Key interactions of RNA polymerase with 6S RNA and secondary channel factors during pRNA synthesis.

Small non-coding 6S RNA mimics DNA promoters and binds to the σ70 holoenzyme of bacterial RNA polymerase (RNAP) to suppress transcription of various genes mainly during the stationary phase of cell growth or starvation. This inhibition can be relieved upon synthesis of short product RNA (pRNA) performed by RNAP from the 6S RNA template. Here, we have shown that pRNA synthesis depends on specific contacts of 6S RNA with RNAP and interactions of the σ finger with the RNA template in the active site of RNAP, and is also modulated by the secondary channel factors. We have adapted a molecular beacon assay with fluorescently labeled σ70 to analyze 6S RNA release during pRNA synthesis. We found the kinetics of 6S RNA release to be oppositely affected by mutations in the σ finger and in the CRE pocket of core RNAP, similarly to the reported role of these regions in promoter-dependent transcription. Secondary channel factors, DksA and GreB, inhibit pRNA synthesis and 6S RNA release from RNAP, suggesting that they may contribute to the 6S RNA-mediated switch in transcription during stringent response. Our results demonstrate that pRNA synthesis depends on a similar set of contacts between RNAP and 6S RNA as in the case of promoter-dependent transcription initiation and reveal that both processes can be regulated by universal transcription factors acting on RNAP.

Stringent Response Factor DksA Contributes to Fatty Acid Degradation Function to Influence Cell Membrane Stability and Polymyxin B Resistance of Yersinia enterocolitica.

DksA is a proteobacterial regulator that binds directly to the secondary channel of RNA polymerase with (p)ppGpp and is responsible for various bacterial physiological activities. While (p)ppGpp is known to be involved in the regulation and response of fatty acid metabolism pathways in many foodborne pathogens, the role of DksA in this process has yet to be clarified. This study aimed to characterize the function of DksA on fatty acid metabolism and cell membrane structure in Yersinia enterocolitica. Therefore, comparison analysis of gene expression, growth conditions, and membrane permeabilization among the wide-type (WT), DksA-deficient mutant (YEND), and the complemented strain was carried out. It confirmed that deletion of DksA led to a more than four-fold decrease in the expression of fatty acid degradation genes, including fadADEIJ. Additionally, YEND exhibited a smaller growth gap compared to the WT strain at low temperatures, indicating that DksA is not required for the growth of Y. enterocolitica in cold environments. Given that polymyxin B is a cationic antimicrobial peptide that targets the cell membrane, the roles of DksA under polymyxin B exposure were also characterized. It was found that DksA positively regulates the integrity of the inner and outer membranes of Y. enterocolitica under polymyxin B, preventing the leakage of intracellular nucleic acids and proteins and ultimately reducing the sensitivity of Y. enterocolitica to polymyxin B. Taken together, this study provides insights into the functions of DksA and paves the way for novel fungicide development.

Transcriptomic Analysis Reveals Key Roles of (p)ppGpp and DksA in Regulating Metabolism and Chemotaxis in Yersinia enterocolitica.

The stringent response is a rapid response system that is ubiquitous in bacteria, allowing them to sense changes in the external environment and undergo extensive physiological transformations. However, the regulators (p)ppGpp and DksA have extensive and complex regulatory patterns. Our previous studies demonstrated that (p)ppGpp and DksA in Yersinia enterocolitica positively co-regulated motility, antibiotic resistance, and environmental tolerance but had opposite roles in biofilm formation. To reveal the cellular functions regulated by (p)ppGpp and DksA comprehensively, the gene expression profiles of wild-type, ΔrelA, ΔrelAΔspoT, and ΔdksAΔrelAΔspoT strains were compared using RNA-Seq. Results showed that (p)ppGpp and DksA repressed the expression of ribosomal synthesis genes and enhanced the expression of genes involved in intracellular energy and material metabolism, amino acid transport and synthesis, flagella formation, and the phosphate transfer system. Additionally, (p)ppGpp and DksA inhibited amino acid utilization (such as arginine and cystine) and chemotaxis in Y. enterocolitica. Overall, the results of this study unraveled the link between (p)ppGpp and DksA in the metabolic networks, amino acid utilization, and chemotaxis in Y. enterocolitica and enhanced the understanding of stringent responses in Enterobacteriaceae.

