PapB paralogues and their effect on the phase variation of type 1 fimbriae in Escherichia coli.

Recent work has demonstrated that expression of type 1 fimbriae is repressed by PapB, a regulator of pyelonephritis-associated pili (P-pili). PapB belongs to family of related adhesin regulators, for which consensus residues required for DNA binding and oligomerization have been identified. Of the regulators tested in this study, PapB, SfaB (S-fimbriae) and PefB (Salmonella enterica serovar Typhimurium--plasmid-encoded fimbriae) repressed FimB-promoted off-to-on inversion of the fim switch, although complete repression was only demonstrated by PapB. DaaA, FaeB, FanA, FanB and ClpB had no effect on fim switching. In addition, only PapB stimulated FimE-promoted on-to-off inversion. Deletion analysis demonstrated that this specificity resides in the carboxy terminal of the protein, and not the amino terminal, with the central region being homologous among the family members. Exchange of Leu(82) and Ile(83) of PapB for the equivalent residues from the DaaA protein (Phe and Gln) within the carboxy terminal virtually abolished cross-talk activity. Whereas PapB can bind to a region around the left inverted repeat of the fim switch, DaaA and the PapB double mutant were effectively unable to bind this region. A previously characterized PapB DNA binding mutant also failed to bind to this region and failed to inhibit FimB activity at the fim switch. Thus, repression of fim expression appears unique to PapB and SfaB within E. coli and requires DNA binding involving amino acid residues located both within the homologous core and in the heterogeneous carboxy terminus. The variation in the carboxy terminus between the PapB family members explains their differential effects on fim. This mechanism of cross-talk seems restricted to the P and S family adhesins with type 1 fimbriae and may ensure variable and sequential expression of adhesins during urinary tract infections.

Heteromeric interactions among nucleoid-associated bacterial proteins: localization of StpA-stabilizing regions in H-NS of Escherichia coli.

The nucleoid-associated proteins H-NS and StpA in Escherichia coli bind DNA as oligomers and are implicated in gene regulatory systems. There is evidence for both homomeric and heteromeric H-NS-StpA complexes. The two proteins show differential turnover, and StpA was previously found to be subject to protease-mediated degradation by the Lon protease. We investigated which regions of the H-NS protein are able to prevent degradation of StpA. A set of truncated H-NS derivatives was tested for their ability to mediate StpA stability and to form heteromers in vitro. The data indicate that H-NS interacts with StpA at two regions and that the presence of at least one of the H-NS regions is necessary for StpA stability. Our results also suggest that a proteolytically stable form of StpA, StpA(F21C), forms dimers, whereas wild-type StpA in the absence of H-NS predominantly forms tetramers or oligomers, which are more susceptible to proteolysis.

Silencing and activation of ClyA cytotoxin expression in Escherichia coli.

Cytolysin A (ClyA) is a pore-forming cytotoxic protein encoded by the clyA gene of Escherichia coli K-12. Genetic analysis suggested that clyA is silenced by the nucleoid protein H-NS. Purified H-NS protein showed preferential binding to clyA sequences in the promoter region, as evidenced by DNase I footprinting and gel mobility shift assays. Transcriptional derepression and activation of a chromosomal clyA::luxAB operon fusion were seen under conditions of H-NS deficiency and SlyA overproduction, respectively. In H-NS-deficient bacteria neither the absence nor the overproduction of SlyA affected the derepressed ClyA expression any further. Therefore, we suggest that overproduction of SlyA in hns(+) E. coli derepresses clyA transcription by counteracting H-NS. The cyclic AMP receptor protein (CRP) was required for ClyA expression, and it interacted with a predicted, albeit suboptimal, CRP binding site in the clyA upstream region. Site-specific alterations of the CRP binding site to match the consensus resulted in substantially higher levels of ClyA expression, while alterations that were predicted to reduce CRP binding reduced ClyA expression. During anaerobic growth the fumarate and nitrate reduction regulator (FNR) was important for ClyA expression, and the clyA gene could be activated by overexpression of FNR. A major clyA transcript having its 5' end (+1) located 72 bp upstream of the translational start codon and 61 bp downstream of the CRP-FNR binding site was detected in the absence of H-NS. The clyA promoter was characterized as a class I promoter that could be transcriptionally activated by CRP and/or FNR. According to DNA bending analyses, the clyA promoter region has high intrinsic curvature. We suggest that it represents a regulatory region which is particularly susceptible to H-NS silencing, and its features are discussed in relation to regulation of other silenced operons.

