Global regulators and environmental adaptation in Gram-negative pathogens.

A powerful combination of single-gene studies and whole genome approaches has provided a wealth of information about the regulatory circuits used by bacteria to adapt to the environmental changes that are encountered during infection. The facultative intracellular pathogen Salmonella enterica will be used to illustrate how global regulators such as the nucleoid-associated proteins Fis and H-NS collaborate with fluctuations in the superhelicity of the DNA template to modify the gene expression profile of the bacterial cell outside and inside the host.

The gyr genes of Salmonella enterica serovar Typhimurium are repressed by the factor for inversion stimulation, Fis.

The DNA sequence of the gyr genes from Salmonella enterica serovar Typhimurium revealed strong similarity between gyrB and its counterpart in Escherichia coli. However, the gyrA gene showed similarity to the E. coli homologue only downstream from the Pribnow box of the promoter, with the sequence upstream diverging markedly. Since this region encompasses the binding sites for the Fis DNA binding protein in E. coli, we investigated the possibility that the gyrA genes in the two species might differ in their responses to this regulatory protein. Fis was found to act as a transcriptional repressor of both gyr genes in S. enterica. In electrophoretic mobility shift assays, Fis was found to bind to both the gyrA and gyrB promoters of S. enterica, despite the strong divergence from the E. coli sequence on the part of the former. The binding sites were mapped by DNase I protection assays, and the results are consistent with conservation of the mechanism of Fis-mediated repression between the two bacterial species.

Shigella flexneri 2a strain 2457T expresses three members of the H-NS-like protein family: characterization of the Sfh protein.

Shigella flexneri 2a is known to express the H-NS nucleoid-structuring protein and the paralogous protein StpA. Using bioinformatic analysis we have now discovered a third member of the H-NS protein family, Sfh (Shigella flexneri H-NS-like protein), in strain 2457T. This protein is encoded by the sfh gene, which is located on a high-molecular-mass plasmid that is closely related to the self-transmissible plasmid R27. When expressed in Escherichia coli, the Sfh protein can complement an hns null mutation, restoring wild-type Bgl, porin protein, and mucoidy phenotypes, and wild-type expression of the fliC and proU genes. While a knockout mutation in the sfh gene alone had no effect on the expression of virulence genes in S. flexneri, an additive effect on virulence gene derepression was seen when the sfh lesion was combined with a mutation in hns. Over-expression of the sfh gene repressed expression of the VirB virulence regulatory protein and transcription of a VirB-dependent structural gene promoter. The purified Sfh protein bound specifically to DNA sequences containing the promoters of the virF and virB virulence regulatory genes. These findings show that Sfh has the ability to influence genetic events beyond the genetic element that encodes it, including the expression of the S. flexneri virulence genes. They raise the possibility of a triangular relationship among three closely related proteins with broad consequences for genetic events in the bacterium that harbours them.

Requirement for the molecular adapter function of StpA at the Escherichia coli bgl promoter depends upon the level of truncated H-NS protein.

Truncated derivatives of the Escherichia coli nucleoid-associated protein H-NS that lack the DNA-binding domain remain competent for silencing of the cryptic bgl operon in vivo. Previous studies have provided evidence for the involvement of either the homologous nucleoid protein StpA or the alternative sigma factor RpoS in this unusual silencing mechanism. Here, we rationalize this apparent discrepancy. We show that two hns alleles (hns-205::Tn10 and hns60), which produce virtually identical amino-terminal fragments of H-NS, have very different requirements for StpA to mediate bgl silencing. The hns60 allele produces a high level of truncated H-NS, which can overcome the absence of StpA, whereas the lower level expressed by hns-205::Tn10 requires StpA for silencing. Reversing the relative levels of the two H-NS fragments reverses their requirement for StpA to silence bgl transcription. This suggests that the amino-terminal fragment of H-NS can be targeted to DNA to mediate silencing by multiple protein-protein interactions. The high-specificity interaction with StpA can function at low levels of truncated H-NS, whereas an alternative mechanism, perhaps involving lower specificity interactions with another protein(s), is only functional when truncated H-NS is abundant. These findings have important implications for the involvement of other proteins in H-NS-dependent transcriptional repression.