DksA, ppGpp, and RegAB Regulate Nitrate Respiration in Paracoccus denitrificans.

The periplasmic (NAP) and membrane-associated (Nar) nitrate reductases of Paracoccus denitrificans are responsible for nitrate reduction under aerobic and anaerobic conditions, respectively. Expression of NAP is elevated in cells grown on a relatively reduced carbon and energy source (such as butyrate); it is believed that NAP contributes to redox homeostasis by coupling nitrate reduction to the disposal of excess reducing equivalents. Here, we show that deletion of either dksA1 (one of two dksA homologs in the P. denitrificans genome) or relA/spoT (encoding a bifunctional ppGpp synthetase and hydrolase) eliminates the butyrate-dependent increase in nap promoter and NAP enzyme activity. We conclude that ppGpp likely signals growth on a reduced substrate and, together with DksA1, mediates increased expression of the genes encoding NAP. Support for this model comes from the observation that nap promoter activity is increased in cultures exposed to a protein synthesis inhibitor that is known to trigger ppGpp synthesis in other organisms. We also show that, under anaerobic growth conditions, the redox-sensing RegAB two-component pair acts as a negative regulator of NAP expression and as a positive regulator of expression of the membrane-associated nitrate reductase Nar. The dksA1 and relA/spoT genes are conditionally synthetically lethal; the double mutant has a null phenotype for growth on butyrate and other reduced substrates while growing normally on succinate and citrate. We also show that the second dksA homolog (dksA2) and relA/spoT have roles in regulation of expression of the flavohemoglobin Hmp and in biofilm formation. IMPORTANCE Paracoccus denitrificans is a metabolically versatile Gram-negative bacterium that is used as a model for studies of respiratory metabolism. The organism can utilize nitrate as an electron acceptor for anaerobic respiration, reducing it to dinitrogen via nitrite, nitric oxide, and nitrous oxide. This pathway (known as denitrification) is important as a route for loss of fixed nitrogen from soil and as a source of the greenhouse gas nitrous oxide. Thus, it is important to understand those environmental and genetic factors that govern flux through the denitrification pathway. Here, we identify four proteins and a small molecule (ppGpp) which function as previously unknown regulators of expression of enzymes that reduce nitrate and oxidize nitric oxide.

Protein-Ligand Interactions in Scarcity: The Stringent Response from Bacteria to Metazoa, and the Unanswered Questions.

The stringent response, originally identified in Escherichia coli as a signal that leads to reprogramming of gene expression under starvation or nutrient deprivation, is now recognized as ubiquitous in all bacteria, and also as part of a broader survival strategy in diverse, other stress conditions. Much of our insight into this phenomenon derives from the role of hyperphosphorylated guanosine derivatives (pppGpp, ppGpp, pGpp; guanosine penta-, tetra- and tri-phosphate, respectively) that are synthesized on starvation cues and act as messengers or alarmones. These molecules, collectively referred to here as (p)ppGpp, orchestrate a complex network of biochemical steps that eventually lead to the repression of stable RNA synthesis, growth, and cell division, while promoting amino acid biosynthesis, survival, persistence, and virulence. In this analytical review, we summarize the mechanism of the major signaling pathways in the stringent response, consisting of the synthesis of the (p)ppGpp, their interaction with RNA polymerase, and diverse factors of macromolecular biosynthesis, leading to differential inhibition and activation of specific promoters. We also briefly touch upon the recently reported stringent-like response in a few eukaryotes, which is a very disparate mechanism involving MESH1 (Metazoan SpoT Homolog 1), a cytosolic NADPH phosphatase. Lastly, using ppGpp as an example, we speculate on possible pathways of simultaneous evolution of alarmones and their multiple targets.

Similar and Divergent Roles of Stringent Regulator (p)ppGpp and DksA on Pleiotropic Phenotype of Yersinia enterocolitica.