Nucleoid proteins stimulate stringently controlled bacterial promoters: a link between the cAMP-CRP and the (p)ppGpp regulons in Escherichia coli.

We report that the H-NS nucleoid protein plays a positive role in the expression of stringently regulated genes in Escherichia coli. Bacteria lacking both H-NS and the paralog StpA show reduced growth rate. Colonies displaying an increased growth rate were isolated, and mapping of a suppressor mutation revealed a base pair substitution in the spoT gene. The spoT(A404E) mutant showed low ppGpp synthesizing ability. The crp gene, which encodes the global regulator CRP, was subject to negative stringent regulation. The stable RNA/protein ratio in an hns, stpA strain was decreased, whereas it was restored in the suppressor strain. Our findings provide evidence of a direct link between the cAMP-CRP modulon and the stringent response.

Cytocidal and apoptotic effects of the ClyA protein from Escherichia coli on primary and cultured monocytes and macrophages.

Cytolysin A (ClyA) is a newly discovered cytolytic protein of Escherichia coli K-12 that mediates a hemolytic phenotype. We show here that highly purified ClyA and ClyA-expressing E. coli were cytotoxic and apoptogenic to fresh as well as cultured human and murine monocytes/macrophages.

Regulatory cross-talk between adhesin operons in Escherichia coli: inhibition of type 1 fimbriae expression by the PapB protein.

Pathogenic Escherichia coli often carry determinants for several different adhesins. We show a direct communication between two adhesin gene clusters in uropathogenic E.coli: type 1 fimbriae (fim) and pyelonephritis-associated pili (pap). A regulator of pap, PapB, is a key factor in this cross-talk. FimB recombinase turns on type 1 fimbrial expression, and PapB inhibited phase transition by FimB in both off-to-on and on-to-off directions. On-to-off switching requiring FimE was increased by PapB. By analysis of FimB- and FimE-LacZ translational fusions it was concluded that the increase in on-to-off transition rates was via an increase in FimE expression. Inhibition of FimB-promoted switching was via a different mechanism: PapB inhibited FimB-promoted in vitro recombination, indicating that FimB activity was blocked at the fim switch. In vitro analyses showed that PapB bound to several DNA regions of the type 1 fimbrial operon, including the fim switch region. These data show that Pap expression turns off type 1 fimbriae expression in the same cell. Such cross-talk between adhesin gene clusters may bring about appropriate expression at the single cell level.

Transfer RNA modification, temperature and DNA superhelicity have a common target in the regulatory network of the virulence of Shigella flexneri: the expression of the virF gene.

