Maurizio Falconi

Thermoregulation of Shigella and Escherichia coli EIEC pathogenicity. A temperature-dependent structural transition of DNA modulates accessibility of virF promoter to transcriptional repressor H-NS

The expression of plasmid‐borne virF of Shigella encoding a transcriptional regulator of the AraC family, is required to initiate a cascade of events resulting in activation of several operons encoding invasion functions. H‐NS, one of the main nucleoid‐associated proteins, controls the temperature‐dependent expression of the virulence genes by repressing the in vivo transcription of virF only below a critical temperature (∼32°C). This temperature‐dependent transcriptional regulation has been reproduced in vitro and the targets of H‐NS on the virF promoter were identified as two sites centred around −250 and −1 separated by an intrinsic DNA curvature. H‐NS bound cooperatively to these two sites below 32°C, but not at 37°C. DNA supercoiling within the virF promoter region did not influence H‐NS binding but was necessary for the H‐NS‐mediated transcriptional repression. Electrophoretic analysis between 4 and 60°C showed that the virF promoter fragment, comprising the two H‐NS sites, undergoes a specific and temperature‐dependent conformational transition at ∼32°C. Our results suggest that this modification of the DNA target may modulate a cooperative interaction between H‐NS molecules bound at two distant sites in the virF promoter region and thus represents the physical basis for the H‐NS‐dependent thermoregulation of virulence gene expression.

The oligomeric structure of nucleoid protein H‐NS is necessary for recognition of intrinsically curved DNA and for DNA bending

Escherichia coli hns, encoding the abundant nucleoid protein H‐NS, was subjected to site‐directed mutagenesis either to delete Pro115 or to replace it with alanine. Unlike the wild‐type protein, hyperproduction of the mutant proteins did not inhibit macromolecular syntheses, was not toxic to cells and caused a less drastic compaction of the nucleoid. Gel shift and ligase‐mediated circularization tests demonstrated that the mutant proteins retained almost normal affinity for non‐curved DNA, but lost the wild‐type capacity to recognize preferentially curved DNA and to actively bend non‐curved DNA, a property of wild‐type H‐NS demonstrated here for the first time. DNase I footprinting and in vitro transcription experiments showed that the mutant proteins also failed to recognize the intrinsically bent site of the hns promoter required for H‐NS transcription autorepression and to inhibit transcription from the same promoter. The failure of the Pro115 mutant proteins to recognize curved DNA and to bend DNA despite their near normal affinity for non‐curved DNA can be attributed to a defect in protein–protein interaction resulting in a reduced capacity to form oligomers observed in vitro and by a new in vivo test based on functional replacement by H‐NS of the oligomerization domain (C‐domain) of bacteriophage λ cI repressor.

Lethal overproduction of the Escherichia coli nucleoid protein H-NS: ultramicroscopic and molecular autopsy.

SummaryThe Escherichia coli hns gene, which encodes the nucleoid protein H-NS, was deprived of its natural promoter and placed under the control of the inducible lambda PL promoter. An hns mutant yielding a protein (H-NSΔ12) with a deletion of four amino acids (Gly112-Arg-Thr-Pro115) was also obtained. Overproduction of wild-type (wt) H-NS, but not of H-NSΔ12, resulted in a drastic loss of cell viability. The molecular events and the morphological alterations eventually leading to cell death were investigated. A strong and nearly immediate inhibition of both RNA and protein synthesis were among the main effects of overproduction of wt H-NS, while synthesis of DNA and cell wall material was inhibited to a lesser extent and at a later time. Upon cryofixation of the cells, part of the overproduced protein was found in inclusion bodies, while the rest was localized by immunoelectron microscopy to the nucleoids. The nucleoids appeared condensed in cells expressing both forms of H-NS, but the morphological alterations were particularly dramatic in those overproducing wt H-NS; their nucleoids appeared very dense, compact and almost perfectly spherical. These results provide direct evidence for involvement of H-NS in control of the organization and compaction of the bacterial nucleoid in vivo and suggest that it may function, either directly or indirectly, as transcriptional repressor and translational inhibitor.

