM AUTHOR]

چکیده مقدمه: سیپروفلوکسازین، آنتی‌بیوتیکی با طیف اثر وسیع از خانوادۀ فلوروکینولون‌هاست که هدف اصلی آن در اشریشیا کلی، DNAژیراز است. سیپروفلوکسازین سبب القای پاسخ SOS از طریق RecA می‌شود. حضور پروتئین RecF‌ برای فعالیت RecA ضروری است. recF در فاز نمایی به‌واسطۀ پروموتور اپرون خود و در فاز سکون به‌وسیلۀ پروموتور اختصاصی‌اش افزایش بیان می‌یابد؛ فعالیت پروموتور اختصاصی به rpoS وابسته است. هدف مطالعۀ حاضر، بررسی میزان بیان rpoS در جهش‌یافتۀ مقاوم به سیپروفلوکسازین است. مواد و روش‏‏ها: به‌منظور بررسی بیان rpoS، RNA سلول در اوایل و میانۀ فاز نمایی رشد استخراج شد. پس‌از سنتز cDNA، میزان بیان نسبی ژن‌ در سویه‌های تیپ وحشی و جهش‌یافته به روش Real time PCR سنجیده شد. نتایج: نتایج، آلوده‌نبودن RNAهای استخراج‌شده و کیفیت مناسب آنها برای تولید cDNA را نشان دادند؛ همچنین بیان rpoS در اوایل و میانۀ فاز نمایی پس‌از تیمار با سیپروفلوکسازین در جهش‌یافتۀ دارای مقاومت زیاد به سیپروفلوکسازین تقریباً مشابه تیپ وحشی بود. بحث و نتیجه‏گیری: به نظر می‌رسد افزایش بیان recFبه‌وسیلۀ پروموتور اپرون است و تیمار با سیپروفلوکسازین سبب ایجاد شرایط مشابه با فاز سکون نمی‌شود.

The ppk1 gene encodes polyphosphate kinase (PPK1), which is the major catalytic enzyme that Escherichia coli utilizes to synthesize inorganic polyphosphate (polyP). The aim of this study was to explore the role of PPK1 in the pathogenesis of Enterohemorrhagic E. coli O157:H7 (EHEC O157:H7). An isogenic in-frame ppk1 deletion mutant (Δppk1) and ppk1 complemented mutant (Cppk1) were constructed and characterized in comparison to wild-type (WT) EHEC O157:H7 strain EDL933w by microscope observation and growth curve analysis. Survival rates under heat stress and acid tolerance, both of which the bacteria would face during pathogenesis, were compared among the three strains. LoVo cells and a murine model of intestinal colitis were used as the in vitro and in vivo models, respectively, to evaluate the effect of PPK1 on adhesion and invasion during the process of pathogenesis. Real-time reverse-transcription PCR of regulatory gene rpoS, adhesion gene eae, and toxin genes stx1 and stx2 was carried out to corroborate the results from the in vitro and in vivo models. The ppk1 deletion mutant exhibited disrupted polyP levels, but not morphology and growth characteristics. The survival rate of the Δppk1 strain under stringent environmental conditions was lower as compared with WT and Cppk1. The in vitro assays showed that deletion of the ppk1 gene reduced the adhesion, formation of attaching and effacing (A/E) lesions, and invasive ability of EHEC O157:H7. Moreover, the virulence of the Δppk1 in BALB/c mice was weaker as compared with the other two strains. Additionally, mRNA expression of rpoS, eae, stx1 and stx2 were consistent with the in vitro and in vivo results. In conclusion: EHEC O157:H7 requires PPK1 for both survival under harsh environmental conditions and virulence in vivo.

bb0616 of Borrelia burgdorferi , the Lyme disease pathogen, encodes a hypothetical protein of unknown function. In this study, we showed that BB0616 was not surface-exposed or associated with the membrane through localization analyses using proteinase K digestion and cell partitioning assays. The expression of bb0616 was influenced by a reduced pH but not by growth phases, elevated temperatures, or carbon sources during in vitro cultivation. A transcriptional start site for bb0616 was identified by using 5' rapid amplification of cDNA ends, which led to the identification of a functional promoter in the 5' regulatory region upstream of bb0616 . By analyzing a bb0616 -deficient mutant and its isogenic complemented counterparts, we found that the infectivity potential of the mutant was significantly attenuated. The inactivation of bb0616 displayed no effect on borrelial growth in the medium or resistance to oxidative stress, but the mutant was significantly more susceptible to osmotic stress. In addition, the production of global virulence regulators such as BosR and RpoS as well as virulence-associated outer surface lipoproteins OspC and DbpA was reduced in the mutant. These phenotypes were fully restored when gene mutation was complemented with a wild-type copy of bb0616 . Based on these findings, we concluded that the hypothetical protein BB0616 is required for the optimal infectivity of B. burgdorferi , potentially by impacting B. burgdorferi virulence gene expression as well as survival of the spirochete under stressful conditions., Competing Interests: The authors declare no conflict of interest.