Stringent response plays an important role in the response of Enterobacteriaceae pathogens to rapid environmental changes. It has been shown that synergistic and antagonistic actions exist between the signaling molecules (p)ppGpp and DksA in several foodborne pathogens; however, the biological function of these molecules and their interactions in Yersinia are still unclear. This study systematically investigated the role of stringent response in Yersinia enterocolitica, a typical foodborne Enterobacteriaceae pathogen, by deleting the (p)ppGpp and DksA biosynthesis genes. (p)ppGpp and DksA copositively regulated most phenotypes, such as motility, antibiotic resistance, and tolerance to oxidative stress, whereas they exhibited independent and/or divergent roles in the growth and biofilm synthesis of Y. enterocolitica. Gene expression analysis revealed that (p)ppGpp- and DksA-deficiency reduced the transcription of flagellar synthesis genes (fliC and flgD) and biofilm synthesis genes (bssS and hmsHFRS), which could potentially contribute to changes in motility and biofilm formation. These results indicate that stringent response regulators (p)ppGpp and DksA have a synergistic role and independent or even completely opposite biological functions in regulating genes and phenotypes of Y. enterocolitica. Our findings revealed the biofunctional relationships between (p)ppGpp and DksA and the underlying molecular mechanisms in the regulation of the pathogenic phenotype of Y. enterocolitica. IMPORTANCE The synergetic actions between the stringent response signaling molecules, (p)ppGpp and DksA, have been widely reported. However, recent transcriptomic and phenotypic studies have suggested that independent or even opposite actions exist between them. In this study, we demonstrated that the knockout of (p)ppGpp and DksA affects the polymorphic phenotype of Yersinia enterocolitica. Although most of the tested phenotypes, such as motility, antibiotic resistance, and tolerance to oxidative stress, were copositively regulated by (p)ppGpp and DksA, it also showed inconsistencies in biofilm formation ability as well as some independent phenotypes. This study deepens our understanding of the strategies of foodborne pathogens to survive in complex environments, so as to provide theoretical basis for the control and treatment of these microorganisms.

Bacterial conversion of a host weapon into a nutritional signal.

Bacteria engulfed by phagocytic cells must resist oxidation damage and adapt to cellular hypoxia, but the mechanisms involved in this process are not completely elucidated. Recent work by Kim et al. in the Journal of Biological Chemistry investigated how the intracellular pathogen Salmonella enterica activates gene expression required to counteract oxidative damage. The authors show that this bacterium utilizes host oxidative molecules to activate regulatory proteins that enhance the production of effector molecules, counteracting the host weapon NADPH oxidase and inducing a protective response.

Oxidative stress activates transcription of Salmonella pathogenicity island-2 genes in macrophages.

The type III secretion system encoded in the Salmonella pathogenicity island-2 (SPI-2) gene cluster facilitates intracellular growth of nontyphoidal Salmonella by interfering with the maturation of Salmonella-containing vacuoles along the degradative pathway. SPI-2 gene products also protect Salmonella against the antimicrobial activity of reactive oxygen species (ROS) synthesized by the phagocyte NADPH oxidase 2 (NOX2). However, a potential relationship between inflammatory ROS and the activation of transcription of SPI-2 genes by intracellular Salmonella is unclear. Here, we show that ROS engendered in the innate host response stimulate SPI-2 gene transcription. We found that the expression of SPI-2 genes in Salmonella-sustaining oxidative stress conditions involves DksA, a protein otherwise known to regulate the stringent response of bacteria to nutritional stress. We also demonstrate that the J and zinc-2-oxidoreductase domains of DnaJ as well as the ATPase activity of the DnaK chaperone facilitate loading of DksA onto RNA polymerase complexed with SPI-2 promoters. Furthermore, the DksA-driven transcription of SPI-2 genes in Salmonella experiencing oxidative stress is contingent on upstream OmpR, PhoP, and SsrB signaling events that participate in the removal of nucleoid proteins while simultaneously recruiting RNA polymerase to SPI-2 promoter regions. Taken together, our results suggest the activation of SPI-2 gene transcription in Salmonella subjected to ROS produced by the respiratory burst of macrophages protects this intracellular pathogen against NOX2-mediated killing. We propose that Salmonella have co-opted inflammatory ROS to induce SPI-2-mediated protective responses against NOX2 host defenses.

