Repressor activity of the RpoS/σS-dependent RNA polymerase requires DNA binding.

The RpoS/σ(S) sigma subunit of RNA polymerase (RNAP) activates transcription of stationary phase genes in many Gram-negative bacteria and controls adaptive functions, including stress resistance, biofilm formation and virulence. In this study, we address an important but poorly understood aspect of σ(S)-dependent control, that of a repressor. Negative regulation by σ(S) has been proposed to result largely from competition between σ(S) and other σ factors for binding to a limited amount of core RNAP (E). To assess whether σ(S) binding to E alone results in significant downregulation of gene expression by other σ factors, we characterized an rpoS mutant of Salmonella enterica serovar Typhimurium producing a σ(S) protein proficient for Eσ(S) complex formation but deficient in promoter DNA binding. Genome expression profiling and physiological assays revealed that this mutant was defective for negative regulation, indicating that gene repression by σ(S) requires its binding to DNA. Although the mechanisms of repression by σ(S) are likely specific to individual genes and environmental conditions, the study of transcription downregulation of the succinate dehydrogenase operon suggests that σ competition at the promoter DNA level plays an important role in gene repression by Eσ(S).

Spacing requirements for Class I transcription activation in bacteria are set by promoter elements.

The Escherichia coli cAMP receptor protein (CRP) activates transcription initiation at many promoters by binding upstream of core promoter elements and interacting with the C-terminal domain of the RNA polymerase α subunit. Previous studies have shown stringent spacing is required for transcription activation by CRP. Here we report that this stringency can be altered by the nature of different promoter elements at target promoters. Several series of CRP-dependent promoters were constructed with CRP moved to different upstream locations, and their activities were measured. The results show that (i) a full UP element, located immediately downstream of the DNA site for CRP, relaxes the spacing requirements for activation and increases the recruitment of RNAP and open complex formation; (ii) the distal UP subsite plays the key role in this relaxation; (iii) modification of the extended -10 element also affects the spacing requirements for CRP-dependent activation. From these results, we conclude that the spacing requirements for CRP-dependent transcription activation vary according to the sequence of different promoter elements, and our results are important for understanding the organization of promoters in many different bacteria which are controlled by transcription factors that use activatory mechanisms similar to CRP.

Crl binds to domain 2 of σ(S) and confers a competitive advantage on a natural rpoS mutant of Salmonella enterica serovar Typhi.

The RpoS sigma factor (σ(S)) is the master regulator of the bacterial response to a variety of stresses. Mutants in rpoS arise in bacterial populations in the absence of stress, probably as a consequence of a subtle balance between self-preservation and nutritional competence. We characterized here one natural rpoS mutant of Salmonella enterica serovar Typhi (Ty19). We show that the rpoS allele of Ty19 (rpoS(Ty19)) led to the synthesis of a σ(S)(Ty19) protein carrying a single glycine-to-valine substitution at position 282 in σ(S) domain 4, which was much more dependent than the wild-type σ(S) protein on activation by Crl, a chaperone-like protein that increases the affinity of σ(S) for the RNA polymerase core enzyme (E). We used the bacterial adenylate cyclase two-hybrid system to demonstrate that Crl bound to residues 72 to 167 of σ(S) domain 2 and that G282V substitution did not directly affect Crl binding. However, this substitution drastically reduced the ability of σ(S)(Ty19) to bind E in a surface plasmon resonance assay, a defect partially rescued by Crl. The modeled structure of the Eσ(S) holoenzyme suggested that substitution G282V could directly disrupt a favorable interaction between σ(S) and E. The rpoS(Ty19) allele conferred a competitive fitness when the bacterial population was wild type for crl but was outcompeted in Δcrl populations. Thus, these results indicate that the competitive advantage of the rpoS(Ty19) mutant is dependent on Crl and suggest that crl plays a role in the appearance of rpoS mutants in bacterial populations.

A proteomic analysis reveals differential regulation of the σ(S)-dependent yciGFE(katN) locus by YncC and H-NS in Salmonella and Escherichia coli K-12.

