rcsB Gene Research Articles

A Novel LysR Family Factor STM0859 is Associated with The Responses of Salmonella Typhimurium to Environmental Stress and Biofilm Formation.

Salmonella enterica subsp. enterica serovar Typhimurium (ST) is an intracellularly parasitic bacterium. This zoonotic pathogen causes food poisoning and thus imposes a severe threat to food safety. Here, to understand the regulatory roles of the novel transcription factor STM0859 on the response of ST to environmental stress and biofilm formation, the STM0859 gene-deficient strain and the complementation strain ΔSTM0859/STM0859 were generated, respectively. Then, its capacity of responding to environmental stresses and biofilm (BF) formation ability under different stresses, including acid, alkali, high salt, cholate, and oxidative stresses was tested. We further analyzed the interaction between the STM0859 protein and the promoter of the acid stress response-related gene rcsB by performing an electrophoresis mobility shift assay (EMSA). The results showed that acid resistance and BF formation capacities of ST-ΔSTM0859 strain were significantly weaker, as compared with those of Salmonella Typhimurium SL1344 (ST-SL1344) wild strain (p < 0.01). Quantitative qRT-PCR analysis showed that the expression levels of acid stress and BF formation-related genes, rcsB and rpoS, of ST-ΔSTM0859 strain were significantly reduced at the transcription levels, while the transcription levels of these genes were fully restored in complementation strain ST-ΔSTM0859/STM0859. The results of EMSA showed that STM0859 was capable of binding the promoter DNA fragments of the rcsB gene, suggesting that STM0859 can promote the transcription of the rcsB gene through interaction with its promoter, thereby exerting an indirectly regulatory role on the adaptive responses to acid stress and BF formation of ST. This study provided new insights into the regulatory mechanisms of the LysR family factors on the tolerances of ST under adverse environmental stresses.

CpxR regulates the Rcs phosphorelay system in controlling the Ysc-Yop type III secretion system in Yersinia pseudotuberculosis.

The CpxRA two-component regulatory system and the Rcs phosphorelay system are both employed by the Enterobacteriaceae family to preserve bacterial envelope integrity and function when growing under stress. Although both systems regulate several overlapping physiological processes, evidence demonstrating a molecular connection between Cpx and Rcs signalling outputs is scarce. Here, we show that CpxR negatively regulates the transcription of the rcsB gene in the Rcs phosphorelay system in Yersinia pseudotuberculosis. Interestingly, transcription of rcsB is under the control of three promoters, which were all repressed by CpxR. Critically, synthetic activation of Cpx signalling through mislocalization of the NlpE lipoprotein to the inner membrane resulted in an active form of CpxR that repressed activity of rcsB promoters. On the other hand, a site-directed mutation of the phosphorylation site at residue 51 in CpxR generated an inactive non-phosphorylated variant that was unable to regulate output from these rcsB promoters. Importantly, CpxR-mediated inhibition of rcsB transcription in turn restricted activation of the Ysc-Yop type III secretion system (T3SS). Moreover, active CpxR blocks zinc-mediated activation of Rcs signalling and the subsequent activation of lcrF transcription. Our results demonstrate a novel regulatory cascade linking CpxR-RcsB-LcrF to control production of the Ysc-Yop T3SS.

MCloverBlaster: A tool to make markerless deletions and fusions using lambda red and I-SceI in Gram-negative bacterial genomes

This study introduces mCloverBlaster as a genetic tool to create deletions and transcriptional and translational fusions in bacterial genomes using recombineering. The major advantage of this system is that it can be used to make deletions and fusions without leaving a selectable marker on the chromosome. mCloverBlaster has a kanamycin resistance cassette with an I-SceI restriction site flanked by fragments of the gene for the mClover3 fluorescent protein including direct repeats of mClover3 sequence on both sides of the kanamycin resistance gene. The mCloverBlaster sequence is introduced into the chromosome using lambda red recombineering, expression of I-SceI creates a double stranded break in the kanamycin resistance cassette that initiates a recombination event that can occur in the mClover3 repeats. This recombination results in the simultaneous removal of the kanamycin resistance gene and the restoration of a functional mClover3 gene that can be used as a reporter. Here, this system was used to replace the rcsB stress response gene in Serratia marcescens. The resulting strain was tested for mClover3 fluorescence as a reporter for rcsB gene expression and evaluated for pigment biosynthesis. In summary, mCloverBlaster is a molecular genetic tool to make markerless mClover3 fusions and gene deletions.

