RpoN1 and RpoN2 play different regulatory roles in virulence traits, flagellar biosynthesis, and basal metabolism in Xanthomonas campestris.

Homologous regulatory factors are widely present in bacteria, but whether homologous regulators synergistically or differentially regulate different biological functions remains mostly unknown. Here, we report that the homologous regulators RpoN1 and RpoN2 of the plant pathogen Xanthomonas campestris pv. campestris (Xcc) play different regulatory roles with respect to virulence traits, flagellar biosynthesis, and basal metabolism. RpoN2 directly regulated Xcc fliC and fliQ to modulate flagellar synthesis in X. campestris, thus affecting the swimming motility of X. campestris. Mutation of rpoN2 resulted in reduced production of biofilms and extracellular polysaccharides in Xcc. These defects may together cause reduced virulence of the rpoN2 mutant against the host plant. Moreover, we demonstrated that RpoN1 could regulate branched-chain fatty acid production and modulate the synthesis of diffusible signal factor family quorum sensing signals. Although RpoN1 and RpoN2 are homologues, the regulatory roles and biological functions of these proteins were not interchangeable. Overall, our report provides new insights into the two different molecular roles that form the basis for the transcriptional specialization of RpoN homologues.

Bacillus cereus ATCC 14579 RpoN (Sigma 54) Is a Pleiotropic Regulator of Growth, Carbohydrate Metabolism, Motility, Biofilm Formation and Toxin Production.

Sigma 54 is a transcriptional regulator predicted to play a role in physical interaction of bacteria with their environment, including virulence and biofilm formation. In order to study the role of Sigma 54 in Bacillus cereus, a comparative transcriptome and phenotypic study was performed using B. cereus ATCC 14579 WT, a markerless rpoN deletion mutant, and its complemented strain. The mutant was impaired in many different cellular functions including low temperature and anaerobic growth, carbohydrate metabolism, sporulation and toxin production. Additionally, the mutant showed lack of motility and biofilm formation at air-liquid interphase, and this correlated with absence of flagella, as flagella staining showed only WT and complemented strain to be highly flagellated. Comparative transcriptome analysis of cells harvested at selected time points during growth in aerated and static conditions in BHI revealed large differences in gene expression associated with loss of phenotypes, including significant down regulation of genes in the mutant encoding enzymes involved in degradation of branched chain amino acids, carbohydrate transport and metabolism, flagella synthesis and virulence factors. Our study provides evidence for a pleiotropic role of Sigma 54 in B. cereus supporting its adaptive response and survival in a range of conditions and environments.

BosR (BB0647) controls the RpoN-RpoS regulatory pathway and virulence expression in Borrelia burgdorferi by a novel DNA-binding mechanism.

In Borrelia burgdorferi (Bb), the Lyme disease spirochete, the alternative σ factor σ54 (RpoN) directly activates transcription of another alternative σ factor, σ(S) (RpoS) which, in turn, controls the expression of virulence-associated membrane lipoproteins. As is customary in σ54-dependent gene control, a putative NtrC-like enhancer-binding protein, Rrp2, is required to activate the RpoN-RpoS pathway. However, recently it was found that rpoS transcription in Bb also requires another regulator, BosR, which was previously designated as a Fur or PerR homolog. Given this unexpected requirement for a second activator to promote σ54-dependent gene transcription, and the fact that regulatory mechanisms among similar species of pathogenic bacteria can be strain-specific, we sought to confirm the regulatory role of BosR in a second virulent strain (strain 297) of Bb. Indeed, BosR displayed the same influence over lipoprotein expression and mammalian infectivity for strain Bb 297 that were previously noted for Bb strain B31. We subsequently found that recombinant BosR (rBosR) bound to the rpoS gene at three distinct sites, and that binding occurred despite the absence of consensus Fur or Per boxes. This led to the identification of a novel direct repeat sequence (TAAATTAAAT) critical for rBosR binding in vitro. Mutations in the repeat sequence markedly inhibited or abolished rBosR binding. Taken together, our studies provide new mechanistic insights into how BosR likely acts directly on rpoS as a positive transcriptional activator. Additional novelty is engendered by the facts that, although BosR is a Fur or PerR homolog and it contains zinc (like Fur and PerR), it has other unique features that clearly set it apart from these other regulators. Our findings also have broader implications regarding a previously unappreciated layer of control that can be involved in σ54-dependent gene regulation in bacteria.