Regulation of virulence gene expression in Shigella flexneri, a facultative intracellular pathogen.

Shigella flexneri and its close relatives are facultative intracellular pathogens of humans and are the etiological agents of bacillary dysentery. These bacteria secrete proteins that enable them to enter human epithelial cells via an elaborate and fascinating cell biology. This behaviour depends on a complicated regulon of virulence genes, whose expression is controlled in response to a multiplicity of environmental signals. This review describes and attempts to interpret these gene control mechanisms.

A role for the Escherichia coli H-NS-like protein StpA in OmpF porin expression through modulation of micF RNA stability.

When a wild-type strain of Escherichia coli and its stpA, hns and stpA hns mutant derivatives were compared by two-dimensional protein gel electrophoresis, the levels of expression of several proteins were found to vary. One of these was identified as the outer membrane porin protein, OmpF. In the stpA hns double mutant, the level of OmpF was downregulated dramatically, whereas in hns or stpA single mutants, it was affected only slightly. Transcription from the ompF promoter was reduced by 64% in the double mutant; however, the level of ompF mRNA was reduced by 96%. This post-transcriptional expression was found to result from a strong reduction in the half-life of ompF message in the double mutant. The micF antisense RNA was shown to be involved in OmpF regulation by StpA using a strain deleted for micF. Moreover, micF antisense RNA accumulated considerably in an stpA hns background. Transcriptional data from a micF-lacZ fusion and measurements of micF RNA half-life confirmed that this was caused by transcriptional derepression of micF as a result of the hns lesion and increased micF RNA stability due to the absence of StpA (a known RNA chaperone). These data suggest a novel facet to the regulation of OmpF expression, namely destabilization of micF RNA by StpA.

The virulence plasmid of Salmonella typhimurium contains an autoregulated gene, rlgA, that codes for a resolvase-like DNA binding protein.

The virulence plasmid of Salmonella typhimurium contains a gene, rlgA, that shows strong homology to several reported resolvase-like proteins. This gene maps 5 kb upstream of spv locus, the major virulence determinant on the plasmid. Regulation of rlgA was studied using a lacZ transcriptional reporter fusion. The rlgA gene was found to be repressed at the level of transcription by its own product and to be expressed maximally in the late exponential phase of growth. The transcription start site of the rlgA gene was determined and the RlgA binding site was mapped and found to overlap with the transcription initiation signals. A derivative of the virulence plasmid was constructed with a knockout mutation in rlgA. This mutation did not alter the stability of the virulence plasmid nor did it affect the ability of S. typhimurium to cause systemic disease in mice.

DNA topology and adaptation of Salmonella typhimurium to an intracellular environment.

The expression of genes coding for determinants of DNA topology in the facultative intracellular pathogen Salmonella typhimurium was studied during adaptation by the bacteria to the intracellular environment of J774A.1 macrophage-like cells. A reporter plasmid was used to monitor changes in DNA supercoiling during intracellular growth. Induction of the dps and spv genes, previously shown to be induced in the macrophage, was detected, as was expression of genes coding for DNA gyrase, integration host factor and the nucleoid-associated protein H-NS. The topA gene, coding for the DNA relaxing enzyme topoisomerase I, was not induced. Reporter plasmid data showed that bacterial DNA became relaxed following uptake of S. typhimurium cells by the macrophage. These data indicate that DNA topology in S. typhimurium undergoes significant changes during adaptation to the intracellular environment. A model describing how this process may operate is discussed.

Effects of local transcription and H-NS on inversion of the fim switch of Escherichia coli.

