Gamblers: An Antibiotic-Induced Evolvable Cell Subpopulation Differentiated by Reactive-Oxygen-Induced General Stress Response.

Antibiotics can induce mutations that cause antibiotic resistance. Yet, despite their importance, mechanisms of antibiotic-promoted mutagenesis remain elusive. We report that the fluoroquinolone antibiotic ciprofloxacin (cipro) induces mutations by triggering transient differentiation of a mutant-generating cell subpopulation, using reactive oxygen species (ROS). Cipro-induced DNA breaks activate the Escherichia coli SOS DNA-damage response and error-prone DNA polymerases in all cells. However, mutagenesis is limited to a cell subpopulation in which electron transfer together with SOS induce ROS, which activate the sigma-S (σS) general-stress response, which allows mutagenic DNA-break repair. When sorted, this small σS-response-"on" subpopulation produces most antibiotic cross-resistant mutants. A U.S. Food and Drug Administration (FDA)-approved drug prevents σS induction, specifically inhibiting antibiotic-promoted mutagenesis. Further, SOS-inhibited cell division, which causes multi-chromosome cells, promotes mutagenesis. The data support a model in which within-cell chromosome cooperation together with development of a "gambler" cell subpopulation promote resistance evolution without risking most cells.

Bacterial Multicellularity: The Biology of Escherichia coli Building Large-Scale Biofilm Communities.

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.

IraM remodels the RssB segmented helical linker to stabilize σs against degradation by ClpXP.

Upon Mg2+ starvation, a condition often associated with virulence, enterobacteria inhibit the ClpXP-dependent proteolysis of the master transcriptional regulator, σs, via IraM, a poorly understood antiadaptor that prevents RssB-dependent loading of σs onto ClpXP. This inhibition results in σs accumulation and expression of stress resistance genes. Here, we report on the structural analysis of RssB bound to IraM, which reveals that IraM induces two folding transitions within RssB, amplified via a segmented helical linker. These conformational changes result in an open, yet inhibited RssB structure in which IraM associates with both the C-terminal and N-terminal domains of RssB and prevents binding of σs to the 4-5-5 face of the N-terminal receiver domain. This work highlights the remarkable structural plasticity of RssB and reveals how a stress-specific RssB antagonist modulates a core stress response pathway that could be leveraged to control biofilm formation, virulence, and the development of antibiotic resistance.

The regulatory network comprising ArcAB-RpoS-RssB influences motility in Vibrio cholerae.

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.

The alternative sigma factor RpoS regulates Pseudomonas aeruginosa quorum sensing response by repressing the pqsABCDE operon and activating vfr.

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.

RpoS acts as a global repressor of virulence gene expression in E. coli O104:H4 and enteroaggregative E. coli.

In 2011, in Germany, Escherichia coli O104:H4 caused the enterohemorrhagic E. coli (EHEC) outbreak with the highest incidence rate of hemolytic uremic syndrome. This pathogen carries an exceptionally potent combination of EHEC- and enteroaggregative E. coli (EAEC)-specific virulence factors. Here, we identified an E. coli O104:H4 isolate that carried a single nucleotide polymorphism (SNP) in the start codon (ATG > ATA) of rpoS, encoding the alternative sigma factor S. The rpoS ATG > ATA SNP was associated with enhanced EAEC-specific virulence gene expression. Deletion of rpoS in E. coli O104:H4 Δstx2 and typical EAEC resulted in a similar effect. Both rpoS ATG > ATA and ΔrpoS strains exhibited stronger virulence-related phenotypes in comparison to wild type. Using promoter-reporter gene fusions, we demonstrated that wild-type RpoS repressed aggR, encoding the main regulator of EAEC virulence. In summary, our work demonstrates that RpoS acts as a global repressor of E. coli O104:H4 virulence, primarily through an AggR-dependent mechanism.

Structure of phosphorylated-like RssB, the adaptor delivering σs to the ClpXP proteolytic machinery, reveals an interface switch for activation.