Expression of Kingella kingae type IV pili is regulated by sigma54, PilS, and PilR.

Kingella kingae is a member of the Neisseriaceae and is being recognized increasingly as an important cause of serious disease in children. Recent work has demonstrated that K. kingae expresses type IV pili that mediate adherence to respiratory epithelial and synovial cells and are selected against during invasive disease. In the current study, we examined the genome of K. kingae strain 269-492 and identified homologs of the rpoN and the pilS and pilR genes that are essential for pilus expression in Pseudomonas aeruginosa but not in the pathogenic Neisseria species. The disruption of either rpoN or pilR in K. kingae resulted in a marked reduction in the level of transcript for the major pilus subunit (pilA1) and eliminated piliation. In contrast, the disruption of pilS resulted in only partial reduction in the level of pilA1 transcript and a partial decrease in piliation. Furthermore, the disruption of pilS in colony variants with high-density piliation resulted in variants with low-density piliation. Mutations in the promoter region of pilA1 and gel shift analysis demonstrated that both sigma(54) and PilR act directly at the pilA1 promoter, with PilR binding to two repetitive elements. These data suggest that the regulation of K. kingae type IV pilus expression is complex and multilayered, influenced by both the genetic state and environmental cues.

Characterizing the regulation of the Pu promoter in Acinetobacter baylyi ADP1.

Effective gene trapping and screening requires sensory and regulatory compatibility of both host and exogenous systems. The naturally competent bacterium Acinetobacter baylyi ADP1 is able to efficiently take up and integrate exogenous DNA into the chromosome, making it an attractive host system for a wide range of metagenomic applications. To test the ability of A. baylyi ADP1 to express the XylR-regulated Pu promoter from Pseudomonas putida mt-2, we have constructed and examined an A. baylyi ADP1 strain, ADPWH-Pu-lux-xylR. The Pu promoter in ADPWH-Pu-lux-xylR was specifically induced by toluene, m-, p- and o-xylene. The substrate-induced Pu promoter was highly dependent on the growth medium: it was repressed in rich media until stationary phase, but was immediately induced in minimal medium with glucose as the sole carbon source (MMG). However, the Pu promoter was repressed in MMG when it was supplemented with 5 g l(-1) yeast extract. Further investigation showed that the Pu promoter in MMG was repressed by 0.5 g l(-1) aspartic acid or asparagine, but not repressed by glutamine. Changing the carbon/nitrogen ratios by addition of ammonia did not significantly affect the Pu promoter activity but addition of nitrate did. These results show that A. baylyi ADP1 reproduced characteristics of the XylR-regulated Pu promoter observed in its original host. It demonstrates that A. baylyi could provide an excellent genetic host for a wide range of functional metagenomic applications.

Role of NtrC in biofilm formation via controlling expression of the gene encoding an ADP-glycero-manno-heptose-6-epimerase in the pathogenic bacterium, Vibrio vulnificus.

To identify the genetic elements required for biofilm formation, we screened a pool of random Vibrio vulnificus mutants for their ability to form biofilms. One mutant displaying significantly decreased biofilm-forming activity was found to contain a transposon insertion in the ntrC gene. The ntrC gene encodes a well-known transcriptional activator. We examined how this regulator modulates a biofilm-forming process in V. vulnificus by searching for NtrC target gene(s). Comparison of the proteomes of ntrC mutant and wild-type strains grown under planktonic and biofilm stages revealed that synthesis of the protein homologous to GmhD (ADP-glycero-manno-heptose-6-epimerase) was elevated during the growth period for biofilm formation and was strongly influenced by NtrC. A luxAB-transcriptional fusion with the gmhD promoter region indicated that gmhD expression was positively regulated by both NtrC and RpoN. The function of the gmhD gene product in V. vulnificus was assessed by constructing and phenotypic analyses of an isogenic mutant. The gmhD mutant was defective in production of mature lipopolysaccharide (LPS) and exopolysaccharides (EPS), and demonstrated an attenuated ability to form a biofilm. These results suggest that NtrC acts as a key regulator of both LPS and EPS biosyntheses and, thereby, modulates critical steps in biofilm development of V. vulnificus.

rpoN gene of Pseudomonas aeruginosa alters its susceptibility to quinolones and carbapenems.