The fim switch of Escherichia coli is responsible for phase-variable expression of type 1 fimbriae. Switching in the ON-to-OFF and OFF-to-ON directions is promoted by the FimB recombinase, while the FimE recombinase directs switching predominantly in the ON-to-OFF direction. The effects of local promoter activity and the H-NS nucleoid-associated protein on inversion of the switch were assessed. In contrast to FimB-mediated inversion, inversion of the switch by the FimE recombinase was unaffected by the H-NS status of the cell. Transcription towards the switch from within a translationally inactivated fimE gene was found to bias the switch strongly in the OFF direction, creating a FimE+-like phenotype in the absence of the FimE protein. This biasing was H-NS dependent and was also contingent on transcription from within the switch. These data show that local transcription and a nucleoid-associated protein both contribute to the modulation of a site-specific recombination event on the bacterial chromosome.

Interaction of the FimB integrase with the fimS invertible DNA element in Escherichia coli in vivo and in vitro.

The FimB protein is a site-specific recombinase that inverts the fimS genetic switch in Escherichia coli. Based on amino acid sequence analysis alone, FimB has been assigned to the integrase family of tyrosine recombinases. We show that amino acid substitutions at positions R47, H141, R144, and Y176, corresponding to highly conserved members of the catalytic motif of integrase proteins, render FimB incapable of inverting the fimS element in vivo. The arginine substitutions reduced the ability of FimB to bind to fimS in vivo or in vitro, while the substitution R144Q resulted in a protein unable to bind independently to the half sites located at the left end of fimS in phase-on bacteria. These data confirm that FimB is an integrase and suggest that residue R144 has a role in binding to a specific component of the fim switch.

Use of the stationary phase inducible promoters, spv and dps, to drive heterologous antigen expression in Salmonella vaccine strains.

We have investigated the ability of the growth phase regulated promoters dps and spv, to drive expression of heterologous antigens in Salmonella vaccine strains. Reporter plasmids were constructed which directed beta-galactosidase expression from dps (pDpslacZ) or spv (pSpvlacZ) and these were introduced independently into the Salmonella typhimurium vaccine strain SL3261 (aroA(-)). beta-galactosidase expression was induced 20-fold and 100-fold when broth cultures of SL3261 (pDpslacZ) or SL3261 (pSpvlacZ) respectively, entered the stationary phase of growth. Within macrophages, beta-galactosidase expression was induced 3.5-fold with SL3261 (pDpslacZ) and 7-fold with SL3261 (pSpvlacZ). The spv and dps promoters were used to drive independent expression of the C fragment domain of tetanus toxin (TetC) from plasmids harboured in S. typhimurium SL3261. Levels of anti-TetC antibodies were significantly higher in the sera of BALB/c mice perorally inoculated with SL3261 (pSpvtetC) or SL3261 (pDpstetC) compared to unvaccinated controls. This suggests that these promoter systems may be used to drive foreign antigen expression in live oral Salmonella vaccines.

Environmentally constrained mutation and adaptive evolution in Salmonella.

The relationship between environment and mutation is complex [1]. Claims of Lamarkian mutation [2] have proved unfounded [3-5]; it is apparent, however, that the external environment can influence the generation of heritable variation, through either direct effects on DNA sequence [6] or DNA maintenance and copying mechanisms [7-10], or as a consequence of evolutionary processes [11-16]. The spectrum of mutational events subject to environmental influence is unknown [6] and precisely how environmental signals modulate mutation is unclear. Evidence from bacteria suggests that a transient recombination-dependent hypermutational state can be induced by starvation [5]. It is also apparent that changes in the mutability of specific loci can be influenced by alterations in DNA topology [10,17]. Here we describe a remarkable instance of adaptive evolution in Salmonella which is caused by a mutation that occurs in intermediate-strength osmotic environments. We show that the mutation is not 'directed' and describe its genetic basis. We also present compelling evidence in support of the hypothesis that the mutational event is constrained by signals transmitted from the external environment via changes in the activity of DNA gyrase.

A role for the leucine-responsive regulatory protein and integration host factor in the regulation of the Salmonella plasmid virulence (spv ) locus in Salmonella typhimurium.