Full expression of the virulence genes of Shigella flexneri requires the presence of two modified nucleosides in the tRNA [queuosine, Q34, present in the wobble position (position 34) and 2-methylthio-N6-isopentenyladenosine (ms2i6A37, adjacent to and 3' of the anticodon)]. The synthesis of these two nucleosides depends on the products of the tgt and miaA genes respectively. We have shown that the intracellular concentration of the virulence-related transcriptional regulator VirF is reduced in the absence of either of these modified nucleosides. The intracellular concentration of VirF is correlated with the expression of the virulence genes. Overproduction of VirF in the tgt and the miaA mutants suppressed the less virulent (tgt) or the avirulent (miaA) phenotypes respectively, caused by the tRNA modification deficiency. This suggests that the primary result of undermodification of the tRNA is a poor translation of virF mRNA and not of any other mRNA whose product acts downstream of the action of VirF. Shigella showed no virulence phenotypes at 30 degrees C, but forced synthesis of VirF at 30 degrees C induced the virulence phenotype at this low temperature. In addition, removal of the known gene silencer H-NS by a mutation in its structural gene hns increased the synthesis of VirF at low temperature and thus induced a virulent phenotype at 30 degrees C. Conversely, decreased expression of VirF at 37 degrees C induced by the addition of novobiocin, a known inhibitor of gyrase, led to an avirulent phenotype. We conclude that tRNA modification, temperature and superhelicity have the same target - the expression of VirF - to influence the expression of the central regulatory gene virB and thereby the virulence of Shigella. These results further strengthen the suggestion that the concentration of VirF is the critical factor in the regulation of virulence in Shigella. In addition, they emphasize the role of the bacterial translational machinery in the regulation of the expression of virulence genes which appears here quantitatively as important as the well-established regulation on the transcriptional level.

Differential protease-mediated turnover of H-NS and StpA revealed by a mutation altering protein stability and stationary-phase survival of Escherichia coli.

The Escherichia coli proteins H-NS is recognized as an important component among the major nucleoid-associated proteins. In studies of E. coli strains with defects in H-NS, we discovered a mutant that phenotypically restored stationary-phase viability (Rsv) of such strains. The Rsv phenotype was the result of a mutation that led to severalfold higher levels of the functionally and structurally related StpA protein. This mutation was a base pair change in the stpA structural gene, and the amino acid substitution in the StpA protein altered its turnover properties, suggesting a role for this residue in a cleavage site for proteolysis. We determined the stability of the StpA and the H-NS proteins and found that the StpA protein was degraded relatively rapidly in strains lacking functional H-NS, whereas H-NS remained stable irrespective of the presence/absence of StpA. Using protease-deficient mutants, we obtained evidence that the Lon protease was responsible for the degradation of StpA. The differential turnover of the nucleoid-associated proteins is suggested to contribute to the regulation of their stoichiometry and ratio in terms of homo- and heteromer formation. We conclude that StpA, in contrast to H-NS, is present mainly in heteromeric form in E. coli.

Mutational analysis of the PapB transcriptional regulator in Escherichia coli. Regions important for DNA binding and oligomerization.

PapB is a transcriptional regulator in the control of pap operon expression in Escherichia coli. There are PapB homologous proteins encoded by many fimbrial gene systems that are involved in the regulation of fimbriae-adhesin production, and previous studies suggested that PapB binds DNA through minor groove contact. Both deletion and alanine-scanning mutagenesis were used to identify functionally important regions of the PapB protein. Mutations altering Arg61 or Cys65 caused deficiency in DNA binding, indicating that these residues are critical for PapB binding to DNA. Alanine substitutions at positions 35-36, 53-56, and 74-76 resulted in mutants that were impaired in oligomerization. All these amino acid residues are conserved among the PapB homologous proteins, suggesting their importance in the whole family of regulatory proteins. The transcriptional efficiency of all the mutants was clearly reduced as compared with that of wild-type PapB. Taken together, we have localized regions in the PapB protein that are involved in DNA binding and oligomerization, and our results show that both functions are required for its activity as a transcriptional regulator.

Molecular analysis of the cytolytic protein ClyA (SheA) from Escherichia coli.

