Sigma Factor RpoH Research Articles

New genetic markers for <i>Burkholderia pseudomallei</i> strains typing

Burkholderia pseudomallei is the causative agent of melioidosis, a serious infectious disease in humans and animals that may be asymptomatic long-term and quickly develop to pneumonia and septicemia in immunocompromised state and due to predisposing factors. Melioidosis is endemic in countries with tropical and subtropical climates, where B. pseudomallei is a part of the soil and water microbiota of stagnant water bodies, as well as the plant rhizosphere. The recording of imported melioidosis cases in countries with temperate climate zone, along with the continued threat of using this pathogen as a bioterrorism agent, indicate the relevance of research aimed at developing modern methods for its diagnosing and typing. Current research in the intraspecific differentiation of pathogenic strains of infectious diseases tends to use two or more types of molecular markers. A promising direction for the B. pseudomallei strains genotyping is based on using a combination of VNTR loci, which provides a high discriminating ability of the method of multi-locus variable tandem repeat number analysis (MLVA), and slowly evolving single nucleotide polymorphisms (SNPs). The aim of this work was to identify new VNTR and SNP loci suitable for use as genetic markers in molecular typing of the melioidosis causative agent. 20 strains of B. pseudomallei from the collection of the Volgograd Plague Control Research Institute and whole genome sequences of 85 B. pseudomallei strains from the GenBank NCBI database were analyzed. While typing melioidosis causative agent strains from the GenBank NCBI database for 4 VNTR loci, 74 genotypes were identified, of which 64 were unique (HunterGaston index 0.997). It was found that the high allelic polymorphism of VNTR loci limited a potential to determine the geographical and phylogenetic relationships of B. pseudomallei isolates by using the MLVA-4 scheme, and the identified null alleles increased the risk of homoplasia. In this regard, the MLVA-4 scheme was supplemented with a VNTR marker in the BPSS1974 locus encoding a collagen-like protein as well as with SNP markers in the genes of lipid A biosynthesis lauroyl acyltransferase, RNA polymerase sigma factor RpoH, and glutamine amidotransferase. For amplification of the select loci, primers and probes were designed as part of the developed scheme, which were tested in the typing of B. pseudomallei collection strains. According to the results of a cluster analysis for 105 strains of the melioidosis causative agent, an integrated approach turned out to be optimal, allowing differentiation of strains within SNP groups based on VNTR profiles. The methodological approach developed for B. pseudomallei genetic typing, based on a comprehensive analysis of 4 SNP- and 5 MLVA-markers in our modification, allowed to differentiate strains according to the geographical regions of their origin and establish the clonal origin of isolates upon revealing cases of melioidosis.

RNase III Participates in the Adaptation to Temperature Shock and Oxidative Stress in Escherichia coli.

Bacteria thrive in ever-changing environments by quickly remodeling their transcriptome and proteome via complex regulatory circuits. Regulation occurs at multiple steps, from the transcription of genes to the post-translational modification of proteins, via both protein and RNA regulators. At the post-transcriptional level, the RNA fate is balanced through the binding of ribosomes, chaperones and ribonucleases. We aim to decipher the role of the double-stranded-RNA-specific endoribonuclease RNase III and to evaluate its biological importance in the adaptation to modifications of the environment. The inactivation of RNase III affects a large number of genes and leads to several phenotypical defects, such as reduced thermotolerance in Escherichia coli. In this study, we reveal that RNase III inactivation leads to an increased sensitivity to temperature shock and oxidative stress. We further show that RNase III is important for the induction of the heat shock sigma factor RpoH and for the expression of the superoxide dismutase SodA.

Increased mistranslation protects E.coli from protein misfolding stress due to activation of a RpoS-dependent heat shock response.

The misincorporation of an incorrect amino acid into a polypeptide during protein synthesis is considered a detrimental phenomenon. A mistranslated protein is often misfolded and degraded or nonfunctional and results in an increased cost to quality control machinery. Despite these costs, errors during protein synthesis are common in bacteria. Here, we report that mistranslation in Escherichia coli increase the protein level of the heat shock sigma factor RpoH and protect cells against heat stress. Surprisingly, this increase in RpoH due to mistranslation is dependent on the presence of the general stress response sigma factor RpoS. This report provides evidence for a protective function of mistranslation and suggests a novel regulatory role of RpoS in the heat shock response.