The Salmonella plasmid virulence (spv ) genes of Salmonella typhimurium are activated at the level of transcription as the bacteria enter stationary phase in vitro or in response to signals received during intracellular growth. Activation requires the LysR-like transcription factor SpvR and the alternative sigma factor RpoS. In this report, we show by biochemical and genetic analyses that two chromosomally encoded DNA-binding proteins contribute to the control of spv expression. These are the integration host factor (IHF), which binds to DNA sequences upstream of the spvR regulatory gene, and the leucine-responsive regulatory protein (Lrp), which binds to sequences upstream of the spvABCD operon. Under all conditions tested, inactivation of IHF expression reduces the level of spvR transcription by twofold. It also alters the response of the spv regulon to loss of DNA gyrase activity, consistent with a role for IHF in organizing DNA structure in the vicinity of the spvR promoter. Lrp represses spvA gene expression by up to fivefold and Lrp-mediated repression is antagonized by leucine. The Lrp binding site upstream of the spvA gene overlaps one of the binding sites for the positive regulator SpvR, suggesting a mechanism by which Lrp repression is exerted. This is a first demonstration of a role for Lrp in controlling genes that are also subject to intracellular regulation. These data show that the spv virulence genes belong simultaneously to several regulons in the cell, raising the possibility that spv expression can be fine-tuned in response to multiple environmental inputs.

Functional analysis of the FimE integrase of Escherichia coli K-12: isolation of mutant derivatives with altered DNA inversion preferences.

Phase variable expression of type 1 fimbriae in Escherichia coli arises from a site-specific recombination event that inverts a short segment of chromosomal DNA carrying the promoter for transcription of the gene encoding the fimbrial subunit protein. Two integrase-like recombinases are involved in switching. The FimB recombinase inverts the DNA segment in either orientation, whereas the FimE protein inverts it predominantly in the ON-to-OFF direction. In this paper, we report the isolation of a FimE mutant protein that has enhanced bidirectional switching activity. This protein has an arginine-to-lysine substitution at position 59, and this confers a FimB-like switching character on FimE without altering its ability to bind to DNA. The arginine was not a member of the arginine-histidine-arginine-tyrosine catalytic tetrad that is common to all integrase-like recombinases. The catalytic tetrad members of FimE were identified at positions 41, 136, 139 and 171 and shown to be essential for FimE function. In addition, other amino acid residues that make important contributions to the DNA binding activity of FimE or its ON-to-OFF inversion efficiency were identified.

Domain organization and oligomerization among H-NS-like nucleoid-associated proteins in bacteria.

The bacterial nucleoid-associated proteins H-NS and StpA can form homomeric or heteromeric complexes, a parallel with protein HU. Thus, functional modulation of H-NS and StpA by one another and by other proteins with appropriate interaction domains is possible. This has implications for bacterial pathogenesis and adaptation to environmental stress.

In vivo analysis of the interactions of the LysR-like regulator SpvR with the operator sequences of the spvA and spvR virulence genes of Salmonella typhimurium.

The interaction of the Salmonella typhimurium virulence gene regulator, SpvR, with its operator sites upstream of the spvA and spvR genes was analysed in vivo by dimethyl sulphate (DMS) footprinting and site-directed mutagenesis. DMS methylation protection assays showed that, in vivo, SpvR forms direct protein-DNA contacts with nucleotides clustered in two regions (+1 to -27 and -51 to -71) of the spvA regulatory region. These regions were subjected to site-directed mutagenesis and the effects on SpvR binding and gene activation assessed. Mutations that prevented occupancy of the promoter distal site (-51 to -71) in vivo also prevented occupancy of the promoter proximal site (+1 to -27), whereas mutations in the proximal site affected binding only at the proximal site and not the distal site. SpvR binding at the promoter proximal site was an essential prerequisite for transcription activation. These findings demonstrated a hierarchy of SpvR binding in which the promoter distal site is dominant to the proximal. The spvR gene was found to possess an operator site that resembled closely the distal SpvR binding site of the spvA operator. Nonetheless, SpvR interaction with the spvR operator was difficult to detect in vivo. When the nucleotide sequence of the spvR operator was altered at two nucleotides so that it corresponded more precisely to that of the distal site of the spvA operator, strong SpvR-DNA interactions were detected, with nucleotides in the region -31 to -67 being protected from DMS methylation in vivo. However, despite the improved interaction with the transcriptional activator, the altered regulatory region was poorer at promoting spvR gene transcription than the wild type. We describe a two-step model for activation of the spvA promoter and discuss the possibility that a specific cofactor in addition to sigma factor RpoS is required for SpvR action at this promoter in vivo.