Suppressor mutations in Escherichia coli RNA polymerase alter transcription initiation but do not affect translesion RNA synthesis in vitro.

Bacterial RNA polymerase (RNAP) coordinates transcription with DNA repair and replication. Many RNAP mutations have pleiotropic phenotypes with profound effects on transcription-coupled processes. One class of RNAP mutations (rpo∗) has been shown to suppress mutations in regulatory factors responsible for changes in gene expression during stationary phase or starvation, as well as in factors involved in the restoration of replication forks after DNA damage. These mutations were suggested to affect the ability of RNAP to transcribe damaged DNA and to decrease the stability of transcription complexes, thus facilitating their dislodging during DNA replication and repair, although this was not explicitly demonstrated. Here, we obtained nine mutations of this class located around the DNA/RNA binding cleft of Escherichia coli RNAP and analyzed their transcription properties in vitro. We found that these mutations decreased promoter complex stability to varying degrees, and all decreased the activity of rRNA promoters. However, they did not have strong effects on elongation complex stability. Some mutations were shown to stimulate transcriptional pauses or decrease intrinsic RNA cleavage by RNAP, but none altered the ability of RNAP to transcribe DNA templates containing damaged nucleotides. Thus, we conclude that the suppressor phenotypes of the mutations are unlikely to result from direct effects on DNA lesion recognition by RNAP but may be primarily explained by changes in transcription initiation. Further analysis of the effects of these mutations on the genomic distribution of RNAP and its interactions with regulatory factors will be essential for understanding their diverse phenotypes in vivo.

Homologs of the Escherichia coli F Element Protein TraR, Including Phage Lambda Orf73, Directly Reprogram Host Transcription.

Bacterial cells and their associated plasmids and bacteriophages encode numerous small proteins of unknown function. One example, the 73-amino-acid protein TraR, is encoded by the transfer operon of the conjugative F plasmid of Escherichia coli. TraR is a distant homolog of DksA, a protein found in almost all proteobacterial species that is required for ppGpp to regulate transcription during the stringent response. TraR and DksA increase or decrease transcription initiation depending on the kinetic features of the promoter by binding directly to RNA polymerase without binding to DNA. Unlike DksA, whose full activity requires ppGpp as a cofactor, TraR is fully active by itself and unaffected by ppGpp. TraR belongs to a family of divergent proteins encoded by proteobacterial bacteriophages and other mobile elements. Here, we experimentally addressed whether other members of the TraR family function like the F element-encoded TraR. Purified TraR and all 5 homologs that were examined bound to RNA polymerase, functioned at lower concentrations than DksA, and complemented a dksA-null strain for growth on minimal medium. One of the homologs, λ Orf73, encoded by bacteriophage lambda, was examined in greater detail. λ Orf73 slowed host growth and increased phage burst size. Mutational analysis suggested that λ Orf73 and TraR have a similar mechanism for inhibiting rRNA and r-protein promoters. We suggest that TraR and its homologs regulate host transcription to divert cellular resources to phage propagation or conjugation without induction of ppGpp and a stringent response. IMPORTANCE TraR is a distant homolog of the transcription factor DksA and the founding member of a large family of small proteins encoded by proteobacterial phages and conjugative plasmids. Reprogramming transcription during the stringent response requires the interaction of DksA not only with RNA polymerase but also with the stress-induced regulatory nucleotide ppGpp. We show here that five phage TraR homologs by themselves, without ppGpp, regulate transcription of host promoters, mimicking the effects of DksA and ppGpp together. During a stringent response, ppGpp independently binds directly to, and inhibits the activities of, many proteins in addition to RNA polymerase, including translation factors, enzymes needed for ribonucleotide biosynthesis, and other metabolic enzymes. Here, we suggest a physiological role for TraR-like proteins: bacteriophages utilize TraR homologs to reprogram host transcription in the absence of ppGpp induction and thus without inhibiting host enzymes needed for phage development.

The ancestral stringent response potentiator, DksA has been adapted throughout Salmonella evolution to orchestrate the expression of metabolic, motility, and virulence pathways.