Targeting sigma factor controlled signaling pathways to modulate biofilm growth and composition

Stress‐responsive transcription factors (TFs) and alternative sigma factor TFs control a multitude of downstream targets, some of which may have a direct impact on the formation of bacterial biofilm and the composition of the extracellular polymeric matrix (EPS) surrounding the biofilm. We explore the effect of moderate overexpression of several different alternative sigma factor TFs on biofilm growth using a variety of biochemical and analytical assays. Biofilm growth is monitored by a dye‐binding microtiter plate assay, and the EPS composition, specifically the concentration of proteins and polysaccharides, is monitored by colorimetric assays and FT‐IR spectroscopy. We monitor the known downstream targets of these TFs using quantitative real‐time PCR (qPCR) to confirm TF overexpression. Currently, we are exploring the impact of overexpressing sigma factors rpoH, normally upregulated upon heat shock, and rpoF, an alternative sigma factor responsible for the expression of chemotaxis receptors and flagellar assembly, on biofilm formation and composition.Support or Funding InformationThis work was supported by Oberlin College.

In vivo trapping of FtsH substrates by label-free quantitative proteomics.

FtsH is the only membrane-bound and essential protease in Escherichia coli. It is responsible for the degradation of regulatory proteins and enzymes such as the heat-shock sigma factor RpoH or LpxC, the key enzyme of lipopolysaccharide biosynthesis. To find new FtsH targets, we trapped substrates in E. coli cells from exponential and stationary growth phase by using a proteolytically inactive FtsH variant. Subsequent analysis of the isolated FtsH-substrate complexes by label-free nanoLC-MS/MS revealed more than 50 putative FtsH substrates, among them five already known substrates. Four out of thirty-seven tested candidates were found to be novel FtsH substrates as shown by in vivo degradation experiments. Six other candidates were degraded by one or more other protease(s). The FtsH substrates SecD and ExbD are involved in transport processes across the membrane, whereas the physiological roles of YlaC and YhbT are yet unknown. The presence of the previously identified YfgM degron in two of the novel substrates suggests general rules for substrate recognition of this unique protease.

Novel strategy for biofilm inhibition by using small molecules targeting molecular chaperone DnaK.

Biofilms are complex communities of microorganisms that attach to surfaces and are embedded in a self-produced extracellular matrix. Since these cells acquire increased tolerance against antimicrobial agents and host immune systems, biofilm-associated infectious diseases tend to become chronic. We show here that the molecular chaperone DnaK is important for biofilm formation and that chemical inhibition of DnaK cellular functions is effective in preventing biofilm development. Genetic, microbial, and microscopic analyses revealed that deletion of the dnaK gene markedly reduced the production of the extracellular functional amyloid curli, which contributes to the robustness of Escherichia coli biofilms. We tested the ability of DnaK inhibitors myricetin (Myr), telmisartan, pancuronium bromide, and zafirlukast to prevent biofilm formation of E. coli. Only Myr, a flavonol widely distributed in plants, inhibited biofilm formation in a concentration-dependent manner (50% inhibitory concentration [IC50] = 46.2 μM); however, it did not affect growth. Transmission electron microscopy demonstrated that Myr inhibited the production of curli. Phenotypic analyses of thermosensitivity, cell division, intracellular level of RNA polymerase sigma factor RpoH, and vulnerability to vancomycin revealed that Myr altered the phenotype of E. coli wild-type cells to make them resemble those of the isogenic dnaK deletion mutant, indicating that Myr inhibits cellular functions of DnaK. These findings provide insights into the significance of DnaK in curli-dependent biofilm formation and indicate that DnaK is an ideal target for antibiofilm drugs.