RpoS, an alternative sigma factor of RNA polymerase, regulates the expression of a great deal of genes involved in stationary-phase survival and stress response. To identify the function of RpoS homologue in Serratia marcescens FS14, in-frame deletion mutant of rpoS was constructed. It was found that RpoS activates the biosynthesis of prodigiosin in FS14 which is just opposite to what was observed in Serratia sp. ATCC 39006. We also demonstrated that RpoS positively regulates the prodigiosin production by activating the transcription of pig cluster in FS14, and the transcription of pig cluster is RpoS-dependent. Further study showed that the differences in the promoters of pig clusters in FS14 and 39006 lead to the different selection of the sigma factors and result in the different regulation mechanisms. The -10 element and the spacer region between -10 and -35 elements of the pig cluster in FS14 are vital for the RpoS recognition in FS14. [ABSTRACT FROM AUTHOR]

Background: Nowadays, recombinant therapeutic proteins have been widely produced and consumed. For the safety and effectiveness of the protein production, an auto-inducible expression vector is required to replace inducer interference, which is uneconomic and could be harmful. In this research, an auto-inducible expression plasmid, pCAD2_sod (a pBR322 derivate plasmid), which was under dps (RpoS-dependent gene) promoter control, was modified to provide RpoS at earlier phase. Hence, accumulates more target protein and resulting a new plasmid, pCAD2+_sod. pCAD2_sod had been constructed to automatically induces the expression of recombinant superoxide dismutase (SOD) from Staphylococcus equorum (rMnSODSeq) in the stationary growth phase of Escherichia coli. This work aimed to obtain pCAD2+_sod and determine the expression level of rMnSODSeq on mRNA and protein level. Method and results: A synthetic rpoS coding region under rpoD promoter control (prpoD_rpoS) was inserted to pCAD2_sod and generated pCAD2+_sod. The rMnSODSeq (24.3 kDa) produced from pCAD2+_sod was ~ 1.5 fold higher at 37 °C and more intense at 43 °C compared to that from pCAD2_sod, likewise shifted to earlier phase (after 1 h of incubation), as shown in the SDS-PAGE. The dismutase activity was also retained after zymography assay. The mRNA level from pCAD2+_sod was determined by qPCR and gave quantification cycle (Cq) values of cDNA lowest among others. It made the relative quantification (RQ) of the mRNA expression towards rho reference gene were high. Conclusions: The prpoD_rpoS insertion shifts and increases the rMnSODSeq production from stationary to exponential phase. The pCAD2+_sod plasmid is potential for further recombinant protein productions. [ABSTRACT FROM AUTHOR]

Bacterial behavior has been studied under microgravity conditions, but very little is known about it under lunar and Martian gravitational regimes. An Earth-based approach was designed and implemented using inclined clinostats and an in-house-developed code to determine the optimal clinorotation angular speed for bacterial liquid cultures of 5 RPM. With this setup, growth dynamics, phenotypic changes, and sensitivity to antibiotics (minimum inhibitory concentration (MIC) of two different classes of antibiotics) for three Escherichia coli strains (including uropathogenic) were examined under simulated micro-, lunar, and Martian gravities. The results included increased growth under simulated micro- and lunar gravities for some strains, and higher concentrations of antibiotics needed under simulated lunar gravity with respect to simulated micro- and Martian gravities. Clinostat-produced results can be considered suggestive but not determinative of what might be expected in altered gravity, as there is still a need to systematically verify these simulation devices’ ability to accurately replicate phenomena observed in space. Nevertheless, this approach serves as a baseline to start interrogating key cellular and molecular aspects relevant to microbial processes on the lunar and Martian surfaces.