The Shigella virulence gene regulatory cascade: a paradigm of bacterial gene control mechanisms.

Shigella flexneri is the causative agent of bacillary dysentery and is a facultative intracellular pathogen. Its virulence regulon is subject to tight control by several mechanisms involving the products of over 20 genes and an array of environmental signals. The reguIon is carried on a plasmid that is prone to instability and to integration into the chromosome, with associated silencing of the virulence genes. Closely related regulons are found in other species of Shigella and in enteroinvasive Escherichia coli. A wealth of detailed information is now available on the Shigella virulence gene control circuits, and it is becoming clear that these share many features with regulatory systems found in other bacterial pathogens. All of this makes the S. flexneri virulence gene control system a very attractive topic for those interested in the nature of gene regulatory networks in bacteria.

Two highly related regulatory proteins, Shigella flexneri VirF and enterotoxigenic Escherichia coli Rns, have common and distinct regulatory properties.

The Rns protein of enterotoxigenic Escherichia coli (ETEC) and the VirF protein of Shigella flexneri are members of the AraC family of transcription regulators. Rns is required for positive activation of the CS1 fimbrial genes, while VirF is a positive regulator of an invasion gene regulon. The amino acid sequences of the proteins are 36% identical, and both proteins activate transcription in response to increases in temperature. Here, we show that Rns is capable of complementing fully a null mutation in the S. flexneri virF gene. However, the VirF protein cannot replace Rns as an activator of CS1 gene expression in ETEC. This failure is not due to the absence from ETEC of a co-factor required by VirF since it also occurs when the CS1 system is moved into an S. flexneri genetic background. Nor is it a function of growth medium composition or a failure in virF gene expression. Instead, these findings point to important differences in the mechanisms by which these related transcription factors regulate gene expression in Gram-negative pathogens.

The StpA protein functions as a molecular adapter to mediate repression of the bgl operon by truncated H-NS in Escherichia coli.

The mechanism of repression of the beta-glucoside utilization (bgl) operon of Escherichia coli by a carboxy-terminally truncated derivative of the nucleoid-associated protein H-NS which is defective in DNA binding was investigated. The DNA-binding function of the H-NS-like protein StpA was found to be necessary for repression, which is consistent with a role for StpA as a DNA-binding adapter for mutant derivatives of H-NS.

Positive regulation of Shigella flexneri virulence genes by integration host factor.

In Shigella flexneri, expression of the plasmid-encoded virulence genes is regulated via a complex cascade involving DNA topology, specific transactivators, and the nucleoid-associated protein H-NS, which represses transcription under inappropriate environmental conditions. We have investigated the involvement of a second nucleoid-associated protein, integration host factor (IHF), in virulence gene expression. We found that transcription of the invasion-specific genes is repressed in a strain harboring an ihfA mutation, particularly on entry into the stationary phase. Expression of the virB gene, whose product is required for the activation of these structural genes, is also enhanced by IHF in the stationary phase. In contrast, the virF gene, which encodes an activator of virB, is stimulated by IHF in both the logarithmic and early stationary phases of growth, as is another virF-regulated gene, icsA. We have identified regions of the virF, virB, and icsA promoters which form IHF-dependent protein-DNA complexes in vitro and have located sequences within these regions with similarity to the consensus IHF binding site. Moreover, results from experiments in which the virF or virB gene was expressed constitutively confirm that IHF has a direct input at the level of both virF and virB transcription. Finally, we provide evidence that at the latter promoter, the primary role of IHF may be to overcome repression by the H-NS protein. To our knowledge, this is the first report of a role for IHF in controlling gene expression in S. flexneri.