대장균에서 프로판올 스트레스에 관한 전사분석

대장균 야생주와 프로판올 내성 변이주에서 프로판올 스트레스에 의해 발현이 크게 변화하는 유전자를 DNA microarray 기술을 이용하여 비교 분석하였다. 프로판올 첨가한 야생주와 무첨가한 야생주 사이의 RNA 발현 연관값은 0.949이며, 50개의 유전자 발현이 2배 이상 변화하였다. 프로판올을 첨가한 내성변이주와 무첨가한 변이주 사이의 연관값은 0.952이며, 71개의 유전자 발현이 크게 변화하였다. 그러나, 야생주와 변이주 사이의 연관값은 프로판올 무첨가한 조건과 첨가한 조건에서 각각 0.992 및 0.974로 매우 높았으며, 2배 이상의 발현차이를 나타내는 유전자는 각각 1개 및 2개로, 두 균주는 매우 유사한 발현양상을 보였다. 야생주 또는 변이주에서 프로판올 스트레스에 반응하는 대표적인 유전자들의 특징은 많은 열충격 반응에 관여하는 유전자들의 발현이 크게 증가하였으며, 리보소옴 합성에 필요한 많은 유전자들의 발현이 감소하였다. 또한, 전사조절인자들인 ArcA, CRP, FNR, H-NS, GatR, PurR에 의해 조절받는 유전자들의 발현이 크게 변화하였으며, 시그마인자들 중에서는 RpoH에 의해서 발현되는 유전자들의 발현이 크게 증가하였다. rpoH가 정상적으로 발현되지 못하는 변이주와 야생주를 이용한 프로판올 내성정도를 측정한 결과, RpoH는 대장균에서 프로판올 스트레스에 적응하는데 중요한 기능을 하는 것으로 확인되었다. We compared the transcriptome in response to propanol stress in wild-type and propanol-resistant mutant Escherichia coli using the DNA microarray technique. The correlation value of RNA expression between the propanol-treated wild type and the untreated-one was about 0.949, and 50 genes were differentially expressed by more than twofold in both samples. The correlation value of RNA expression between the propanol-treated mutant and the untreated one was about 0.951, and 71 genes in two samples showed differential expression patterns. However, the values between the wild type and mutant, regardless of propanol addition, were 0.974-0.992 and only 1-2 genes were differentially expressed in the two strains. The representative characteristics among differentially expressed genes in W3110 or P19 treated with propanol compared to untreated samples were up-regulation of hest shock response genes and down-regulation of genes relating to ribosome biosynthesis. In addition, many genes were regulated by transcription regulation factors such as ArcA, CRP, FNR, H-NS, GatR, or PurR and overexpressed by sigma factor RpoH. We confirmed that RpoH mediated an important host defense function in propanol stress in E. coli W3110 and P19 by comparison of cell growth rate among the wild type, rpoH disruptant mutant, and rpoH-complemented strain.

Stress induced cross-protection against environmental challenges on prokaryotic and eukaryotic microbes.

Prokaryotic and eukaryotic microbes thrive successfully in stressful environments such as high osmolarity, acidic or alkali, solar heat and u.v. radiation, nutrient starvation, oxidative stress, and several others. To live under these continuous stress conditions, these microbes must have mechanisms to protect their proteins, membranes, and nucleic acids, as well as other mechanisms that repair nucleic acids. The stress responses in bacteria are controlled by master regulators, which include alternative sigma factors, such as RpoS and RpoH. The sigma factor RpoS integrates multiple signals, such as the general stress response regulators and the sigma factor RpoH regulates the heat shock proteins. These response pathways extensively overlap and are induced to various extents by the same environmental stresses. In eukaryotes, two major pathways regulate the stress responses: stress proteins, termed heat shock proteins (HSP), which appear to be required only for growth during moderate stress, and stress response elements (STRE), which are induced by different stress conditions and these elements result in the acquisition of a tolerant state towards any stress condition. In this review, the mechanisms of stress resistance between prokaryotic and eukaryotic microbes will be described and compared.

Overlapping Alternative Sigma Factor Regulons in the Response to Singlet Oxygen inRhodobacter sphaeroides

Organisms performing photosynthesis in the presence of oxygen have to cope with the formation of highly reactive singlet oxygen ((1)O(2)) and need to mount an adaptive response to photooxidative stress. Here we show that the alternative sigma factors RpoH(I) and RpoH(II) are both involved in the (1)O(2) response and in the heat stress response in Rhodobacter sphaeroides. We propose RpoH(II) to be the major player in the (1)O(2) response, whereas RpoH(I) is more important for the heat stress response. Mapping of the 5' ends of RpoH(II)- and also RpoH(I)/RpoH(II)-dependent transcripts revealed clear differences in the -10 regions of the putative promoter sequences. By using bioinformatic tools, we extended the RpoH(II) regulon, which includes genes induced by (1)O(2) exposure. These genes encode proteins which are, e.g., involved in methionine sulfoxide reduction and in maintaining the quinone pool. Furthermore, we identified small RNAs which depend on RpoH(I) and RpoH(II) and are likely to contribute to the defense against photooxidative stress and heat stress.