In enterobacteria such as Escherichia coli, the general stress response is mediated by σs, the stationary phase dissociable promoter specificity subunit of RNA polymerase. σs is degraded by ClpXP during active growth in a process dependent on the RssB adaptor, which is thought to be stimulated by the phosphorylation of a conserved aspartate in its N-terminal receiver domain. Here we present the crystal structure of full-length RssB bound to a beryllofluoride phosphomimic. Compared to the structure of RssB bound to the IraD anti-adaptor, our new RssB structure with bound beryllofluoride reveals conformational differences and coil-to-helix transitions in the C-terminal region of the RssB receiver domain and in the interdomain segmented helical linker. These are accompanied by masking of the α4-ß5-α5 (4-5-5) "signaling" face of the RssB receiver domain by its C-terminal domain. Critically, using hydrogen-deuterium exchange mass spectrometry, we identify σs-binding determinants on the 4-5-5 face, implying that this surface needs to be unmasked to effect an interdomain interface switch and enable full σs engagement and hand-off to ClpXP. In activated receiver domains, the 4-5-5 face is often the locus of intermolecular interactions, but its masking by intramolecular contacts upon phosphorylation is unusual, emphasizing that RssB is a response regulator that undergoes atypical regulation.

Assessment of the hypothetical protein BB0616 in the murine infection of Borrelia burgdorferi.

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.

Defining the regulatory mechanisms of sigma factor RpoS degradation in Azotobacter vinelandii and Pseudomonas aeruginosa.

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.

How many Mutations are needed to Evolve the Chemical Makeup of a Synthetic Cell?

The chemical evolution of a synthetic cell endowed with a synthetic amino acid as building block, analog to tryptophan, required the emergence of key mutations in genes involved in, inter alia, the general stress response, amino acid metabolism, stringent response, and chemotaxis. Understanding adaptation mechanisms to non-canonical biomass components will inform strategies for engineering synthetic metabolic pathways and cells.

RpoS role in antibiotic resistance, tolerance and persistence in E. coli natural isolates.

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 ß -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.

rpoS involved in immune response of Macrobrachium nipponens to Vibrio mimicus infection.

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 ΔrpoS strain V. mimicus infection were investigated. Over the entire infection period, the ΔrpoS 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 ΔrpoS strain at 24 hpi were selected for RNA-seq sequencing. The results revealed a significant change in the transcriptomes of the hepatopancreases infected with ΔrpoS strain. In contrast to the wild-type infected group, the ΔrpoS 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.

Function of Burkholderia pseudomallei RpoS and RpoN2 in bacterial invasion, intracellular survival, and multinucleated giant cell formation in mouse macrophage cell line.

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.

Common food preservatives impose distinct selective pressures on Salmonella Typhimurium planktonic and biofilm populations.

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.

The Rrp2-RpoN-RpoS pathway plays an important role in the blood-brain barrier transmigration of the Lyme disease pathogen.

Lyme disease, caused by Borrelia (or Borreliella) burgdorferi, is a complex multisystemic disorder that includes Lyme neuroborreliosis resulting from the invasion of both the central and peripheral nervous systems. However, factors that enable the pathogen to cross the blood-brain barrier (BBB) and invade the central nervous system (CNS) are still not well understood. The objective of this study was to identify the B. burgdorferi factors required for BBB transmigration. We utilized a transwell BBB model based on human brain-microvascular endothelial cells and focused on investigating the Rrp2-RpoN-RpoS pathway, a central regulatory pathway that is essential for mammalian infection by B. burgdorferi. Our results demonstrated that the Rrp2-RpoN-RpoS pathway is crucial for BBB transmigration. Furthermore, we identified OspC, a major surface lipoprotein controlled by the Rrp2-RpoN-RpoS pathway, as a significant contributor to BBB transmigration. Constitutive production of OspC in a mutant defective in the Rrp2-RpoN-RpoS pathway did not rescue the impairment in BBB transmigration, indicating that this pathway controls additional factors for this process. Two other major surface lipoproteins controlled by this pathway, DbpA/B and BBK32, appeared to be dispensable for BBB transmigration. In addition, both the surface lipoprotein OspA and the Rrp1 pathway, which are required B. burgdorferi colonization in the tick vector, were found not required for BBB transmigration. Collectively, our findings using in vitro transwell assays uncover another potential role of the Rrp2-RpoN-RpoS pathway in BBB transmigration of B. burgdorferi and invasion into the CNS.