DksA is a conserved RNA polymerase-binding protein known to play a key role in the stringent response of proteobacteria species, including many gastrointestinal pathogens. Here, we used RNA-sequencing of Escherichia coli, Salmonella bongori and Salmonella enterica serovar Typhimurium, together with phenotypic comparison to study changes in the DksA regulon, during Salmonella evolution. Comparative RNA-sequencing showed that under non-starved conditions, DksA controls the expression of 25%, 15%, and 20% of the E. coli, S. bongori, and S. enterica genes, respectively, indicating that DksA is a pleiotropic regulator, expanding its role beyond the canonical stringent response. We demonstrate that DksA is required for the growth of these three enteric bacteria species in minimal medium and controls the expression of the TCA cycle, glycolysis, pyrimidine biosynthesis, and quorum sensing. Interestingly, at multiple steps during Salmonella evolution, the type I fimbriae and various virulence genes encoded within SPIs 1, 2, 4, 5, and 11 have been transcriptionally integrated under the ancestral DksA regulon. Consequently, we show that DksA is necessary for host cells invasion by S. Typhimurium and S. bongori and for intracellular survival of S. Typhimurium in bone marrow-derived macrophages (BMDM). Moreover, we demonstrate regulatory inversion of the conserved motility-chemotaxis regulon by DksA, which acts as a negative regulator in E. coli, but activates this pathway in S. bongori and S. enterica. Overall, this study demonstrates the regulatory assimilation of multiple horizontally acquired virulence genes under the DksA regulon and provides new insights into the evolution of virulence genes regulation in Salmonella spp.

DksA Modulates Antimicrobial Susceptibility of Acinetobacter baumannii.

The stringent response regulators, (p)ppGpp and DksA, modulate various genes involved in physiological processes, virulence, and antimicrobial resistance in pathogenic bacteria. This study investigated the role of DksA in the antimicrobial susceptibility of Acinetobacter baumannii. The ∆dksA mutant (KM0248D) of A. baumannii ATCC 17978 and its complemented strain (KM0248C) were used, in addition to the ∆dksA mutant strain (NY0298D) of clinical 1656-2 strain. The microdilution assay was used to determine the minimum inhibitory concentrations (MICs) of antimicrobial agents. Quantitative real-time PCR was performed to analyze the expression of genes associated with efflux pumps. The KM0248D strain exhibited an increase of MICs to quinolones and tetracyclines, whereas KM0248D and NY0298D strains exhibited a decrease of MICs to aminoglycosides. The expression of genes associated with efflux pumps, including adeB, adeI/J, abeM, and/or tetA, was upregulated in both ∆dksA mutant strains. The deletion of dksA altered bacterial morphology in the clinical 1656-2 strain. In conclusion, DksA modulates the antimicrobial susceptibility of A. baumannii. The ∆dksA mutant strains of A. baumannii upregulate efflux pump gene expression, whereas (p)ppGpp-deficient mutants downregulate efflux pump gene expression. (p)ppGpp and DksA conduct opposite roles in the antimicrobial susceptibility of A. baumannii via efflux pump gene regulation.

Global regulator DksA modulates virulence of Acinetobacter baumannii.

DksA with (p)ppGpp regulates a wide range of gene transcriptions during the stringent response. The aim of this study was to identify a DksA ortholog in Acinetobacter baumannii and clarify the roles of DksA in bacterial physiology and virulence. The ∆dksA mutant and its complemented strains were constructed using A. baumannii ATCC 17978. The AlS_0248 in A. baumannii ATCC 17978 was identified to dksA using sequence homology, protein structure prediction, and gene expression patterns under different culture conditions. The ∆dksA mutant strain showed a filamentous morphology compared with the wild-type (WT) strain. Bacterial growth was decreased in the ∆dksA mutant strain under static conditions. Surface motility was decreased in the ∆dksA mutant strain compared with the WT strain. In contrast, biofilm formation was increased and biofilm-associated genes, such as bfmR/S and csuC/D/E, were upregulated in the ∆dksA mutant strain. The ∆dksA mutant strain produced less autoinducers than the WT strain. The expression of abaI and abaR was significantly decreased in the ∆dksA mutant strain. Furthermore, the ∆dksA mutant strain showed less bacterial burden and milder histopathological changes in the lungs of mice than the WT strain. Mice survival was also significantly different between the ∆dksA mutant and WT strains. Conclusively, DksA is directly or indirectly involved in regulating a wide range of genes associated with bacterial physiology and virulence, which contributes to the pathogenesis of A. baumannii. Thus, DksA is a potential anti-virulence target for A. baumannii infection.