The alternative sigma factor sigma54 has been implicated in diverse functions within the cells. In this study, we have constructed an rpoN mutant of Pseudomonas aeruginosa and investigated its importance as a target for antimicrobial agents, such as quinolones and carbapenems. The stationary-phase cells of the rpoN mutant displayed a survival rate approximately 15 times higher than that of the wild-type cells in the presence of quinolones and carbapenems. The stationary phase led to substantial production of pyoverdine by the P. aeruginosa rpoN mutant. Pyoverdine synthesis correlated with decreased susceptibility to antimicrobial agents. Quantitative real-time PCR revealed that stationary-phase cells of the rpoN mutant grown without an antimicrobial agent had approximately 4- to 140- and 2- to 14-fold-higher levels of transcripts of the pvdS and vqsR genes, respectively, than the wild-type strain. In the presence of an antimicrobial agent, levels of pvdS and vqsR transcripts were elevated 400- and 5-fold, respectively, in comparison to the wild-type levels. Flow cytometry assays using a green fluorescent protein reporter demonstrated increased expression of the vqsR gene in the rpoN mutant throughout growth. A pvdS mutant of P. aeruginosa, deficient in pyoverdine production, was shown to be susceptible to biapenem. These findings suggest that rpoN is involved in tolerance to antimicrobial agents in P. aeruginosa and that its tolerant effect is partly dependent on increased pyoverdine production and vqsR gene expression.

Evidence that RpoS (sigmaS) in Borrelia burgdorferi is controlled directly by RpoN (sigma54/sigmaN).

The alternative sigma factor (RpoN-RpoS) pathway controls the expression of key virulence factors in Borrelia burgdorferi. However, evidence to support whether RpoN controls rpoS directly or, perhaps, indirectly via a transactivator has been lacking. Herein we provide biochemical and genetic evidence that RpoN directly controls rpoS in B. burgdorferi.

A sigma54-dependent promoter in the regulatory region of the Escherichia coli rpoH gene.

The Escherichia coli rpoH gene is transcribed from four known and differently regulated promoters: P1, P3, P4 and P5. This study demonstrates that the conserved consensus sequence of the sigma54 promoter in the regulatory region of the rpoH gene, described previously, is a functional promoter, P6. The evidence for this conclusion is: (i) the specific binding of the sigma54-RNAP holoenzyme to P6, (ii) the location of the transcription start site at the predicted position (C, 30 nt upstream of ATG) and (iii) the dependence of transcription on sigma54 and on an ATP-dependent activator. Nitrogen starvation, heat shock, ethanol and CCCP treatment did not activate transcription from P6 under the conditions examined. Two activators of sigma54 promoters, PspF and NtrC, were tested but neither of them acted specifically. Therefore, PspFDeltaHTH, a derivative of PspF, devoid of DNA binding capability but retaining its ATPase activity, was used for transcription in vitro, taking advantage of the relaxed specificity of ATP-dependent activators acting in solution. In experiments in vivo overexpression of PspFDeltaHTH from a plasmid was employed. Thus, the sigma54-dependent transcription capability of the P6 promoter was demonstrated both in vivo and in vitro, although the specific conditions inducing initiation of the transcription remain to be elucidated. The results clearly indicate that the closed sigma54-RNAP-promoter initiation complex was formed in vitro and in vivo and needed only an ATP-dependent activator to start transcription.

The sigma factor RpoN (sigma54) is involved in osmotolerance in Listeria monocytogenes.

Listeria monocytogenes is able to grow under conditions of high osmolarity. We constructed a deletion mutant of rpoN, encoding the alternative sigma factor RpoN, and analyzed its response to osmotic stress. In a minimal medium with 4% NaCl and 1 mM betaine, the mutant showed a similar growth to that of the parental strain, EGD. In the same medium with 4% NaCl and 1 M carnitine, the growth rate of the mutant was greatly reduced, when the optical density at 600 nm (OD600) at the starting point of growth, was 0.15. However, when growth of the culture was started at an OD600 of 0.025, the growth of the mutant was similar to that of EGD. The mutant's expression of two betaine transporter genes, betL and gbuB, and the carnitine transporter gene opuCA, was osmotically induced at a level similar to EGD, and its rate of carnitine uptake was similar to that of EGD. These results suggest that the growth defect from the rpoN mutant is caused not by the transcriptional regulation of opuCA or by a decrease in carnitine uptake, but possibly by larger amounts of carnitine being needed for growth of the mutant in minimal medium when NaCl is present.

Regulation of type IV fimbrial biogenesis in Dichelobacter nodosus.