The stationary phase sigma factor σ(S) (RpoS) controls a regulon required for general stress resistance of the closely related enterobacteria Salmonella and Escherichia coli. The σ(S)-dependent yncC gene encodes a putative DNA binding regulatory protein. Application of the surface-enhanced laser desorption/ionization-time of flight (SELDI-TOF) ProteinChip technology for proteome profiling of wild-type and mutant strains of Salmonella enterica serovar Typhimurium revealed potential protein targets for YncC regulation, which were identified by mass spectrometry, and subsequently validated. These proteins are encoded by the σ(S)-dependent operon yciGFEkatN and regulation of their expression by YncC operates at the transcriptional level, as demonstrated by gene fusion analyses and by in vitro transcription and DNase I footprinting experiments with purified YncC. The yciGFE genes are present (without katN) in E. coli K-12 but are poorly expressed, compared with the situation in Salmonella. We report that the yciGFE(katN) locus is silenced by the histone-like protein H-NS in both species, but that σ(S) efficiently relieves silencing in Salmonella but not in E. coli K-12. In Salmonella, YncC acts in concert with σ(S) to activate transcription at the yciG promoter (pyciG). When overproduced, YncC also activated σ(S)-dependent transcription at pyciG in E. coli K-12, but solely by countering the negative effect of H-NS. Our results indicate that differences between Salmonella and E. coli K-12, in the architecture of cis-acting regulatory sequences upstream of pyciG, contribute to the differential regulation of the yciGFE(katN) genes by H-NS and YncC in these two enterobacteria. In E. coli, this locus is subject to gene rearrangements and also likely to horizontal gene transfer, consistent with its repression by the xenogeneic silencer H-NS.

Identification of conserved amino acid residues of the Salmonella sigmaS chaperone Crl involved in Crl-sigmaS interactions.

Proteins that bind sigma factors typically attenuate the function of the sigma factor by restricting its access to the RNA polymerase (RNAP) core enzyme. An exception to this general rule is the Crl protein that binds the stationary-phase sigma factor sigma(S) (RpoS) and enhances its affinity for the RNAP core enzyme, thereby increasing expression of sigma(S)-dependent genes. Analyses of sequenced bacterial genomes revealed that crl is less widespread and less conserved at the sequence level than rpoS. Seventeen residues are conserved in all members of the Crl family. Site-directed mutagenesis of the crl gene from Salmonella enterica serovar Typhimurium and complementation of a Deltacrl mutant of Salmonella indicated that substitution of the conserved residues Y22, F53, W56, and W82 decreased Crl activity. This conclusion was further confirmed by promoter binding and abortive transcription assays. We also used a bacterial two-hybrid system (BACTH) to show that the four substitutions in Crl abolish Crl-sigma(S) interaction and that residues 1 to 71 in sigma(S) are dispensable for Crl binding. In Escherichia coli, it has been reported that Crl also interacts with the ferric uptake regulator Fur and that Fur represses crl transcription. However, the Salmonella Crl and Fur proteins did not interact in the BACTH system. In addition, a fur mutation did not have any significant effect on the expression level of Crl in Salmonella. These results suggest that the relationship between Crl and Fur is different in Salmonella and E. coli.

Characterization of regulatory element Rp3 of regulation of beta-lactamases from Ralstonia pickettii.

The two chromosomally encoded beta-lactamases, OXA-22 and OXA-60, from Ralstonia pickettii are inducible by beta-lactam molecules. Disruption of RP3 abolished induction of both beta-lactamases and the resistance to pH, osmolarity and survival in the stationary phase, suggesting that RP3 might be a global regulator. Interactions between RP3, OXA-22 and OXA-60 were investigated at a transcript and protein level using 5'-rapid amplification of cDNA ends experiments, real-time reverse transcription (RT)-PCR and footprinting assays. The rp3 gene was actively transcribed and the promoter sequences corresponded to a nontypical sigma(70)-type promoter. RT-PCR analysis showed that rp3 expression as well as that of the bla(OXA) genes was positively regulated: the level of transcripts of rp3, bla(OXA-22) and bla(OXA-60) genes were, respectively, increased 20-, 100- and 2000-fold upon imipenem induction. DNAse I footprinting showed that RP3 specifically bound to tandem repeats centered at positions -55.5 and -73.5 upstream from the bla(OXA-22) and bla(OXA-60) transcriptional start sites. Interestingly, the binding site at bla(OXA-60) overlapped the -35 region of the rp3 promoter, although the region essential for induction lies at the beginning of the orf-rp3. This result indicates that RP3 is most probably only one component of a novel regulatory system involved in the expression of beta-lactamases in R. pickettii.