Virus-Host Interaction Gets Curiouser and Curiouser. PART II: Functional Transcriptomics of the E. coli DksA-Deficient Cell upon Phage P1vir Infection.

The virus-host interaction requires a complex interplay between the phage strategy of reprogramming the host machinery to produce and release progeny virions, and the host defense against infection. Using RNA sequencing, we investigated the phage-host interaction to resolve the phenomenon of improved lytic development of P1vir phage in a DksA-deficient E. coli host. Expression of the ant1 and kilA P1vir genes in the wild-type host was the highest among all and most probably leads to phage virulence. Interestingly, in a DksA-deficient host, P1vir genes encoding lysozyme and holin are downregulated, while antiholins are upregulated. Gene expression of RepA, a protein necessary for replication initiating at the phage oriR region, is increased in the dksA mutant; this is also true for phage genes responsible for viral morphogenesis and architecture. Still, it seems that P1vir is taking control of the bacterial protein, sugar, and lipid metabolism in both, the wild type and dksA- hosts. Generally, bacterial hosts are reacting by activating their SOS response or upregulating the heat shock proteins. However, only DksA-deficient cells upregulate their sulfur metabolism and downregulate proteolysis upon P1vir infection. We conclude that P1vir development is enhanced in the dksA mutant due to several improvements, including replication and virion assembly, as well as a less efficient lysis.

Virus-Host Interaction Gets Curiouser and Curiouser. PART I: Phage P1vir Enhanced Development in an E. coli DksA-Deficient Cell.

Bacteriophage P1 is among the best described bacterial viruses used in molecular biology. Here, we report that deficiency in the host cell DksA protein, an E. coli global transcription regulator, improves P1 lytic development. Using genetic and microbiological approaches, we investigated several aspects of P1vir biology in an attempt to understand the basis of this phenomenon. We found several minor improvements in phage development in the dksA mutant host, including more efficient adsorption to bacterial cell and phage DNA replication. In addition, gene expression of the main repressor of lysogeny C1, the late promoter activator Lpa, and lysozyme are downregulated in the dksA mutant. We also found nucleotide substitutions located in the phage immunity region immI, which may be responsible for permanent virulence of phage P1vir. We suggest that downregulation of C1 may lead to a less effective repression of lysogeny maintaining genes and that P1vir may be balancing between lysis and lysogeny, although finally it is able to enter the lytic pathway only. The mentioned improvements, such as more efficient replication and more "gentle" cell lysis, while considered minor individually, together may account for the phenomenon of a more efficient P1 phage development in a DksA-deficient host.

The DnaK/DnaJ Chaperone System Enables RNA Polymerase-DksA Complex Formation in Salmonella Experiencing Oxidative Stress.