Photooxidative stress‐induced and abundant small RNAs in Rhodobacter sphaeroides

Exposure to oxygen and light generates photooxidative stress by the bacteriochlorophyll a mediated formation of singlet oxygen ((1)O(2)) in Rhodobacter sphaeroides. Our study reports the genome-wide search for small RNAs (sRNAs) involved in the regulatory response to (1)O(2). By using 454 pyrosequencing and Northern blot analysis, we identified 20 sRNAs from R. sphaeroides aerobic cultures or following treatment with (1)O(2) or superoxide (O(-)(2)). One sRNA was specifically induced by (1)O(2) and its expression depends on the extracytoplasmic function sigma factor RpoE. Two sRNAs induced by (1)O(2) and O(-)(2) were cotranscribed with upstream genes preceded by promoters with target sequences for the alternative sigma factors RpoH(I) and RpoH(II). The most abundant sRNA was processed in the presence of (1)O(2) but not by O(-)(2). From this and a second sRNA a conserved 3'-segment accumulated from a larger precursor. Absence of the RNA chaperone Hfq changed the half-lives, abundance and processing of (1)O(2)-affected sRNAs. Orthologues of three sRNA genes are present in different alpha-proteobacteria, but the majority was unique to R. sphaeroides or Rhodobacterales species. Our discovery that abundant sRNAs are affected by (1)O(2) exposure extends the knowledge on the role of sRNAs and Hfq in the regulatory response to oxidative stress.

Promoter Characterization in the AT-Rich Genome of the Obligate Endosymbiont “ Candidatus Blochmannia floridanus”

The characterization of transcriptional start sites of 14 genes encoded by the extremely AT-rich genome of "Candidatus Blochmannia floridanus" revealed a high degree of conservation with the RpoD promoter consensus sequence of the free-living relative Escherichia coli. Moreover, in agreement with the presence of the alternative heat shock sigma factor RpoH in "Ca. Blochmannia," typical RpoH-dependent promoters were identified. However, no heat shock response resembling that of E. coli could be detected in "Ca. Blochmannia."

Region C of the Escherichia coli heat shock sigma factor RpoH (σ32) contains a turnover element for proteolysis by the FtsH protease

Transcription of most heat shock genes in Escherichia coli is initiated by the alternative sigma factor sigma(32) (RpoH). At physiological temperatures, RpoH is rapidly degraded by chaperone-mediated FtsH-dependent proteolysis. Several RpoH residues critical for degradation are located in the highly conserved region 2.1. However, additional residues were predicted to be involved in this process. We introduced mutations in region C of RpoH and found that a double mutation (A131E, K134V) significantly stabilized RpoH against degradation by the FtsH protease. Single-point mutations at these positions only showed a slight effect on RpoH stability. Both double and single amino acid substitutions did not impair sigma factor activity as demonstrated by a groE-lacZ reporter gene fusion, Western blot analysis of heat shock gene expression and increased heat tolerance in the presence of these proteins. Combined mutations in regions 2.1 and C further stabilized RpoH. We also demonstrate that an RpoH fragment composed of residues 37-147 (including regions 2.1 and C) is degraded in an FtsH-dependent manner. We conclude that in addition to the previously described turnover element in region 2.1, a previously postulated second region important for proteolysis of RpoH by FtsH lies in region C of the sigma factor.

MtrR Modulates rpoH Expression and Levels of Antimicrobial Resistance in Neisseria gonorrhoeae

The MtrR transcriptional-regulatory protein is known to repress transcription of the mtrCDE operon, which encodes a multidrug efflux pump possessed by Neisseria gonorrhoeae that is important in the ability of gonococci to resist certain hydrophobic antibiotics, detergents, dyes, and host-derived antimicrobials. In order to determine whether MtrR can exert regulatory action on other gonococcal genes, we performed a whole-genome microarray analysis using total RNA extracted from actively growing broth cultures of isogenic MtrR-positive and MtrR-negative gonococci. We determined that, at a minimum, 69 genes are directly or indirectly subject to MtrR control, with 47 being MtrR repressed and 22 being MtrR activated. rpoH, which encodes the general stress response sigma factor RpoH (sigma 32), was found by DNA-binding studies to be directly repressed by MtrR, as it was found to bind to a DNA sequence upstream of rpoH that included sites within the rpoH promoter. MtrR also repressed the expression of certain RpoH-regulated genes, but this regulation was likely indirect and a reflection of MtrR control of rpoH expression. Inducible expression of MtrR was found to repress rpoH expression and to increase gonococcal susceptibility to hydrogen peroxide (H(2)O(2)) and an antibiotic (erythromycin) recognized by the MtrC-MtrD-MtrE efflux pump system. We propose that, apart from its ability to control the expression of the mtrCDE-encoded efflux pump operon and, as a consequence, levels of gonococcal resistance to host antimicrobials (e.g., antimicrobial peptides) recognized by the efflux pump, the ability of MtrR to regulate the expression levels of rpoH and RpoH-regulated genes also modulates levels of gonococcal susceptibility to H(2)O(2).