Assays for transcription factor activity.

Transcription factors interact at promoters to modulate the transcription of genes. This chapter describes three in vitro methods that can be used to monitor their activity: transcript assays, abortive initiation assays, and potassium permanganate footprinting. These techniques have been developed using bacterial systems, and can be used to study the kinetics of transcription initiation, and hence to unravel regulatory mechanisms.

IHF-binding sites inhibit DNA loop formation and transcription initiation.

Transcriptional activation of enhancer and sigma(54)-dependent promoters requires efficient interactions between enhancer-binding proteins (EBP) and promoter bound sigma(54)-RNA polymerase (E sigma(54)) achieved by DNA looping, which is usually facilitated by the integration host factor (IHF). Since the lengths of the intervening region supporting DNA-loop formation are similar among IHF-dependent and IHF-independent promoters, the precise reason(s) why IHF is selectively important for the frequency of transcription initiation remain unclear. Here, using kinetic cyclization and in vitro transcription assays we show that, in the absence of IHF protein, the DNA fragments containing an IHF-binding site have much less looping-formation ability than those that lack an IHF-binding site. Furthermore, when an IHF consensus-binding site was introduced into the intervening region between promoter and enhancer of the target DNA fragments, loop formation and DNA-loop-dependent transcriptional activation are significantly reduced in a position-independent manner. DNA-looping-independent transcriptional activation was unaffected. The binding of IHF to its consensus site in the target promoters clearly restored efficient DNA looping formation and looping-dependent transcriptional activation. Our data provide evidence that one function for the IHF protein is to release a communication block set by intrinsic properties of the IHF DNA-binding site.

Binding of the unorthodox transcription activator, Crl, to the components of the transcription machinery.

The small regulatory protein Crl binds to sigmaS, the RNA polymerase stationary phase sigma factor. Crl facilitates the formation of the sigmaS-associated holoenzyme (EsigmaS) and thereby activates sigmaS-dependent genes. Using a real time surface plasmon resonance biosensor, we characterized in greater detail the specificity and mode of action of Crl. Crl specifically forms a 1:1 complex with sigmaS, which results in an increase of the association rate of sigmaS to core RNA polymerase without any effect on the dissociation rate of EsigmaS. Crl is also able to associate with preformed EsigmaS with a higher affinity than with sigmaS alone. Furthermore, even at saturating sigmaS concentrations, Crl significantly increases EsigmaS association with the katN promoter and the productive isomerization of the EsigmaS-katN complex, supporting a direct role of Crl in transcription initiation. Finally, we show that Crl does not bind to sigma70 itself but is able at high concentrations to form a weak and transient 1:1 complex with both core RNA polymerase and the sigma70-associated holoenzyme, leaving open the possibility that Crl might also exert a side regulatory role in the transcriptional activity of additional non-sigmaS holoenzymes.

The role of FIS protein in the physiological control of the expression of the Escherichia coli meta-hpa operon.

Expression from the Escherichia coli W meta-hpa operon promoter (Pg) is under a strict catabolic repression control mediated by the cAMP-catabolite repression protein (CRP) complex in a glucose-containing medium. The Pg promoter is also activated by the integration host factor (IHF) and repressed by the specific transcriptional regulator HpaR when 4-hydroxyphenylacetate (4HPA) is not present in the medium. Expression from the hpa promoter is also repressed in undefined rich medium such as LB, but the molecular basis of this mechanism is not understood. We present in vitro and in vivo studies to demonstrate the involvement of FIS protein in this catabolic repression. DNase I footprinting experiments show that FIS binds to multiple sites within the Pg promoter. FIS-site I overlaps the CRP-binding site. By using an electromobility shift assay, we demonstrated that FIS efficiently competes with CRP for binding to the Pg promoter, suggesting an antagonist/competitive mechanism. RT-PCR showed that the Pg repression effect is relieved in a FIS deleted strain. The repression role of FIS at Pg was further demonstrated by in vitro transcription assays. These results suggest that FIS contributes to silencing the Pg promoter in the exponential phase of growth in an undefined rich medium when FIS is predominantly expressed.