Integration of multiple stimuli-sensing systems to regulate HrpS and type III secretion system in Erwinia amylovora.

The bacterial enhancer binding protein (bEBP) HrpS is essential for Erwinia amylovora virulence by activating the type III secretion system (T3SS). However, how the hrpS gene is regulated remains poorly understood in E. amylovora. In this study, 5' rapid amplification of cDNA ends and promoter deletion analyses showed that the hrpS gene contains two promoters driven by HrpX/HrpY and the Rcs phosphorelay system, respectively. Electrophoretic mobility shift and gene expression assays demonstrated that integration host factor IHF positively regulates hrpS expression through directly binding the hrpX promoter and positively regulating hrpX/hrpY expression. Moreover, hrpX expression was down-regulated in the relA/spoT ((p)ppGpp-deficient) mutant and the dksA mutant, but up-regulated when the wild-type strain was treated with serine hydroxamate, which induced (p)ppGpp-mediated stringent response. Furthermore, the csrA mutant showed significantly reduced transcripts of major hrpS activators, including the hrpX/hrpY, rcsA and rcsB genes, indicating that CsrA is required for full hrpS expression. On the other hand, the csrB mutant exhibited up-regulation of the rcsA and rcsB genes, and hrpS expression was largely diminished in the csrB/rcsB mutant, indicating that the Rcs system is mainly responsible for the increased hrpS expression in the csrB mutant. These findings suggest that E. amylovora recruits multiple stimuli-sensing systems, including HrpX/HrpY, the Rcs phosphorelay system and the Gac-Csr system, to regulate hrpS and T3SS gene expression.

Engineering Escherichia coli BL21 genome to improve the heptanoic acid tolerance by using CRISPR-Cas9 system

Acid tolerance is one of the critical factors to determine the quality of the industrial production strains. Therefore, we have investigated the introduction of the acid tolerance genes into the genome of Escherichia coli BL21 by using CRISPR-Cas9 system. The dsrA and rcsB genes of E. coli K-12, which are involved in the heptanoic acid tolerance, were inserted into the genome of E. coli BL21 without scar. The native transcription unit (TU) of dsrA and the synthetic TU of rcsB were integrated in E. coli BL21 genome. We found that the position of genomic coordinate of 1,300,270 was more efficient to integrate dsrA and rcsB than genomic coordinate of 3,876,428. Furthermore, the rcsB was successfully expressed in the resulting engineered strains (i.e., rcsB+ or dsrA+rcsB+ strains). The engineered strains expressing dsrA and/or rcsB showed the higher survival rate and specific growth rate under n-heptanoic acid stress than wild-type E. coli BL21. These results indicate that the newly introduced acid-tolerance systems were active in the E. coli BL21 strain.

Disruption of rcsB by a duplicated sequence in a curli-producing Escherichia coli O157:H7 results in differential gene expression in relation to biofilm formation, stress responses and metabolism

BackgroundEscherichia coli O157:H7 (O157) strain 86–24, linked to a 1986 disease outbreak, displays curli- and biofilm-negative phenotypes that are correlated with the lack of Congo red (CR) binding and formation of white colonies (CR−) on a CR-containing medium. However, on a CR medium this strain produces red isolates (CR+) capable of producing curli fimbriae and biofilms.ResultsTo identify genes controlling differential expression of curli fimbriae and biofilm formation, the RNA-Seq profile of a CR+ isolate was compared to the CR− parental isolate. Of the 242 genes expressed differentially in the CR+ isolate, 201 genes encoded proteins of known functions while the remaining 41 encoded hypothetical proteins. Among the genes with known functions, 149 were down- and 52 were up-regulated. Some of the upregulated genes were linked to biofilm formation through biosynthesis of curli fimbriae and flagella. The genes encoding transcriptional regulators, such as CsgD, QseB, YkgK, YdeH, Bdm, CspD, BssR and FlhDC, which modulate biofilm formation, were significantly altered in their expression. Several genes of the envelope stress (cpxP), heat shock (rpoH, htpX, degP), oxidative stress (ahpC, katE), nutrient limitation stress (phoB-phoR and pst) response pathways, and amino acid metabolism were downregulated in the CR+ isolate. Many genes mediating acid resistance and colanic acid biosynthesis, which influence biofilm formation directly or indirectly, were also down-regulated. Comparative genomics of CR+ and CR− isolates revealed the presence of a short duplicated sequence in the rcsB gene of the CR+ isolate. The alignment of the amino acid sequences of RcsB of the two isolates showed truncation of RcsB in the CR+ isolate at the insertion site of the duplicated sequence. Complementation of CR+ isolate with rcsB of the CR− parent restored parental phenotypes to the CR+ isolate.ConclusionsThe results of this study indicate that RcsB is a global regulator affecting bacterial survival in growth-restrictive environments through upregulation of genes promoting biofilm formation while downregulating certain metabolic functions. Understanding whether rcsB inactivation enhances persistence and survival of O157 in carrier animals and the environment would be important in developing strategies for controlling this bacterial pathogen in these niches.