RtxA1 is a major cytotoxin of Vibrio vulnificus (V. vulnificus) causing fatal septicemia and necrotic wound infections. Our previous work has shown that RpoS regulates the expression and secretion of V. vulnificus RtxA1 toxin. This study was conducted to further investigate the potential mechanisms of RpoS on RtxA1 secretion. First, V. vulnificus TolCV1 and TolCV2 proteins, two Escherichia coli TolC homologs, were measured at various time points by Western blotting. The expression of TolCV1 was increased time-dependently, whereas that of TolCV2 was decreased. Expression of both TolCV1 and TolCV2 was significantly downregulated in an rpoS deletion mutation. Subsequently, we explored the roles of TolCV1 and TolCV2 in V. vulnificus pathogenesis. Western blot analysis showed that RtxA1 toxin was exported by TolCV1, not TolCV2, which was consistent with the cytotoxicity results. Furthermore, the expression of TolCV1 and TolCV2 was increased after treatment of the host signal bile salt and the growth of tolCV1 mutant was totally abolished in the presence of bile salt. A tolCV1 mutation resulted in significant reduction of V. vulnificus induced-virulence in mice. Taken together, TolCV1 plays key roles in RtxA1 secretion, bile salt resistance, and mice lethality of V. vulnificus, suggesting that TolCV1 could be an attractive target for the design of new medicines to treat V. vulnificus infections.

RtxA1 is a major cytotoxin of Vibrio vulnificus (V. vulnificus) causing fatal septicemia and necrotic wound infections. Our previous work has shown that RpoS regulates the expression and secretion of V. vulnificus RtxA1 toxin. This study was conducted to further investigate the potential mechanisms of RpoS on RtxA1 secretion. First, V. vulnificus TolCV1 and TolCV2 proteins, two Escherichia coli TolC homologs, were measured at various time points by Western blotting. The expression of TolCV1 was increased time-dependently, whereas that of TolCV2 was decreased. Expression of both TolCV1 and TolCV2 was significantly downregulated in an rpoS deletion mutation. Subsequently, we explored the roles of TolCV1 and TolCV2 in V. vulnificus pathogenesis. Western blot analysis showed that RtxA1 toxin was exported by TolCV1, not TolCV2, which was consistent with the cytotoxicity results. Furthermore, the expression of TolCV1 and TolCV2 was increased after treatment of the host signal bile salt and the growth of tolCV1 mutant was totally abolished in the presence of bile salt. A tolCV1 mutation resulted in significant reduction of V. vulnificus induced-virulence in mice. Taken together, TolCV1 plays key roles in RtxA1 secretion, bile salt resistance, and mice lethality of V. vulnificus , suggesting that TolCV1 could be an attractive target for the design of new medicines to treat V. vulnificus infections. [ABSTRACT FROM AUTHOR]

Legionellapneumophila (Lp) is a waterborne bacterium able to infect human alveolar macrophages, causing Legionnaires' disease. Lp can survive for several months in water, while searching for host cells to grow in, such as ciliates and amoeba. In Lp, the sigma factor RpoS is essential for survival in water. A previous transcriptomic study showed that RpoS positively regulates the small regulatory RNA Lpr10. In the present study, deletion of lpr10 results in an increased survival of Lp in water. Microarray analysis and RT‐qPCR revealed that Lpr10 negatively regulates the expression of RpoS in the postexponential phase. Electrophoretic mobility shift assay and in‐line probing showed that Lpr10 binds to a region upstream of the previously identified transcription start sites (TSS) of rpoS. A third putative transcription start site was identified by primer extension analysis, upstream of the Lpr10 binding site. In addition, nlpD TSS produces a polycistronic mRNA including the downstream gene rpoS, indicating a fourth TSS for rpoS. Our results suggest that the transcripts from the third and fourth TSS are negatively regulated by the Lpr10 sRNA. Therefore, we propose that Lpr10 is involved in a negative regulatory feedback loop to maintain expression of RpoS to an optimal level. [ABSTRACT FROM AUTHOR]

In the model organism Escherichia coli and related species, the general stress response relies on tight regulation of the intracellular levels of the promoter specificity subunit RpoS. RpoS turnover is exclusively dependent on RssB, a two‐domain response regulator that functions as an adaptor that delivers RpoS to ClpXP for proteolysis. Here, we report crystal structures of the receiver domain of RssB both in its unphosphorylated form and bound to the phosphomimic BeF3−. Surprisingly, we find only modest differences between these two structures, suggesting that truncating RssB may partially activate the receiver domain to a "meta‐active" state. Our structural and sequence analysis points to RssB proteins not conforming to either the Y–T coupling scheme for signaling seen in prototypical response regulators, such as CheY, or to the signaling model of the less understood FATGUY proteins. PDB Code(s): 7L9C and 7LCM; [ABSTRACT FROM AUTHOR]