Effect of growth temperature on Crl-dependent regulation of sigmaS activity in Salmonella enterica serovar Typhimurium.

The small regulatory protein Crl favors association of the stationary-phase sigma factor sigma(S) (RpoS) with the core enzyme polymerase and thereby increases sigma(S) activity. Crl has a major physiological impact at low levels of sigma(S). Here, we report that the Crl effects on sigma(S)-dependent gene expression, the H(2)O(2) resistance of Salmonella enterica serovar Typhimurium, and the resistance of this organism to acidic pH are greater at 28 degrees C than at 37 degrees C. Immunoblot experiments revealed a negative correlation between sigma(S) and Crl levels; the production of Crl was slightly greater at 28 degrees C than at 37 degrees C, whereas the sigma(S) levels were about twofold lower at 28 degrees C than at 37 degrees C. At both temperatures, Crl was present in excess of sigma(S), and increasing the Crl level further did not increase the H(2)O(2) resistance level of Salmonella and the expression of the sigma(S)-dependent gene katE encoding the stationary-phase catalase. In contrast, increasing the sigma(S) level rendered Salmonella more resistant to H(2)O(2) at 28 degrees C, increased the expression of katE, and reduced the magnitude of Crl activation. In addition, the effect of Crl on katE transcription in vitro was not dependent on temperature. These results suggest that the effect of temperature on Crl-dependent regulation of the katE gene and H(2)O(2) resistance are mediated mainly via an effect on sigma(S) levels. In addition, our results revealed that sigma(S) exerts a negative effect on the production of Crl in stationary phase when the cells contain high levels of sigma(S).

The Escherichia coli regulator of sigma 70 protein, Rsd, can up-regulate some stress-dependent promoters by sequestering sigma 70.

The Escherichia coli Rsd protein forms complexes with the RNA polymerase sigma(70) factor, but its biological role is not understood. Transcriptome analysis shows that overexpression of Rsd causes increased expression from some promoters whose expression depends on the alternative sigma(38) factor, and this was confirmed by experiments with lac fusions at selected promoters. The LP18 substitution in Rsd increases the Rsd-dependent stimulation of these promoter-lac fusions. Analysis with a bacterial two-hybrid system shows that the LP18 substitution in Rsd increases its interaction with sigma(70). Our experiments support a model in which the role of Rsd is primarily to sequester sigma(70), thereby increasing the levels of RNA polymerase containing the alternative sigma(38) factor.

Interplay between CRP-cAMP and PII-Ntr systems forms novel regulatory network between carbon metabolism and nitrogen assimilation in Escherichia coli.

In Escherichia coli, utilization of carbon sources is regulated by the phosphoenolpyruvate-dependent phosphotransferase system (PTS), which modulates the intracellular levels of cAMP. The cAMP receptor protein (CRP) controls the transcription of many catabolic genes. The availability of nitrogen is sensed by the PII protein at the level of intracellular glutamine. Glutamine is transported mainly by GlnHPQ, and synthesized by glutamine synthetase (GS) encoded by glnA. Previous studies suggest that CRP affects nitrogen assimilation. Here we showed that at least two mechanisms are involved. First, CRP activates glnHp1 via synergistic binding with sigma 70 RNA polymerase (Esigma(70)) and represses glnHp2. As a consequence, in the presence of glutamine, the overall enhancement of glnHPQ expression alters GlnB signalling and de-activates glnAp2. Second, in vitro studies show that CRP can be recruited by sigma 54 holoenzyme (Esigma(54)) to a site centred at -51.5 upstream of glnAp2. CRP-induced DNA-bending prevents the nitrogen regulation protein C (NtrC) activator from approaching the activator-accessible face of the promoter-bound Esigma(54) closed complex, and inhibits glnAp2. Therefore, as the major transcriptional effector of the 'glucose effect', CRP affects both the signal transduction pathway and the overall geometry of the transcriptional machinery of components of the nitrogen regulon.

Physiological effects of Crl in Salmonella are modulated by sigmaS level and promoter specificity.