Involvement of the Rcs regulon in the persistence of Salmonella Typhimurium in tomatoes.

It is becoming clear that human enteric pathogens, like Salmonella, can efficiently colonize vegetative and reproductive organs of plants. Even though the bacterium's ability to proliferate within plant tissues has been linked to outbreaks of salmonellosis, little is known about regulatory and physiological adaptations of Salmonella, or other human pathogens, to their persistence in plants. A screen of Salmonella deletion mutants in tomatoes identified rcsA and rcsB genes as those under positive selection. In tomato fruits, populations of Salmonella rcsB mutants were as much as 100-fold lower than those of the wild type. In the follow-up experiments, competitive fitness of rcsA and rcsB mutants was strongly reduced in tomatoes. Bioinformatics predictions identified a putative Salmonella RcsAB binding box (TTMGGAWWAABCTYA) and revealed an extensive putative RcsAB regulon, of which many members were differentially fit within tomatoes.

Natural rpoS mutations contribute to population heterogeneity in Escherichia coli O157:H7 strains linked to the 2006 US spinach-associated outbreak

We previously reported significantly different acid resistance between curli variants derived from the same Escherichia coli O157:H7 strain, although the curli fimbriae were not associated with this phenotypic divergence. Here we investigated the underlying molecular mechanism by examining the genes encoding the common transcriptional regulators of curli biogenesis and acid resistance. rpoS null mutations were detected in all curli-expressing variants of the 2006 spinach-associated outbreak strains, whereas a wild-type rpoS was present in all curli-deficient variants. Consequently curli-expressing variants were much more sensitive to various stress challenges than curli-deficient variants. This loss of general stress fitness appeared solely to be the result of rpoS mutation since the stress resistances could be restored in curli-expressing variants by a functional rpoS. Comparative transcriptomic analyses between the curli variants revealed a large number of differentially expressed genes, characterized by the enhanced expression of metabolic genes in curli-expressing variants, but a marked decrease in transcription of genes related to stress resistances. Unlike the curli-expressing variants of the 1993 US hamburger-associated outbreak strains (Applied Environmental Microbiology 78: 7706–7719), all curli-expressing variants of the 2006 spinach-associated outbreak strains carry a functional rcsB gene, suggesting an alternative mechanism governing intra-strain phenotypic divergence in E. coli O157:H7.

Polysialic and colanic acids metabolism in Escherichia coli K92 is regulated by RcsA and RcsB

We have shown previously that Escherichia coli K92 produces two different capsular polymers known as CA (colanic acid) and PA (polysialic acid) in a thermoregulated manner. The complex Rcs phosphorelay is largely related to the regulation of CA synthesis. Through deletion of rscA and rscB genes, we show that the Rcs system is involved in the regulation of both CA and PA synthesis in E. coli K92. Deletion of either rcsA or rcsB genes resulted in decreased expression of cps (CA biosynthesis cluster) at 19°C and 37°C, but only CA production was reduced at 19°C. Concerning PA, both deletions enhanced its synthesis at 37°C, which does not correlate with the reduced kps (PA biosynthesis cluster) expression observed in the rcsB mutant. Under this condition, expression of the nan operon responsible for PA catabolism was greatly reduced. Although RcsA and RcsB acted as negative regulators of PA synthesis at 37°C, their absence did not reestablish PA expression at low temperatures, despite the deletion of rcsB resulting in enhanced kps expression. Finally, our results revealed that RcsB controlled the expression of several genes (dsrA, rfaH, h-ns and slyA) involved in the thermoregulation of CA and PA synthesis, indicating that RcsB is part of a complex regulatory mechanism governing the surface appearance in E. coli.

