Factor RpoN Research Articles

BosR and PlzA reciprocally regulate RpoS function to sustain Borrelia burgdorferi in ticks and mammals.

The RNA polymerase alternative σ factor RpoS in Borrelia burgdorferi (Bb), the Lyme disease pathogen, is responsible for programmatic-positive and -negative gene regulation essential for the spirochete's dual-host enzootic cycle. RpoS is expressed during tick-to-mammal transmission and throughout mammalian infection. Although the mammalian-phase RpoS regulon is well described, its counterpart during the transmission blood meal is unknown. Here, we used Bb-specific transcript enrichment by tick-borne disease capture sequencing (TBDCapSeq) to compare the transcriptomes of WT and ΔrpoS Bb in engorged nymphs and following mammalian host-adaptation within dialysis membrane chambers. TBDCapSeq revealed dramatic changes in the contours of the RpoS regulon within ticks and mammals and further confirmed that RpoS-mediated repression is specific to the mammalian-phase of Bb's enzootic cycle. We also provide evidence that RpoS-dependent gene regulation, including repression of tick-phase genes, is required for persistence in mice. Comparative transcriptomics of engineered Bb strains revealed that the Borrelia oxidative stress response regulator (BosR), a noncanonical Fur family member, and the cyclic diguanosine monophosphate (c-di-GMP) effector PlzA reciprocally regulate the function of RNA polymerase complexed with RpoS. BosR is required for RpoS-mediated transcription activation and repression in addition to its well-defined role promoting transcription of rpoS by the RNA polymerase alternative σ factor RpoN. During transmission, ligand-bound PlzA antagonizes RpoS-mediated repression, presumably acting through BosR.

Transcriptome Analysis Revealed Overlapping and Special Regulatory Roles of RpoN1 and RpoN2 in Motility, Virulence, and Growth of Xanthomonas oryzae pv. oryzae

σ54 factor (RpoN) plays a crucial role in bacterial motility, virulence, growth, and other biological functions. In our previous study, two homologous σ54 factors, RpoN1 and RpoN2, were identified in Xanthomonas oryzae pv. oryzae (Xoo), the causative agent of bacterial leaf blight in rice. However, their functional roles, i.e., whether they exert combined or independent effects, remain unknown. In the current study, rpoN1 or rpoN2 deletion in Xoo significantly disrupted bacterial swimming motility, flagellar assembly, and virulence. Transcriptome analysis led to the identification of 127 overlapping differentially expressed genes (DEGs) regulated by both RpoN1 and RpoN2. Furthermore, GO and KEGG classification demonstrated that these DEGs were highly enriched in flagellar assembly, chemotaxis, and c-di-GMP pathways. Interestingly, ropN1 deletion decreased ropN2 transcription, while rpoN2 deletion did not affect ropN1 transcription. No interaction between the rpoN2 promoter and RpoN1 was detected, suggesting that RpoN1 indirectly regulates rpoN2 transcription. In addition, RpoN1-regulated DEGs were specially enriched in ribosome, carbon, and nitrogen metabolism pathways. Besides, bacterial growth was remarkably repressed in ΔrpoN1 but not in ΔrpoN2. Taken together, this study demonstrates the overlapping and unique regulatory roles of RpoN1 and RpoN2 in motility, virulence, growth and provides new insights into the regulatory mechanism of σ54 factors in Xoo.

The RpoS Gatekeeper in Borrelia burgdorferi: An Invariant Regulatory Scheme That Promotes Spirochete Persistence in Reservoir Hosts and Niche Diversity.

Maintenance of Borrelia burgdorferi within its enzootic cycle requires a complex regulatory pathway involving the alternative σ factors RpoN and RpoS and two ancillary trans-acting factors, BosR and Rrp2. Activation of this pathway occurs within ticks during the nymphal blood meal when RpoS, the effector σ factor, transcribes genes required for tick transmission and mammalian infection. RpoS also exerts a ‘gatekeeper’ function by repressing σ70-dependent tick phase genes (e.g., ospA, lp6.6). Herein, we undertook a broad examination of RpoS functionality throughout the enzootic cycle, beginning with modeling to confirm that this alternative σ factor is a ‘genuine’ RpoS homolog. Using a novel dual color reporter system, we established at the single spirochete level that ospA is expressed in nymphal midguts throughout transmission and is not downregulated until spirochetes have been transmitted to a naïve host. Although it is well established that rpoS/RpoS is expressed throughout infection, its requirement for persistent infection has not been demonstrated. Plasmid retention studies using a trans-complemented ΔrpoS mutant demonstrated that (i) RpoS is required for maximal fitness throughout the mammalian phase and (ii) RpoS represses tick phase genes until spirochetes are acquired by a naïve vector. By transposon mutant screening, we established that bba34/oppA5, the only OppA oligopeptide-binding protein controlled by RpoS, is a bona fide persistence gene. Lastly, comparison of the strain 297 and B31 RpoS DMC regulons identified two cohorts of RpoS-regulated genes. The first consists of highly conserved syntenic genes that are similarly regulated by RpoS in both strains and likely required for maintenance of B. burgdorferi sensu stricto strains in the wild. The second includes RpoS-regulated plasmid-encoded variable surface lipoproteins ospC, dbpA and members of the ospE/ospF/elp, mlp, revA, and Pfam54 paralogous gene families, all of which have evolved via inter- and intra-strain recombination. Thus, while the RpoN/RpoS pathway regulates a ‘core’ group of orthologous genes, diversity within RpoS regulons of different strains could be an important determinant of reservoir host range as well as spirochete virulence.