Functional Characterization and Regulation of gadX, a Gene Encoding an AraC/XylS-Like Transcriptional Activator of the Escherichia coli Glutamic Acid Decarboxylase System

The Escherichia coli chromosome contains two distantly located genes, gadA and gadB, which encode biochemically undistinguishable isoforms of glutamic acid decarboxylase (Gad). The Gad reaction contributes to pH homeostasis by consuming intracellular H(+) and producing gamma-aminobutyric acid. This compound is exported via the protein product of the gadC gene, which is cotranscribed with gadB. Here we demonstrate that transcription of both gadA and gadBC is positively controlled by gadX, a gene downstream of gadA, encoding a transcriptional regulator belonging to the AraC/XylS family. The gadX promoter encompasses the 67-bp region preceding the gadX transcription start site and contains both RpoD and RpoS putative recognition sites. Transcription of gadX occurs in neutral rich medium upon entry into the stationary phase and is increased at acidic pH, paralleling the expression profile of the gad structural genes. However, P(T5)lacO-controlled gadX expression in neutral rich medium results in upregulation of target genes even in exponential phase, i.e., when the gad system is normally repressed. Autoregulation of the whole gad system is inferred by the positive effect of GadX on the gadA promoter and gadAX cotranscription. Transcription of gadX is derepressed in an hns mutant and strongly reduced in both rpoS and hns rpoS mutants, consistent with the expression profile of gad structural genes in these genetic backgrounds. Gel shift and DNase I footprinting analyses with a MalE-GadX fusion protein demonstrate that GadX binds gadA and gadBC promoters at different sites and with different binding affinities.

Antagonistic involvement of FIS and H-NS proteins in the transcriptional control of hns expression

Gel shift and DNase I footprinting experiments showed that Escherichia coli FIS (factor for inversion stimulation) protein binds to at least seven sites in the promoter region of hns. These sites extend from −282 to +25 with two sites, closely flanking the DNA bend located at −150 from the transcriptional startpoint, partly overlapping the H‐NS binding sites involved in the transcriptional autorepression of hns. The interplay between FIS, H‐NS and the hns promoter region were studied by examining the effects of FIS and H‐NS on in vitro transcription of hns–cat fusions, as well as looking at the effect of FIS on preformed complexes containing H‐NS and a DNA fragment derived from the hns promoter region. Taken together, our data suggest that in the cell, FIS and H‐NS interact with the promoter region of hns and influence their respective interactions (possibly competing for the same binding site), eliciting antagonistic effects so that an interplay between these proteins might contribute to the transcriptional control of hns

Involvement of FIS in the H‐NS‐mediated regulation of virF gene of Shigella and enteroinvasive Escherichia coli

The mechanism of pathogenicity in Shigella and enteroinvasive Escherichia coli (EIEC) requires the co‐ordinated expression of several genes located on both the virulence plasmid and the chromosome. We found that cells lacking a functional FIS protein (factor for inversion stimulation) are partially impaired in expressing the virulence genes and that full expression is totally restored when Shigella wild‐type fis gene is offered in trans. We also identified virF, among the virulence genes, as a target of FIS‐mediated activation and showed that FIS binds to four specific sites in the promoter region of virF. Previous studies have demonstrated that the expression of VirF, the first positive activator of a multistep regulatory cascade, is subject to temperature‐dependent regulation by H‐NS, one of the main nucleoid‐associated proteins. We now demonstrate that two of the four FIS sites overlap one of the two H‐NS sites responsible for thermoregulation (H‐NS site I). FIS was found to exercise a direct positive transcriptional control at permissive temperature (37°C), when H‐NS fails to repress virF, as well as an indirect effect by partially counteracting H‐NS inhibition at the transition temperature (32°C). Our data indicate that FIS may be relevant for the rapid increase in virF expression after penetration of bacteria into the host.