The small regulatory protein Crl activates sigma(S) (RpoS), the stationary-phase and general stress response sigma factor. Crl has been reported to bind sigma(S) in vitro and to facilitate the formation of RNA polymerase holoenzyme. In Salmonella enterica serovar Typhimurium, Crl is required for the development of the rdar morphotype and transcription initiation of the sigma(S)-dependent genes csgD and adrA, involved in curli and cellulose production. Here, we examined the expression of other sigma(S)-dependent phenotypes and genes in a Deltacrl mutant of Salmonella. Gene fusion analyses and in vitro transcription assays indicate that the magnitude of Crl activation differs between promoters and is highly dependent on sigma(S) levels. We replaced the wild-type rpoS allele in S. enterica serovar Typhimurium strain ATCC 14028 with the rpoS(LT2) allele that shows reduced expression of sigma(S); the result was an increased Crl activation ratio and larger physiological effects of Crl on oxidative, thermal, and acid stress resistance levels during stationary phase. We also found that crl, rpoS, and crl rpoS strains grew better on succinate than did the wild type and expressed the succinate dehydrogenase sdhCDBA operon more strongly. The crl and rpoS(LT2) mutations also increased the competitive fitness of Salmonella in stationary phase. These results show that Crl contributes to negative regulation by sigma(S), a finding consistent with a role for Crl in sigma factor competition via the facilitation of sigma(S) binding to core RNA polymerase.

Probing the importance of selected phylum-specific amino acids in sigma A of Bacteroides fragilis, a primary sigma factor naturally devoid of an N-terminal acidic region 1.1.

The sigmaA factor of Bacteroides fragilis is the prototype of a novel subgroup of primary sigma factors that are essential for growth and ensure the initiation of transcription of the housekeeping genes. This subgroup is confined to the phyla Bacteroidetes and Chlorobi. Its members carry a specific amino acid signature and are notably characterized by a short, basic N-terminal segment instead of the typical acidic region 1.1. Using in vitro mutagenesis, we investigated the importance of this basic segment and of several residues of the signature for the function of sigmaA. We have shown that the conserved residues Phe-61 and Lys-265, located in the core binding and DNA binding subregions 2.1 and 4.2, respectively, are critical for full function of the B. fragilis holoenzyme. With respect to the unusual subregion composition of sigmaA, we have shown that truncation of the basic N-terminal segment, or reversion of its charge, strongly affects the overall transcriptional activity of B. fragilis RNA polymerase in vitro. Our results indicate that the presence of the intact basic segment is required for the formation of RNA polymerase (RNAP)-promoter open complexes, the correct architecture of the transcription bubble, and efficient promoter clearance.

Regulatory components at the csgD promoter--additional roles for OmpR and integration host factor and role of the 5' untranslated region.

CsgD is a master regulator of multicellular behaviour in Salmonella enterica serovar Typhimurium. Expression of CsgD is highly regulated on the transcriptional level. A nucleo-protein complex had been defined where the global regulators OmpR and integration host factor (IHF) bind up- and downstream of the csgD core promoter. In this study, the nucleo-protein complex of PcsgD was extended through characterization of additional OmpR and IHF binding sites that influence the transcriptional activity of the csgD promoter. Furthermore, the role of the 174 bp long 5'-untranslated region on transcriptional activity was defined.

Crl activates transcription initiation of RpoS-regulated genes involved in the multicellular behavior of Salmonella enterica serovar Typhimurium.

In Salmonella enterica serovar Typhimurium, the stationary-phase sigma factor sigma(S) (RpoS) is required for virulence, stress resistance, biofilm formation, and development of the rdar morphotype. This morphotype is a multicellular behavior characterized by expression of the adhesive extracellular matrix components cellulose and curli fimbriae. The Crl protein of Escherichia coli interacts with sigma(S) and activates expression of sigma(S)-regulated genes, such as the csgBAC operon encoding the subunit of the curli proteins, by an unknown mechanism. Here, we showed using in vivo and in vitro experiments that the Crl protein of Salmonella serovar Typhimurium is required for development of a typical rdar morphotype and for maximal expression of the csgD, csgB, adrA, and bcsA genes, which are involved in curli and cellulose biosynthesis. In vitro transcription assays and potassium permanganate reactivity experiments with purified His(6)-Crl showed that Crl directly activated sigma(S)-dependent transcription initiation at the csgD and adrA promoters. We observed no effect of Crl on sigma(70)-dependent transcription. Crl protein levels increased during the late exponential and stationary growth phases in Luria-Beratani medium without NaCl at 28 degrees C. We obtained complementation of the crl mutation by increasing sigma(S) levels. This suggests that Crl has a major physiological impact at low concentrations of sigma(S).