Our previous biochemical approaches showed that the oxidoreductase activity of the DnaJ protein facilitates the interaction of oxidized DksA with RNA polymerase. Investigations herein demonstrate that under biologically relevant conditions the DnaJ- and DksA-codependent activation of the stringent response in Salmonella undergoing oxidative stress involves the DnaK chaperone. Oxidation of DksA cysteine residues stimulates redox-based and holdase interactions with zinc-binding and C-terminal domains of DnaJ. Genetic and biochemical evidence indicates that His33 in the HPD motif in the J domain of DnaJ facilitates interactions of unfolded DksA with DnaK. A mutation in His33 in the J domain prevents the presentation of unfolded DksA to DnaK without limiting the oxidoreductase activity mapped to DnaJ's zinc-2 site. Thr199 in the ATPase catalytic site of DnaK is required for the formation of the DksA/RNA polymerase complex. The DnaK/DnaJ/DksA complex enables the formation of an enzymatically active RNA polymerase holoenzyme that stimulates transcription of branched-chain amino acid and histidine metabolic genes in Salmonella exposed to reactive oxygen species. The DnaK/DnaJ chaperone protects Salmonella against the cytotoxicity associated with reactive oxygen species generated by the phagocyte NADPH oxidase in the innate host response. The antioxidant defenses associated with DnaK/DnaJ can in part be ascribed to the elicitation of the DksA-dependent stringent response and the protection this chaperone system provides against protein carbonylation in Salmonella undergoing oxidative stress.IMPORTANCE DksA was discovered 30 years ago in a screen for suppressors that reversed the thermosensitivity of Escherichia coli mutant strains deficient in DnaK/DnaJ, raising the possibility that this chaperone system may control DksA function. Since its serendipitous discovery, DksA has emerged as a key activator of the transcriptional program called the stringent response in Gram-negative bacteria experiencing diverse adverse conditions, including nutritional starvation or oxidative stress. DksA activates the stringent response through the allosteric control this regulatory protein exerts on the kinetics of RNA polymerase promoter open complexes. Recent investigations have shown that DksA overexpression protects dnaKJ mutant bacteria against heat shock indirectly via the ancestral chaperone polyphosphate, casting doubt on a possible complexation of DnaK, DnaJ, and DksA. Nonetheless, research presented herein demonstrates that the cochaperones DnaK and DnaJ enable DksA/RNA polymerase complex formation in response to oxidative stress.

Chemical inhibitors of the conserved bacterial transcriptional regulator DksA1 suppressed quorum sensing-mediated virulence of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen whose virulence is dependent on quorum sensing (QS). DksA1, an RNA polymerase-binding transcriptional regulator, plays a role in determining a number of phenotypes, including QS-mediated virulence. We therefore envisioned that DksA1 inhibitors may help to control P. aeruginosa infection. Here, we screened a library of 6970 chemical compounds and identified two compounds (henceforth termed Dkstatins) that specifically suppressed DksA1 activity. Treatment with these two compounds also substantially decreased the production of elastase and pyocyanin, dominant virulence determinants of P. aeruginosa, and protected murine hosts from lethal infection from a prototype strain of P. aeruginosa, PAO1. The Dkstatins also suppressed production of homoserine lactone (HSL)-based autoinducers that activate P. aeruginosa QS. The level of 3-oxo-C12-HSL produced by Dkstatin-treated wildtype PAO1 closely resembled that of the ΔdksA1 mutant. RNA-Seq analysis showed that transcription levels of QS- and virulence-associated genes were markedly reduced in Dkstatin-treated PAO1 cells, indicating that Dkstatin-mediated suppression occurs at the transcriptional level. Importantly, Dkstatins increased the antibiotic susceptibilities of PAO1, particularly to protein synthesis inhibitors, such as tobramycin and tetracycline. Co-immunoprecipitation assays demonstrated that these Dkstatins interfered with DksA1 binding to the ß subunit of RNA polymerase, pointing to a potential mechanism of action. Collectively, our results illustrate that inhibition of P. aeruginosa QS may be achieved via DksA1 inhibitors and that Dkstatins may serve as potential lead compounds to control infection.

Universal functions of the σ finger in alternative σ factors during transcription initiation by bacterial RNA polymerase.

The bacterial σ factor plays the central role in promoter recognition by RNA polymerase (RNAP). The primary σ factor, involved in transcription of housekeeping genes, was also shown to participate in the initiation of RNA synthesis and promoter escape by RNAP. In the open promoter complex, the σ finger formed by σ region 3.2 directly interacts with the template DNA strand upstream of the transcription start site. Here, we analysed the role of the σ finger in transcription initiation by four alternative σ factors in Escherichia coli, σ38, σ32, σ28 and σ24. We found that deletions of the σ finger to various extent compromise the activity of RNAP holoenzymes containing alternative σ factors, especially at low NTP concentrations. All four σs are able to utilize NADH as a noncanonical priming substrate but it has only mild effects on the efficiency of transcription initiation. The mediators of the stringent response, transcription factor DksA and the alarmone ppGpp decrease RNAP activity and promoter complex stability for all four σ factors on tested promoters. For all σs except σ38, deletions of the σ finger conversely increase the stability of promoter complexes and decrease their sensitivity to DksA and ppGpp. The result suggests that the σ finger plays a universal role in transcription initiation by alternative σ factors and sensitizes promoter complexes to the action of global transcription regulators DksA and ppGpp by modulating promoter complex stability.