Genome-Wide Identification of Genes Regulated by the Rcs Phosphorelay System in Erwinia amylovora

The exopolysaccharide amylovoran is one of the major pathogenicity factors in Erwinia amylovora, the causal agent of fire blight of apples and pears. We have previously demonstrated that the RcsBCD phosphorelay system is essential for virulence by controlling amylovoran biosynthesis. We have also found that the hybrid sensor kinase RcsC differentially regulates amylovoran production in vitro and in vivo. To further understand how the Rcs system regulates E. amylovora virulence gene expression, we conducted genome-wide microarray analyses to determine the regulons of RcsB and RcsC in liquid medium and on immature pear fruit. Array analyses identified a total of 648 genes differentially regulated by RcsCB in vitro and in vivo. Consistent with our previous findings, RcsB acts as a positive regulator in both conditions, while RcsC positively controls expression of amylovoran biosynthetic genes in vivo but negatively controls expression in vitro. Besides amylovoran biosynthesis and regulatory genes, cell-wall and cell-envelope (membrane) as well as regulatory genes were identified as the major components of the RcsBC regulon, including many novel genes. We have also demonstrated that transcripts of rcsA, rcsC, and rcsD genes but not the rcsB gene were up-regulated when bacterial cells were grown in minimal medium or following infection of pear fruits compared with those grown in Luria Bertani medium. Furthermore, using the genome of E. amylovora ATCC 49946, a hidden Markov model predicted 60 genes with a candidate RcsB binding site in the intergenic region, 28 of which were identified in the microarray assay. Based on these findings as well as previous reported data, a working model has been proposed to illustrate how the Rcs phosphorelay system regulates virulence gene expression in E. amylovora.

RcsB plays a central role in H-NS-dependent regulation of motility and acid stress resistance in Escherichia coli

In Escherichia coli, hns mutants lack flagellar motility and display an increase in acid stress resistance. Spontaneous phenotypic revertants showed reversion of both H-NS-controlled phenotypes. In the present study, suppressor mutations were identified in the rcsB gene. In addition to RcsA, our experiments establish that H-NS indirectly controlled the RcsB regulator via repression of RcsD. We also show that RcsBD56E, mimicking phosphorylated RcsB, interacts with GadE to form a RcsB-P/GadE complex, a general direct regulator of glutamate-, arginine- and lysine-dependent acid resistance pathways. In addition, we showed that H-NS positively affects motility via the flhDC master operon repression by RcsB. This substantiates the central role of RcsB in H-NS-mediated control of motility and acid stress resistance.

Identification of a new promoter for the response regulatorrcsBexpression inSalmonella entericaserovar Typhimurium

The RcsCDB (Rcs) phosphorelay system regulates capsule synthesis, flagella production and other cellular activities in several enteric bacteria. This system consists of three proteins: the sensor RcsC, the cognate response regulator RcsB and the histidine-containing phosphotransfer protein RcsD (YojN), which is hypothesized to act as an intermediary in the phosphotransfer from RcsC to RcsB. The rcsC gene is convergently transcribed toward rcsB, which follows rcsD in what appears to be a two-gene operon. Here, it is reported that the overproduction of the rcsB gene represses rcsD transcription, but has a weak effect on its own expression. We demonstrated that the differential rcsD and rcsB expression is due to the activity of two promoters to transcribe the rcsB gene: (1) P(rcsDB) located upstream of rcsD and (2) P(rcsB) located within the rcsD coding region. In addition, here it was demonstrated that in Salmonella typhimurium, P(rcsB) is important to activate the rcsB expression during the stationary growth phase.

Repression of the RcsC-YojN-RcsB phosphorelay by the IgaA protein is a requisite for Salmonella virulence.