FIS activates glnAp2 in Escherichia coli: role of a DNA bend centered at -55, upstream of the transcription start site.

A binding site for the Escherichia coli nucleoid binding protein FIS (factor for inversion stimulation) was identified upstream of a sigma54-dependent promoter, glnAp2. The binding and bending center of FIS is positioned at -55 with respect to the transcription start site (+1). Binding of FIS at this site activates the transcription of glnAp2 both in vivo and in vitro. Furthermore, we substituted the FIS-mediated DNA bending with other protein (cAMP receptor protein or integration host factor)-mediated DNA bending, without changing the position of the bending center. In vitro transcription assays indicated that all DNA bends centered at -55 activate transcriptional initiation of glnAp2, especially when linear templates were used.

Protein-induced DNA bending clarifies the architectural organization of the sigma54-dependent glnAp2 promoter.

Sigma54-RNA polymerase (Esigma54) predominantly contacts one face of the DNA helix in the closed promoter complex, and interacts with the upstream enhancer-bound activator via DNA looping. Up to date, the precise face of Esigma54 that contacts the activator to convert the closed complex to an open one remains unclear. By introducing protein-induced DNA bends at precise locations between upstream enhancer sequences and the core promoter of the sigma54-dependent glnAp2 promoter without changing the distance in-between, we observed a strong enhanced or decreased promoter activity, especially on linear DNA templates in vitro. The relative positioning and orientations of Esigma54, DNA bending protein and enhancer-bound activator on linear DNA were determined by in vitro footprinting analysis. Intriguingly, the locations from which the DNA bending protein exerted its optimal stimulatory effects were all found on the opposite face of the DNA helix compared with the DNA bound Esigma54 in the closed complex. Therefore, these results provide evidence that the activator must approach the Esigma54 closed complexes from the unbound face of the promoter DNA helix to catalyse open complex formation. This proposal is further supported by the modelling of activator-promoter DNA-Esigma54 complex.

Binding sites of VanRB and sigma70 RNA polymerase in the vanB vancomycin resistance operon of Enterococcus faecium BM4524.

The vanB operon of Enterococcus faecium BM4524 which confers inducible resistance to vancomycin is composed of the vanR(B)S(B) gene encoding a two-component regulatory system and the vanY(B)WH(B)BX(B) resistance genes that are transcribed from promoters P(RB) and P(YB) respectively. In this study, primer extension revealed transcription start sites at 13 and 48 bp upstream from the start codon of vanR(B) and vanY(B), respectively, that allowed identification of -10 and -35 promoter motifs. The VanR(B) protein was overproduced in Escherichia coli, purified and phosphorylated (VanR(B)-P) non-enzymically with acetylphosphate. VanR(B)-P and VanR(B) specifically bound to P(RB) and P(YB) promoters. VanR(B) bound at a single site at position -32.5 upstream from the P(RB) transcriptional start site and at two sites at positions -33.5 and -55.5 upstream from that of P(YB). The proximal VanR(B) binding site overlapped the -35 region of both promoters. VanR(B) was converted from a monomer to a dimer upon acetylphosphate treatment. VanR(B)-P had higher affinity than VanR(B) for its targets and appeared more efficient than VanR(B) in promoting open complex formation with P(RB) and P(YB). In the absence of regulator, E. coli RNA polymerase was able to interact with P(RB) but not with P(YB). Phosphorylation of VanR(B) significantly increased promoter interaction with RNA polymerase and led to an extended and modified footprint. In vitro transcription assays showed that VanR(B)-P activates P(YB) more strongly than P(RB). Analysis of the protected regions revealed one copy of a 21 bp sequence in the P(RB) promoter and two copies in the P(YB) promoter which may serve as recognition sites for VanR(B) and VanR(B)-P binding that are required for transcriptional activation and expression of vancomycin resistance.