Escherichia coli K-12 carries a gene for a protein denoted ClyA or SheA that can mediate a cytolytic phenotype. The ClyA protein is not expressed at detectable levels in most strains of E. coli, but overproduction suitable for purification was accomplished by cloning the structural gene in an hns mutant strain. Highly purified ClyA protein was cytotoxic to macrophage cells in culture and caused detachment and lysis of the mammalian cells. Results from osmotic protection assays were consistent with the suggestion that the protein formed pores with a diameter of up to 3 nm. Using Acholeplasma laidlawii cells and multilamellar liposomes, we studied the effect of ClyA on membranes with varying compositions and in the presence of different ions. ClyA induced cytolytic release of the fluorescent tracer from carboxyfluorescein-loaded liposomes, and the release was stimulated if cholesterol was present in the membranes whereas addition of calcium had no effect. Pretreatment of the ClyA protein with cholesterol inhibited the pore formation, suggesting that ClyA could bind to cholesterol. Efficient coprecipitation of ClyA with either cholesterol or 1,2,3-trioctadecanoylglycerol in aqueous solutions showed that ClyA directly interacted with the hydrophobic molecular aggregates. We tested the possible functional importance of selected ClyA protein regions by site-directed mutagenesis. Defined mutants of ClyA were obtained with alterations in postulated transmembrane structures in the central part and in a postulated membrane-targeting domain in the C-terminal part. Our results were consistent with the suggestion that particular amphiphilic segments are required for ClyA activity. We propose that these domains are necessary for ClyA to form pores.

Alterations in protein expression caused by the hha mutation in Escherichia coli: influence of growth medium osmolarity.

The Hha protein belongs to a new family of regulators involved in the environmental regulation of virulence factors. The aim of this work was to study the effect of the hha mutation on the overall protein pattern of Escherichia coli cells by two-dimensional polyacrylamide gel electrophoresis. The growth medium osmolarity clearly influenced the effect of the hha mutation. The number of proteins whose expression was altered in hha cells, compared with wild-type cells, was three times larger at a high osmolarity than at a low osmolarity. Among the proteins whose expression was modified by the hha allele, both OmpA and protein IIAGlc of the phosphotransferase system could be identified. As this latter enzyme participates in the regulation of the synthesis of cyclic AMP and hence influences the catabolite repression system, we tested whether the expression of the lacZ gene was also modified in hha mutants. This was the case, suggesting that at least some of the pleiotropic effects of the hha mutation could be caused by its effect on the catabolite repression system.

An apoptotic response by J774 macrophage cells is common upon infection with diarrheagenic Escherichia coli.

Representative strains of the different diarrheagenic Escherichia coli virotypes were tested for their potential cytotoxicity in the J774 macrophage cell line. All the seven virotypes of E. coli were cytotoxic to J774 macrophages, and in most cases the bacteria induced an apoptotic response. With the exception of the enterotoxigenic E. coli (ETEC) strain, all the other six virotypes caused induction of apoptosis as evidenced by quantitative analysis of the characteristic DNA fragmentation at the individual cell level. These results suggest that apoptosis could be one of the mechanisms contributing to the diarrheal disease development.

Oligomeric interaction of the PapB transcriptional regulator with the upstream activating region of pili adhesin gene promoters in Escherichia coli.

Transcriptional regulation of the pap genes, which encode fimbrial adhesins in uropathogenic Escherichia coli, depends on an upstream activating region. This region contains binding sites for a transcription factor, PapB, which is a member of a growing family of putative regulatory proteins found in several virulence-associated fimbrial gene systems. To assess the nature of the PapB binding sites, we studied different naturally occurring variants and a number of in vitro constructed mutant binding sites. DNase I footprinting analysis and visualization of the PapB-DNA complex by atomic force microscopy showed that the protein occupied a DNA region of more than 50 bp. Purified PapB protein was shown to recognize a motif including a 9 bp repeat sequence containing T/A triplets at a conserved position. PapB binding was affected by distamycin, and the results were consistent with the possibility that the binding to DNA occurred through minor groove interaction. From these analyses and estimation of the relative number of PapB proteins per binding site, we suggest that PapB binds the DNA in an oligomeric fashion and may function as an architectural factor in the transcriptional control of adhesin expression.

H-NS and StpA proteins stimulate expression of the maltose regulon in Escherichia coli.