Region 2.1 of the Escherichia coli heat-shock sigma factor RpoH (σ 32) is necessary but not sufficient for degradation by the FtsH protease

The cellular level of the Escherichia coli heat-shock sigma factor RpoH (sigma32) is negatively controlled by chaperone-mediated proteolysis through the essential metalloprotease FtsH. Point mutations in the highly conserved region 2.1 stabilize RpoH in vivo. To assess the importance of this turnover element, hybrid proteins were constructed between E. coli RpoH and Bradyrhizobium japonicum RpoH1, a stable RpoH protein that differs from region 2.1 of E. coli RpoH at several positions. Nine amino acids forming a putative alpha-helix were exchanged between the two proteins. Both hybrids were active sigma factors and showed intermediate protein stability. Introduction of RpoH region 2.1 into the general stress sigma factor RpoS, which is a substrate of the ClpXP protease, did not render RpoS susceptible to FtsH. Hence, region 2.1 alone is not sufficient to confer FtsH sensitivity to other proteins. Region 2.1 is not a major chaperone-binding site since DnaK and DnaJ bound efficiently to all RpoH variants. The in vivo stability of the mutated RpoH proteins correlated with their stability in a purified in vitro degradation system, suggesting that region 2.1 might be directly involved in the interaction with the FtsH protease.

Multiple groESL Operons Are Not Key Targets of RpoH1 and RpoH2 in Sinorhizobium meliloti

Among the rhizobia that establish nitrogen-fixing nodules on the roots of host plants, many contain multiple copies of genes encoding the sigma factor RpoH and the chaperone GroEL/GroES. In Sinorhizobium meliloti there are two rpoH genes, four groESL operons, and one groEL gene. rpoH1 mutants are defective for growth at high temperature and form ineffective nodules, rpoH1 rpoH2 double mutants are unable to form nodules, and groESL1 mutants form ineffective nodules. To explore the roles of RpoH1 and RpoH2, we identified mutants that suppress both the growth and nodulation defects. These mutants do not suppress the nitrogen fixation defect. This implies that the functions of RpoH1 during growth and RpoH1/RpoH2 during the initiation of symbiosis are similar but that there is a different function of RpoH1 needed later during symbiosis. We showed that, unlike in Escherichia coli, overexpression of groESL is not sufficient to bypass any of the RpoH defects. Under free-living conditions, we determined that RpoH2 does not control expression of the groE genes, and RpoH1 only controls expression of groESL5. Finally, we completed the series of groE mutants by constructing groESL3 and groEL4 mutants and demonstrated that they do not display symbiotic defects. Therefore, the only groESL operon required by itself for symbiosis is groESL1. Taken together, these results suggest that GroEL/GroES production alone cannot explain the requirements for RpoH1 and RpoH2 in S. meliloti and that there must be other crucial targets.

Structure-function studies of Escherichia coli RpoH (sigma32) by in vitro linker insertion mutagenesis.

The sigma factor RpoH (sigma(32)) is the key regulator of the heat shock response in Escherichia coli. Many structural and functional properties of the sigma factor are poorly understood. To gain further insight into RpoH regions that are either important or dispensable for its cellular activity, we generated a collection of tetrapeptide insertion variants by a recently established in vitro linker insertion mutagenesis technique. Thirty-one distinct insertions were obtained, and their sigma factor activity was analyzed by using a groE-lacZ reporter fusion in an rpoH-negative background. Our study provides a map of permissive sites which tolerate linker insertions and of functionally important regions at which a linker insertion impairs sigma factor activity. Selected linker insertion mutants will be discussed in the light of known sigma factor properties and in relation to a modeled structure of an RpoH fragment containing region 2.