Type IV fimbriae are expressed by several bacterial pathogens and are essential for virulence in Dichelobacter nodosus, which causes ovine footrot. We have identified a two-component signal transduction system (PilR/S) and an alternative sigma factor (sigma 54) that were shown by insertional inactivation to be required for the regulation of fimbrial biogenesis in D. nodosus. Western blots showed that in both pilR and rpoN mutants, fimbrial subunit production was significantly reduced by a process that was shown to occur at a PilR- and sigma 54-dependent promoter. The mutants lacked surface fimbriae, which were shown to be required for the adherence of D. nodosus cells to tissue culture monolayers. The reduction in fimbrial subunit production in these mutants also resulted in a concomitant loss of the ability to secrete extracellular proteases. A maltose binding protein-PilR fusion protein was purified and was shown to bind specifically to a region located 234 to 594 bp upstream of the fimA transcriptional start point. To determine additional targets of PilR and sigma 54, genome-wide transcriptional profiling was performed using a whole-genome oligonucleotide microarray. The results indicated that PilR and sigma 54 regulated genes other than fimA; these genes appear to encode surface-exposed proteins whose role in virulence is unknown. In conclusion, this study represents a significant advancement in our understanding of how the ability of D. nodosus to cause ovine footrot is regulated, as we have shown that the biogenesis of type IV fimbriae in D. nodosus is regulated by a sigma 54-dependent PilR/S system that also indirectly controls protease secretion.

Sigma L is important for cold shock adaptation of Bacillus subtilis.

Although sigma factor-dependent transcriptional regulation was shown to be essential for adaptation to different environmental stimuli, no such sigma factor has been related to the regulation of the cold shock response in Bacillus subtilis. In this study, we present genetic evidence for participation of sigma(L) (sigma(54)) and the two sigma(L)-dependent transcriptional enhancers BkdR and YplP in the cold shock response of Bacillus subtilis JH642. Single-gene deletion of either sigL, bkdR, or yplP resulted in a cold-sensitive phenotype.

The dctA gene of Pseudomonas chlororaphis O6 is under RpoN control and is required for effective root colonization and induction of systemic resistance.

Transcription from the dctA gene, which encodes an organic acid transporter in the root-colonizing bacterium Pseudomonas chlororaphis O6, is under complex regulatory control. Promoter sequence analysis revealed an RpoN binding site. The regulation of transcript accumulation by the level of ammonium ions in the growth medium confirmed RpoN regulation, even in the presence of glucose. A dctA mutant colonized tobacco roots to a lesser extent than the wild-type mutant during early seedling development. Colonization by the dctA mutant, as compared to the wild type, also reduced the level of systemically induced resistance against the soft rot pathogen Erwinia carotovora SCC1. We ascribe this reduced colonization to the inability of the mutant to utilize certain organic acid components in the root exudates. The dctA mutant failed to grow on succinate and fumarate, and showed reduced growth on malate. All altered properties of the mutant were complemented by the full-length dctA gene. We propose that organic acids in root exudates may provide important nutrient sources for the beneficial root-colonizing pseudomonad.

The flagellar hierarchy of Rhodobacter sphaeroides is controlled by the concerted action of two enhancer-binding proteins.

The expression of the bacterial flagellar genes follows a hierarchical pattern. In Rhodobacter sphaeroides the flagellar genes encoding the hook and basal body proteins are expressed from sigma54-dependent promoters. This type of promoters is always regulated by transcriptional activators that belong to the family of the enhancer-binding proteins (EBPs). We searched for possible EBPs in the genome of R. sphaeroides and mutagenized two open reading frames (ORFs) (fleQ and fleT), which are in the vicinity of flagellar genes. The resulting mutants were non-motile and could only be complemented by the wild-type copy of the mutagenized gene. Transcriptional fusions showed that all the flagellar sigma54-dependent promoters with exception of fleTp, required both transcriptional activators for their expression. Interestingly, transcription of the fleT operon is only dependent on FleQ, and FleT has a negative effect. Both activators were capable of hydrolysing ATP, and were capable of promoting transcription from the flagellar promoters at some extent. Electrophoretic mobility shift assays suggest that only FleQ interacts with DNA whereas FleT improves binding of FleQ to DNA. A four-tiered flagellar transcriptional hierarchy and a regulatory mechanism based on the intracellular concentration of both activators and differential enhancer affinities are proposed.