Bacterial pathogenesis relies on regulators that activate virulence genes. Some of them act, in addition, as repressors of specific genes. Intracellular-growth-attenuator-A (IgaA) is a Salmonella enterica membrane protein that prevents overactivation of the RcsC-YojN-RcsB regulatory system. This negative control is critical for growth because disruption of the igaA gene is only possible in rcsC, yojN or rcsB strains. In this work, we examined the contribution of this regulatory circuit to virulence. Viable igaA point mutant alleles were isolated and characterized. These alleles encode IgaA variants leading to different levels of activation of the RcsC-YojN-RcsB system. IgaA-mediated repression of the RcsB-YojN-RcsC system occurred at the post-translational level, as shown by chromosomal epitope tagging of the rcsC, yojN and rcsB genes. The activity of the RcsC-YojN-RcsB system, monitored with the product of a tagged gmd-3xFLAG gene (positively regulated by RcsC-YojN-RcsB), was totally abolished by wild-type bacteria in mouse target organs. Such tight repression occurred only in vivo and was mediated by IgaA. Shutdown of the RcsC-YojN-RcsB system is a requisite for Salmonella virulence since all igaA point mutant strains were highly attenuated. The degree of attenuation correlated to that of the activation status of RcsC-YojN-RcsB. In some cases, the attenuation recorded was unprecedented, with competitive index (CI) values as low as 10(-6). Strikingly, IgaA is a protein absolutely dispensable for virulence in mutant strains having a non-functional RcsC-YojN-RcsB system. To our knowledge, IgaA exemplifies the first protein that contributes to virulence by exclusively acting as a negative regulator upon host colonization.

Null mutations in the essential gene yrfF ( mucM) are not lethal in rcsB, yojN or rcsC strains of Salmonella enterica serovar Typhimurium

Insertion of factor MudJ in the intergenic region between divergent genes yrfF and yrfE, at centisome 76 in the genome of Salmonella enterica serovar Typhimurium LT2, confers the characteristics recently described for mucM mutants, i.e. mucoidy and resistance to mecillinam. Cloning of the intergenic region plus either the yrfF or the yrfE gene in a multicopy plasmid showed that only the plasmid carrying the yrfF gene complemented mucM mutants, thus suggesting that mucM mutations are in fact yrfF mutations. A null yrfF mutation obtained by insertion of a kanamycin cassette into the yrfF open reading frame ( yrfF28::Kan) produced abortive colonies when transduced to a wild-type strain but was normally accepted by rcsB, rcsC or yojN strains. Neither mutations preventing synthesis of the capsular exopolysaccharide colanic acid ( cps, galE) nor rcsA mutations, which reduce expression of cps genes, conferred tolerance to the lethal yrfF28::Kan mutation. Spontaneous suppressor mutations arose very frequently in abortive yrfF28::Kan colonies, and all of them affected either rcsC, yojN, or rcsB genes. Thus, the lethal effect caused by inactivation of gene yrfF appears to be mediated by a function that is dependent on the rcsC- yojN- rcsB phosphorelay system but does not involve synthesis of colanic acid.

REGULATION AND BIOCHEMISTRY OF EXOPOLYSACCHARIDE SYNTHESIS OF THE FIRE BLIGHT PATHOGEN ERWINIA AMYLOVORA AND PEAR PATHOGENS FROM KOREA AND JAPAN

The fire blight pathogen Erwinia amylovora and the Asian pear pathogens synthesize similar capsular exopolysaccharides (EPS), which are strictly required for pathogenicity. The molecular weight of the EPS is at least 1 MDa. The repeating units of EPS synthesized by Erwinia pyrifoliae from Korea and the Erwinia strains from Japan do not bear a glucose residue as a second side chain found in 60% of the repeating units of amylovoran. Levan, a homopolymer of fructose synthesized by the secreted enzyme levansucrase of E. amylovora, is not produced by the Asian pear pathogens. Amylovoran and levan expression are controlled by regulatory genes and affected differently by the phosphate and chloride concentrations of the environment. Alignments of rcsB genes, and also of the nucleotide sequences upstream of the gene cluster for EPS synthesis, show a significant relationship between the pear pathogens and an evolutionary distance to E. amylovora. Three activator proteins for expression of levansucrase have been identified; RlsA, RlsB, and RlsC. The H-NS protein binds to a DNA fragment with the start of lsc. From a bacteriophage, a DNA fragment was sequenced with genes encoding a lysozyme and an EPS depolymerase. The enzyme was used to degrade amylovoran capsules and shown to interfere with colonization of pear slices by E. amylovora.