DksA coordinates bile-mediated regulation of virulence-associated phenotypes in type three secretion system-positive Vibrio cholerae.

In order to cause disease, pathogenic strains of Vibrio cholerae rely on intricate regulatory networks to orchestrate the transition between their native aquatic environment and the human host. For example, bacteria in a nutrient-starved environment undergo a metabolic shift called the stringent response, which is mediated by the alarmone ppGpp and an RNA-polymerase binding transcriptional factor, DksA. In O1 serogroup strains of V. cholerae, which use the toxin co-regulated pilus (TCP) and cholera toxin (CT) as primary virulence factors, DksA was reported to have additional functions as a mediator of virulence gene expression. However, little is known about the regulatory networks coordinating virulence phenotypes in pathogenic strains that use TCP/CT-independent virulence mechanisms. We therefore investigated whether functions of DksA outside of the stringent response are conserved in type three secretion system (T3SS)-positive V. cholerae. In using the T3SS-positive clinically isolated O39 serogroup strain AM-19226, we observed an increase in dksA expression in the presence of bile at 37 °C. However, DksA was not required for wild-type levels of T3SS structural gene expression, or for colonization in vivo. Rather, data indicate that DksA positively regulates the expression of master regulators in the motility hierarchy. Interestingly, the ΔdksA strain forms a less robust biofilm than the WT parent strain at both 30 and 37 °C. We also found that DksA regulates the expression of hapR, encoding a major regulator of biofilm formation and protease expression. Athough DksA does not appear to modulate T3SS virulence factor expression, its activity is integrated into existing regulatory networks governing virulence-related phenotypes. Strain variations therefore may take advantage of conserved ancestral proteins to expand regulons responding to in vivo signals and thus coordinate multiple phenotypes important for infection.

Identification of Natural Mutations Responsible for Altered Infection Phenotypes of Salmonella enterica Clinical Isolates by Using Cell Line Infection Screens.

The initial steps of Salmonella pathogenesis involve adhesion to and invasion into host epithelial cells. While well-studied for Salmonella enterica serovar Typhimurium, the factors contributing to this process in other, host-adapted serovars remains unexplored. Here, we screened clinical isolates of serovars Gallinarum, Dublin, Choleraesuis, Typhimurium, and Enteritidis for adhesion to and invasion into intestinal epithelial cell lines of human, porcine, and chicken origins. Thirty isolates with altered infectivity were used for genomic analyses, and 14 genes and novel mutations associated with high or low infectivity were identified. The functions of candidate genes included virulence gene expression regulation and cell wall or membrane synthesis and components. The role of several of these genes in Salmonella adhesion to and invasion into cells has not previously been investigated. The genes dksA (encoding a stringent response regulator) and sanA (encoding a vancomycin high-temperature exclusion protein) were selected for further analyses, and we confirmed their roles in adhesion to and invasion into host cells. Furthermore, transcriptomic analyses were performed for S Enteritidis and S Typhimurium, with two highly infective and two marginally infective isolates for each serovar. Expression profiles for the isolates with altered infection phenotypes revealed the importance of type 3 secretion system expression levels in the determination of an isolate's infection phenotype. Taken together, these data indicate a new role in cell host infection for genes or gene variants previously not associated with adhesion to and invasion into the epithelial cells.IMPORTANCESalmonella is a foodborne pathogen affecting over 200 million people and resulting in over 200,000 fatal cases per year. Its adhesion to and invasion into intestinal epithelial cells represent one of the first and key steps in the pathogenesis of salmonellosis. Still, around 35 to 40% of bacterial genes have no experimentally validated function, and their contribution to bacterial virulence, including adhesion and invasion, remains largely unknown. Therefore, the significance of this study is in the identification of new genes or gene allelic variants previously not associated with adhesion and invasion. It is well established that blocking adhesion and/or invasion would stop or hamper bacterial infection; therefore, the new findings from this study could be used in future developments of anti-Salmonella therapy targeting genes involved in these key processes. Such treatment could be a valuable alternative, as the prevalence of antibiotic-resistant bacteria is increasing very rapidly.