Involvement of the transcriptional regulator RpoS in the persistence of viable but non-culturable (VBNC) state has been demonstrated in several species of bacteria. This study investigated the role of the RpoS in the formation and resuscitation of VBNC state in Salmonella enterica serovar Enteritidis CICC 21482 by measuring bacterial survival, morphology, physiological characteristics, and gene expression in wild-type (WT) and rpoS -deletion (ΔrpoS) strains during long-term storage in powdered infant formula (PIF). The ΔrpoS strain was produced by allelic exchange using a suicide plasmid. Bacteria were inoculated into PIF for 635-day storage. Survival, morphology, intracellular reactive oxygen species (ROS) levels and intercellular quorum sensing autoinducer-2 (AI-2) contents were regularly measured. Resuscitation assays were conducted after obtaining VBNC cells. Gene expression was measured using real-time quantitative polymerase chain reaction (qPCR). The results showed that RpoS and low temperature conditions were associated with enhanced culturability and recoverability of Salmonella Enteritidis after desiccation storage in low water activity (a w) PIF. In addition, the synthesis of intracellular ROS and intercellular quorum sensing AI-2 was regulated by RpoS, inducing the formation and resuscitation of VBNC cells. Gene expression of soxS , katG and relA was found strongly associated with RpoS. Due to the lack of RpoS factor, the ΔrpoS strain could not normally synthesize SoxS, catalase and (p)ppGpp, resulting in its early shift to the VBNC state. This study elucidates the role of rpoS in desiccation stress and the formation and resuscitation mechanism of VBNC cells under desiccation stress. It serves as the basis for preventing and controlling the recovery of pathogenic bacteria in VBNC state in low a w foods. • RpoS and low temperature were associated with enhanced culturability and recoverability of VBNC Salmonella Enteritidis. • Sodium pyruvate and catalase had stronger resuscitation effect in low a w PIF. • The synthesis of reactive oxygen species and autoinducer-2 was regulated by RpoS. • Bacterial antioxidant gene soxS , katG and (p)ppGpp synthetase gene relA was strongly associated with rpoS. [ABSTRACT FROM AUTHOR]

Abstract Carotenoids are widely used in functional foods, cosmetics, and health supplements, and their importance and scope of use are continuously expanding. Here, we characterized carotenoid biosynthetic genes of the plant‐pathogenic bacterium Pantoea ananatis, which carries a carotenoid biosynthetic gene cluster (including crtE, X, Y, I, B, and Z) on a plasmid. Reverse transcription–polymerase chain reaction (RT‐PCR) analysis revealed that the crtEXYIB gene cluster is transcribed as a single transcript and crtZ is independently transcribed in the opposite direction. Using splicing by overlap extension with polymerase chain reaction (SOE by PCR) based on asymmetric amplification, we reassembled crtE–B, crtE–B–I, and crtE–B–I–Y. High‐performance liquid chromatography confirmed that Escherichia coli expressing the reassembled crtE–B, crtE–B–I, and crtE–B–I–Y operons produced phytoene, lycopene, and β‐carotene, respectively. We found that the carotenoids conferred tolerance to UV radiation and toxoflavin. Pantoea ananatis shares rice environments with the toxoflavin producer Burkholderia glumae and is considered to be the first reported example of producing and using carotenoids to withstand toxoflavin. We confirmed that carotenoid production by P. ananatis depends on RpoS, which is positively regulated by Hfq/ArcZ and negatively regulated by ClpP, similar to an important regulatory network of E. coli (HfqArcZ →RpoS Ͱ ClpXP). We also demonstrated that Hfq‐controlled quorum signaling de‐represses EanR to activate RpoS, thereby initiating carotenoid production. Survival genes such as those responsible for the production of carotenoids of the plant‐pathogenic P. ananatis must be expressed promptly to overcome stressful environments and compete with other microorganisms. This mechanism is likely maintained by a brake with excellent performance, such as EanR.

Microbial tolerance to organic solvents is critical for efficient production of biofuels. In this study, n-butanol tolerance of Escherichia coli JM109 was improved by overexpressing of genes encoding stress-responsive small RNA-regulator, RNA chaperone, and molecular chaperone. Gene rpoS, coding for sigma S subunit of RNA polymerase, was the most efficient in improving n-butanol tolerance of E. coli. The highest OD600 and the specific growth rate of JM109/pQE80L-rpoS reached 1.692 and 0.144 h–1 respectively at 1.0% (v/v) n-butanol. Double and triple expression of molecular chaperones rpoS, secB, and groS were conducted and optimized. Recombinant strains JM109/pQE80L-secB-rpoS and JM109/pQE80L-groS-secB-rpoS exhibited the highest n-butanol tolerance, with specific growth rates of 0.164 and 0.165 h–1, respectively. Membrane integrity, potentials, and cell morphology analyses demonstrated the high viability of JM109/pQE80L-groS-secB-rpoS. This study provides guidance on employing various molecular chaperones for enhancing the tolerance of E. coli against n-butanol. [ABSTRACT FROM AUTHOR]