Characterization of the rcsB gene from Erwinia amylovora and its influence on exoploysaccharide synthesis and virulence of the fire blight pathogen.

RcsB belongs to a family of positive regulators of exopolysaccharide synthesis in various enterobacteria. The rcsB gene of the fire blight pathogen Erwinia amylovora was cloned by PCR amplification with consensus primers, and its role in exopolysaccharide (EPS) synthesis was investigated. Its overexpression from high-copy-number plasmids stimulated the synthesis of the acidic EPS amylovoran and suppressed expression of the levan-forming enzyme levansucrase. Inactivation of rcsB by site-directed mutagenesis created mutants that were deficient in amylovoran synthesis and avirulent on host plants. In addition, a cosmid which complemented rcsB mutants was selected from a genomic library. The spontaneous E. amylovora mutant E8 has a similar phenotype and was complemented by the cloned rcsB gene. The rcsB region of strain E8 was also amplified by PCR, and the mutation was characterized as a nine-nucleotide deletion at the start of the rcsB gene. Nucleotide sequence analysis of the E. amylovora rcsB region and the predicted amino acid sequence of RcsB revealed extensive homology to rcsB and the encoded protein of other bacteria such as Escherichia coli and Erwinia stewartii. In all three organisms, rcsB is localized adjacent to the rcsC gene, which is transcribed in the opposite direction of rcsB. The E. amylovora rcsB gene has now been shown to strongly affect the formation of disease symptoms of a plant pathogen.

Characterization of the rcsA and rcsB genes from Salmonella typhi: rcsB through tviA is involved in regulation of Vi antigen synthesis

Synthesis of Vi antigen, a capsular polysaccharide expressed by Salmonella typhi, is controlled by the viaA and viaB chromosomal loci. It was previously shown that Vi antigen expression was regulated by a system similar to the rcs regulatory system involved in colanic acid synthesis in Escherichia coli. We have cloned the rcsA, rcsB, and rcsC genes from S. typhi. The predicted amino sequences of the RcsA and RcsB proteins showed a high degree of similarity to their E. coli homologs. The nucleotide sequence of the rcsC gene was partially determined and was shown to be homologous to that of its E. coli counterpart. Complementation experiments indicated that rcsB and rcsC were encompassed within the viaA locus. The RcsA protein was not involved in Vi antigen synthesis. In contrast, the RcsB protein acted as a positive regulator of Vi polysaccharide expression. By mRNA and gene fusion analyses, we studied the role of RcsB and TviA, a via-B-encoded regulatory protein characterized previously, in regulating Vi antigen synthesis. The transcriptional start point of tviA mRNA was not influenced by RcsB or TviA. In the absence of RcsB or TviA protein, transcription of tviA gave rise to only a monocistronic tviA-specific mRNA. The presence of RcsB and TriA not only increased the amount of monocistronic tviA-specific mRNA but also resulted in countranscription of tviA and tviB, which is located immediately downstream of tviA on the viaB locus. In addition, TviA protein did not appear to be subject to degradation by the Lon protease. These results strongly suggest that TviA might act in concert with RcsB at the tviA promoter to activate transcription of the genes involved in Vi polymer synthesis in S. typhi in a Lon-independent manner.

Coexpression of colanic acid and serotype-specific capsular polysaccharides in Escherichia coli strains with group II K antigens

In Escherichia coli K-12, the rcsA and rcsB gene products are positive regulators in expression of the slime polysaccharide colanic acid. We have previously demonstrated the presence of rcsA sequences in E. coli K1 and K5, strains with group II capsular K antigens, and shown that introduction of multicopy rcsA into these strains results in the expression of colanic acid. We report here the presence of rcsB sequences in E. coli K1 and K5 and demonstrate that RcsB also plays a role in the biosynthesis of colanic acid in strains with group II K antigens. In E. coli K1 and K5 grown at 37 degrees C, multicopy rcsB and the resulting induction of colanic acid synthesis had no significant effect on synthesis of the group II K antigens. K-antigen-specific sugar transferase activities were not significantly different in the presence or absence of multicopy rcsB, and introduction of a cps mutation to eliminate colanic acid biosynthesis in a K1-derivative strain did not influence the activity of the polysialyltransferase enzyme responsible for synthesis of the K1 polymer. Furthermore, immunoelectron microscopy showed no detectable difference in the size or distribution of the group II K-antigen capsular layer in cells which produced colanic acid. Colanic acid expression therefore does not appear to significantly affect synthesis of the group II K-antigen capsule and, unlike for group I K antigens, expression of group II K antigens is not positively regulated by the rcs system.