Identification of the heat-shock sigma factor RpoH and a second RpoH-like protein in Sinorhizobium meliloti.

Hybridization to a PCR product derived from conserved sigma-factor sequences led to the identification of two Sinorhizobium meliloti DNA segments that display significant sequence similarity to the family of rpoH genes encoding the sigma(32) (RpoH) heat-shock transcription factors. The first gene, rpoH1, complements an Escherichia coli rpoH mutation. Cells containing an rpoH1 mutation are impaired in growth at 37 degrees C under free-living conditions and are defective in nitrogen fixation during symbiosis with alfalfa. A plasmid-borne rpoH1-gusA fusion increases in expression upon entry of the culture into the stationary phase of growth. The second gene, designated rpoH2, is 42% identical to the S. meliloti rpoH1 gene. Cells containing an rpoH2 mutation have no apparent phenotype under free-living conditions or during symbiosis with the host plant alfalfa. An rpoH2-gusA fusion increases in expression during the stationary phase of growth. The presence of two rpoH-like sequences in S. meliloti is reminiscent of the situation in Bradyrhizobium japonicum, which has three rpoH genes.

Regulation of tRNA5Leu-encoding gene leuX that is associated with a pathogenicity island in the uropathogenic Escherichia coli strain 536.

Pathogenicity islands (PAls) contain virulence determinants and are often linked to tRNA loci. The leuX gene encoding tRNA5Leu is associated with PAI II536 of the uropathogenic E. coli strain 536 (O6:K15:H31) and is important for the expression of virulence factors in this strain. Transcription of leuX differs from that of the majority of tRNA genes including the major leucyl-tRNA gene leuV. Availability of the tRNAsLeu can be linked with the efficient expression of virulence determinants. Transcription of leuX and letV was studied to gain further insights into the regulation of this tRNA gene and its role during the expression of virulence determinants. leuX but not letV transcripts accumulated in stationary phase. While leuX transcription was unchanged or slightly up-regulated by an increase in growth temperature, osmolarity or ethanol concentration, that of leuV was reduced. The heat shock-specific sigma factor RpoH was shown to affect transcription of leuX but not of leuV. These results demonstrate that transcription of a tRNA gene can be specifically influenced by an alternative sigma factor. Our finding expands our knowledge of the regulation of tRNA gene transcription and represents an additional mode of regulation of gene expression that can also affect bacterial virulence.

An internal region of the RpoH heat shock transcription factor is critical for rapid degradation by the FtsH protease

The proteolysis of regulatory proteins plays an important role in the control of gene expression. The Escherichia coli heat shock sigma factor RpoH (σ 32) is highly unstable. Its instability is determined by interactions with the DnaK chaperone machine, RNA polymerase and the ATP-dependent protease FtsH. Bradyrhizobium japonicum expresses three RpoH proteins of which RpoH 1 is highly stable. To determine which regions of E. coli RpoH determine protein lability, we generated a number of truncated versions and hybrid proteins. Truncation of N-terminal amino acids had no, and deletion of C-terminal amino acids only a minor effect on stability of RpoH. A major determinant of RpoH lability was mapped to a region of about 85 amino acids (residues 36–122) roughly comprising the sigma factor region 2. This is the first demonstration of an internal RpoH region being responsible for FtsH-mediated degradation.

Isolation and Characterization of theXanthomonas campestris rpoHGene Coding for a 32-kDa Heat Shock Sigma Factor

Degenerate oligonucleotide primers corresponding to the conserved regions of bacterial heat shock sigma factor RpoH (σ32) were used to amplify a 190-bp fragment by PCR on theX. campestrispv. campestris strain 11 chromosome. Using this fragment as a probe, plasmid pXC57 carrying a 4.7-kb insert was isolated from a genomic library of Xc11. Sequence analysis of a stretch of 2,053 bp from the pXC57 insert revealed an ORF encoding a polypeptide of 291 aa (32,854 dal) which displays 59.6% and 57.3% identity to therpoHgene products ofE. coliandP. aeruginosa,respectively. The Xc11rpoHgene was able to complement the RpoH deficientE. colistrain A7448. Both amino acid and mRNA sequences deduced from the Xc11rpoHgene show structural features characteristic of the corresponding sequences from those of the γ sub-group proteobacteria. The RpoH levels in Xc11 were demonstrated to increase transiently in response to heat shock treatment by immunoblot analysis using the polyclonal antibody raised against the purified Xc11 RpoH.