Enhanced cellular content and lactate fraction of the poly(lactate-co-3-hydroxybutyrate) polyester produced in recombinant Escherichia coli by the deletion of σ factor RpoN

A new approach at the transcriptional level was applied to lactate-based polyester production. Four σ factor disruptants, ΔrpoN, ΔrpoS, ΔfliA and ΔfecI, of Escherichia coli were used as hosts for poly(lactate-co-3-hydroxybutyrate) production from glucose. Among them, ΔrpoN caused dual positive effects of polymer production, enhanced cellular content and lactate fraction.

Metabolite Profiling to Characterize Disease-related Bacteria: GLUCONATE EXCRETION BY PSEUDOMONAS AERUGINOSA MUTANTS AND CLINICAL ISOLATES FROM CYSTIC FIBROSIS PATIENTS

Metabolic footprinting of supernatants has been proposed as a tool for assigning gene function. We used NMR spectroscopy to measure the exometabolome of 86 single-gene transposon insertion mutant strains (mutants from central carbon metabolism and regulatory mutants) of the opportunistic pathogen Pseudomonas aeruginosa, grown on a medium designed to represent the nutritional content of cystic fibrosis sputum. Functionally related genes had similar metabolic profiles. E.g. for two-component system mutants, the cognate response regulator and sensor kinase genes clustered tightly together. Some strains had metabolic phenotypes (metabotypes) that could be related to the known gene function. E.g. pyruvate dehydrogenase mutants accumulated large amounts of pyruvate in the medium. In other cases, the metabolic phenotypes were not easily interpretable. The rpoN mutant, which lacks the alternative σ factor RpoN (σ(54)), accumulated high levels of gluconate in the medium. In addition, endometabolome profiling of intracellular metabolites identified a number of systemic metabolic changes. We linked this to indirect regulation of the catabolite repression protein Crc via the non-coding RNA crcZ and found that a crcZ (but not crc) mutant also shared the high-gluconate phenotype. We profiled an additional set of relevant metabolic enzymes and transporters, including Crc targets, and showed that the Crc-regulated edd mutant (gluconate-6-phosphate dehydratase) had similar gluconate levels as the rpoN mutant. Finally, a set of clinical isolates showed patient- and random amplification of polymorphic DNA (RAPD) type-specific differences in gluconate production, which were associated significantly with resistance across four antibiotics (tobramycin, ciprofloxacin, aztreonam, and imipenem), indicating that this has potential clinical relevance.

The Pseudomonas aeruginosa Sensor Kinase KinB Negatively Controls Alginate Production through AlgW-Dependent MucA Proteolysis

Mucoidy, or overproduction of the exopolysaccharide known as alginate, in Pseudomonas aeruginosa is a poor prognosticator for lung infections in cystic fibrosis. Mutation of the anti-sigma factor MucA is a well-accepted mechanism for mucoid conversion. However, certain clinical mucoid strains of P. aeruginosa have a wild-type (wt) mucA. Here, we describe a loss-of-function mutation in kinB that causes overproduction of alginate in the wt mucA strain PAO1. KinB is the cognate histidine kinase for the transcriptional activator AlgB. Increased alginate production due to inactivation of kinB was correlated with high expression at the alginate-related promoters P(algU) and P(algD). Deletion of alternative sigma factor RpoN (sigma(54)) or the response regulator AlgB in kinB mutants decreased alginate production to wt nonmucoid levels. Mucoidy was restored in the kinB algB double mutant by expression of wt AlgB or phosphorylation-defective AlgB.D59N, indicating that phosphorylation of AlgB was not required for alginate overproduction when kinB was inactivated. The inactivation of the DegS-like protease AlgW in the kinB mutant caused loss of alginate production and an accumulation of the hemagglutinin (HA)-tagged MucA. Furthermore, we observed that the kinB mutation increased the rate of HA-MucA degradation. Our results also indicate that AlgW-mediated MucA degradation required algB and rpoN in the kinB mutant. Collectively, these studies indicate that KinB is a negative regulator of alginate production in wt mucA strain PAO1.

Posttranscriptional regulation of flagellin synthesis in Helicobacter pylori by the RpoN chaperone HP0958.

The Helicobacter pylori protein HP0958 is essential for flagellum biogenesis. It has been shown that HP0958 stabilizes the sigma(54) factor RpoN. The aim of this study was to further investigate the role of HP0958 in flagellum production in H. pylori. Global transcript analysis identified a number of flagellar genes that were differentially expressed in an HP0958 mutant strain. Among these, the transcription of the major flagellin gene flaA was upregulated twofold, suggesting that HP0958 was a negative regulator of the flaA gene. However, the production of the FlaA protein was significantly reduced in the HP0958 mutant, and this was not due to the decreased stability of the FlaA protein. RNA stability analysis and binding assays indicated that HP0958 binds and destabilizes flaA mRNA. The HP0958 mutant was successfully complemented, confirming that the mutant phenotype described was due to the lack of HP0958. We conclude that HP0958 is a posttranscriptional regulator that modulates the amount of the flaA message available for translation in H. pylori.