Characterization of rcsB and rcsC from Escherichia coli O9:K30:H12 and examination of the role of the rcs regulatory system in expression of group I capsular polysaccharides

In Escherichia coli K-12, RcsC and RcsB are thought to act as the sensor and effector components, respectively, of a two-component regulatory system which regulates expression of the slime polysaccharide colanic acid (V. Stout and S. Gottesman, J. Bacteriol. 172:659-669, 1990). Here, we report the cloning and DNA sequence of a 4.3-kb region containing rcsC and rcsB from E. coli O9:K30:H12. This strain does not produce colanic acid but does synthesize a K30 (group I) capsular polysaccharide. The rcsB gene from E. coli K30 (rcsBK30) is identical to the rcsB gene from E. coli K-12 (rcsBK-12). rcsCK30 has 16 nucleotide changes, resulting in six amino acid changes in the predicted protein. To examine the function of the rcs regulatory system in expression of the K30 capsular polysaccharide, chromosomal insertion mutations were constructed in E. coli O9:K30:H12 to independently inactivate rcsBK30 and the auxiliary positive regulator rcsAK30. Strains with these mutations maintained wild-type levels of K30 capsular polysaccharide expression and still produced a K30 capsule, indicating that the rcs system is not essential for expression of low levels of the group I capsular polysaccharide in lon+ E. coli K30. However, K30 synthesis is increased by introduction of a multicopy plasmid carrying rcsBK30. K30 polysaccharide expression is also markedly elevated in an rcsBK30-dependent fashion by a mutation in rcsCK30, suggesting that the rcs system is involved in high levels of synthesis. To determine whether the involvement of the rcs system in E. coli K30 expression is typical of group I (K antigen) capsules, multicopy rcsBK30 was introduced into 22 additional strains with structurally different group I capsules. All showed an increase in mucoid phenotype, and the polysaccharides produced in the presence and absence of multicopy rcsBK30 were examined. It is has been suggested that E. coli strains with group I capsules can be subdivided based on K antigen structure. For the first time, we show that strains with group I capsules can also be subdivided by the ability to produce colanic acid. Group IA contains capsular polysaccharides (including K30) with repeating-unit structures lacking amino sugars, and expression of group IA capsular polysaccharides is increased by multicopy rcsBK30. Group IB capsular polysaccharides all contain amino sugars. In group IB strains, multicopy rcsBK30 activates synthesis of colanic acid.

The rcsB gene, a positive regulator of colanic acid biosynthesis in Escherichia coli, is also an activator of ftsZ expression

Wild-type genes which, when overexpressed, are capable of restoring the growth deficiency of the division mutant ftsZ84 of Escherichia coli on L medium containing no added NaCl have been isolated. One of these genes is rcsB, a positive regulator of colanic acid biosynthesis. A direct relationship between rcsB expression and FtsZ activity was observed, suggesting that RcsB specifically increases transcription of ftsZ, thus accounting for the restoration of colony formation by ftsZ84 mutant cells. Analysis of the 5' upstream sequence of rcsB revealed, in addition to the sigma 54 promoter sequence previously reported, a presumptive sigma 70 promoter and LexA-binding site plus an upstream sequence that is found to be essential for the expression of rcsB on a plasmid. The absence of the sigma 54 factor does not have a negative effect on the transcription of rcsB. The RcsB protein is an activator of its own synthesis, particularly in the presence of NaCl. Evidence which suggests that RcsB can be phosphorylated by a presumably modified EnvZ or PhoM sensor protein leading to a suppression of the growth deficiency of ftsZ84 mutant cells and to an increase in colanic acid production was obtained. We also demonstrated that the level of colanic acid is reduced when the cells carry a multicopy rcsC plasmid, suggesting that the RcsC sensor has phosphatase activity.