Role of Stress-Induced Proteins RpoS and YicC in the Persistence of Salmonella enterica subsp. enterica Serotype Typhimurium in Tomato Plants.

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. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Transcriptional organization and regulation of the Pseudomonas putida K1 type VI secretion system gene cluster.

The type VI secretion system (T6SS) is an antimicrobial molecular weapon that is widespread in Proteobacteria and offers competitive advantages to T6SS-positive micro-organisms. Three T6SSs have recently been described in Pseudomonas putida KT2440 and it has been shown that one, K1-T6SS, is used to outcompete a wide range of phytopathogens, protecting plants from pathogen infections. Given the relevance of this system as a powerful and innovative mechanism of biological control, it is critical to understand the processes that govern its expression. Here, we experimentally defined two transcriptional units in the K1-T6SS cluster. One encodes the structural components of the system and is transcribed from two adjacent promoters. The other encodes two hypothetical proteins, the tip of the system and the associated adapters, and effectors and cognate immunity proteins, and it is also transcribed from two adjacent promoters. The four identified promoters contain the typical features of σ70-dependent promoters. We have studied the expression of the system under different conditions and in a number of mutants lacking global regulators. P. putida K1-T6SS expression is induced in the stationary phase, but its transcription does not depend on the stationary σ factor RpoS. In fact, the expression of the system is indirectly repressed by RpoS. Furthermore, it is also repressed by RpoN and the transcriptional regulator FleQ, an enhancer-binding protein typically acting in conjunction with RpoN. Importantly, expression of the K1-T6SS gene cluster is positively regulated by the GacS-GacA two-component regulatory system (TCS) and repressed by the RetS sensor kinase, which inhibits this TCS. Our findings identified a complex regulatory network that governs T6SS expression in general and P. putida K1-T6SS in particular, with implications for controlling and manipulating a bacterial agent that is highly relevant in biological control.

DNA supercoiling and regulation of intrinsic ß-lactamase in pathogenic Escherichia coli.

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 ß-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 ß-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.

Genotypic and phenotypic characterization of a Salmonella Typhimurium strain resistant to pulsed electric fields.

Pulsed Electric Fields (PEF) technology is regarded as one of the most interesting alternatives to current food preservation methods, due to its capability to inactivate vegetative microorganisms while leaving the product's organoleptic and nutritional properties mostly unchanged. However, many aspects regarding the mechanisms of bacterial inactivation by PEF are still not fully understood. The aim of this study was to obtain further insight into the mechanisms responsible for the increased resistance to PEF of a Salmonella Typhimurium SL1344 variant (SL1344-RS, Sagarzazu et al., 2013), and to quantify the impact that the acquisition of PEF resistance has on other aspects of S. enterica physiology, such as growth fitness, biofilm formation ability, virulence and antibiotic resistance. WGS, RNAseq and qRT-PCR assays indicated that the increased PEF resistance of the SL1344-RS variant is due to a higher RpoS activity caused by a mutation in the hnr gene. This increased RpoS activity also results in higher resistance to multiple stresses (acidic, osmotic, oxidative, ethanol and UV-C, but not to heat and HHP), decreased growth rate in M9-Gluconate (but not in TSB-YE or LB-DPY), increased ability to adhere to Caco-2 cells (but no significant change in invasiveness) and enhanced antibiotic resistance (to six out of eight agents). This study significantly contributes to the understanding of the mechanisms of the development of stress resistance in Salmonellae and underscores the crucial role played by RpoS in this process. Further studies are needed to determine whether this PEF-resistant variant would represent a higher, equal or lower associated hazard than the parental strain.

Unraveling the regulation of pyocyanin synthesis by RsmA through MvaU and RpoS in Pseudomonas aeruginosa ID4365.

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.