The nucleoid-associated protein H-NS is a major component of the chromosome-protein complex, and it is known to influence the regulation of many genes in Escherichia coli. Its role in gene regulation is manifested by the increased expression of several gene products in hns mutant strains. Here we report findings showing that H-NS and the largely homologous protein StpA play a positive role in the expression of genes in the maltose regulon. In studies with hns mutant strains and derivatives also deficient in the stpA gene, we found that expression of the LamB porin was decreased. Our results showed that the amounts of both LamB protein and lamB mRNA were greatly reduced in hns and hns-stpA mutant strains. The same results were obtained when we monitored the amount of transcription from the malEFG operon. The lamB gene is situated in the malKlamBmalM operon, which forms a divergent operon complex together with the malEFG operon. The activation of these genes depends on the action of the maltose regulon activator MalT and the global activator cyclic AMP receptor protein. Using a malT-lacZ translational fusion and antiserum raised against MalT to measure the expression of MalT, we detected reduced MalT expression in hns and hns-stpA mutant strains in comparison with the wild-type strain. Our results suggest that the H-NS and StpA proteins stimulate MalT translation and hence play a positive role in the control of the maltose regulon.

Coordinated and differential expression of histone-like proteins in Escherichia coli: regulation and function of the H-NS analog StpA.

The histone-like protein H-NS has been shown to influence the regulation of gene expression at the transcriptional level in several Escherichia coli operons. We have examined the regulation of the stpA gene, which encodes a protein sharing 58% identity with H-NS, by mRNA analysis and by using stpA-lacZ operon fusions. The expression of stpA is temperature dependent, with 2-fold higher expression at 37 degrees C than at 26 degrees C. In addition, stpA expression is stimulated by the global regulator Lrp. In an hns mutant E.coli derivative stpA expression is derepressed, suggesting that regulation of the two genes is coupled. Overproduction of the StpA protein affects expression from at least four hns regulated operons (the papB, proU, bgl and hns operons), in both the presence and absence of H-NS. The construction of E.coli strains carrying mutations in both stpA and hns demonstrated that the absence of both proteins affects growth rate and viability of the cells. Our work establishes that E.coli can express two H-NS-like proteins with coordinated yet differential regulation. Evidently, these proteins have both overlapping and distinct functions in the cell, and they are both important for normal cell growth and gene control.

In vitro analysis of mRNA processing by RNase E in the pap operon of Escherichia coli.

Differential gene expression from operons encoding fimbrial adhesins in Escherichia coli involves processing and differential decay of polycistronic transcripts. Previous analyses of mRNA processing in vivo using ribonuclease mutants of E. coli have given different results with the different fimbrial gene systems tested. For the pap operon from uropathogenic E. coli, the results suggested that the mRNA processing is dependent on ribonuclease E (RNase E), whereas in other fimbrial operons with similar genetic organisation, the processing was concluded to be RNase E independent. We have developed an in vitro system allowing us to assess the cleavage of pap mRNA, to study the mRNA processing of a fimbrial operon in more detail, and to define the enzymatic activity and target. The results of this study establish that RNase E does indeed cleave the papBA intercistronic transcript. Analysis of the cleavage products reveals that in vitro RNase E can cleave the mRNA at other positions in addition to the site preferentially cleaved in vivo. The specificity of the cleavage pattern was assessed using transcripts derived from mutants with base substitutions near, or within, the major in vivo cleavage site. Such mutants have alternative cleavage sites. A common feature of the different cleavage sites is a high A/U nucleotide content, similar to other known RNase E cleavage sites. Features of the secondary structure of the papBA intercistronic mRNA were investigated using single-strand-specific and double-strand-specific nucleases. The secondary structure model derived from stability calculations and our results from the nuclease-probing experiments indicate that the positions subject to RNase E cleavage are mainly single stranded and flanked by more stable stem-loop structures. The results are consistent with the notion that an mRNA conformation exposing A/U-rich, non-paired regions constitutes the target, i.e. a flexible determinant, for processing by RNase E in the pap transcript. The findings are discussed in relation to the existence of a potential recognition site for RNase E and the analysis of RNase E cleavages in other RNA molecules.