Expression of the gene encoding the major bacterial nucleoid protein H‐NS is subject to transcriptional auto‐repression

Expression of a promoterless cat gene fused to a DNA fragment of approximately 400 bp, beginning at –313 of Escherichia coli hns, was significantly repressed in E. coli and Salmonella typhimurium strains with wild‐type hns but not in mutants carrying hns alleles. CAT expression from fusions containing a shorter (110 bp) segment of hns was essentially unaffected in the same genetic backgrounds. The stage of growth was found to influence the extent of repression which was maximum (approximately 75%) in mid‐log cultures and negligible in cells entering the stationary phase. The level of repression in early‐log phase was lower than in mid‐log phase cultures, probably because of the presence of high levels of Fis protein, which counteracts the H‐NS inhibition by stimulating hns transcription. The effects observed in vivo were mirrored by similar results obtained in vitro upon addition of purified H‐NS and Fis protein to transcriptional systems programmed with the same hns caf fusions. Electrophoretic gel shift assays, DNase I footprinting and cyclic permutation get analyses revealed that H‐NS binds preferentially to the upstream region of its own gene recognizing two rather extended segments of DNA on both sides of a bend centred around –150. When these sites are filled by H‐NS, an additional site between approximately –20 and –65, which partly overlaps the promoter, is also occupied. Binding of H‐NS to this site is probably the ultimate cause of transcriptional auto‐repression.

A role for H-NS in the regulation of the virF gene of Shigella and enteroinvasive Escherichia coli.

We have investigated the role of H-NS, one of the major components of the bacterial nucleoid, in the expression of the virF gene present on the large virulence plasmid of Shigella and enteroinvasive Escherichia coli in response to different environmental conditions. VirF is an AraC-like protein which activates at least two promoters, virB and virG, both repressed by H-NS. Band shift experiments reveal that the affinity of H-NS for the virF and virB promoters is comparable, while the affinity for the virG promoter is higher. Polyacrylamide gel electrophoresis of three DNA fragments containing the virF, the virB and the VirG promoters demonstrates, in agreement with computer predictions, that they have an intrinsically curved structure, confirming the preference of H-NS for bent DNA. In vivo transcriptional analysis of virF mRNA shows that H-NS negatively controls the expression of virF at 30 degrees C. The expression of a virF-lacZ translational fusion in E.coli wild type and in an hns-defective derivative grown at 30 degrees or 37 degrees C and at pH 6.0 or 7.0 indicates that, in the absence of H-NS, virF expression becomes insensitive to temperature and to limited pH changes. Our results strongly suggest that H-NS controls virF expression by binding to the virF promoter and by repressing its expression at low temperature and at low pH.

Antagonistic Role of H-NS and GadX in the Regulation of the Glutamate Decarboxylase-dependent Acid Resistance System in Escherichia coli

One of the most efficient systems of acid resistance in Escherichia coli, the gad system, is based on the coordinated action of two isoforms of glutamate decarboxylase (GadA and GadB) and of a specific glutamate/γ-aminobutyrate antiporter (GadC). The gadA/BC genes, activated in response to acid stress and in stationary phase cells, are subjected to complex circuits of regulation involving σ70, σS, cAMP receptor protein, H-NS, EvgAS, TorRS, GadE, GadX, GadW, and YdeO. Herein, we provide evidence that the nucleoid-associated protein H-NS directly functions as repressor of gadA, one of the structural genes, and gadX, a regulatory gene encoding one of the primary activators of the gad system. Band shift and DNase I footprints reveal that H-NS indeed binds to specific sites in the promoter regions of gadA and gadX and represses the transcription of these genes both in an in vitro system and in vivo. Moreover, we show that a maltose-binding protein MalE-GadX fusion is able to stimulate the promoter activity of gadA/BC, thus indicating that GadX is by itself able to up-regulate the gad genes and that a functional competition between H-NS and GadX takes place at the gadA promoter. Altogether, our results indicate that H-NS directly inhibits gadA and gadX transcription and, by controlling the intracellular level of the activator GadX, indirectly affects the expression of the whole gad system.

Nature and mechanism of the in vivo oligomerization of nucleoid protein H-NS

Two types of two‐hybrid systems demonstrate that the transcriptional repressor, nucleoid‐associated protein H‐NS (histone‐like, nucleoid structuring protein) forms dimers and tetramers in vivo, the latter being the active form of the protein. The H‐NS ‘protein oligomerization’ domain (N‐domain) is unable to oligomerize in the absence of the intradomain linker while the ‘DNA‐binding’ C‐domain clearly displays a protein–protein interaction capacity, which contributes to H‐NS tetramerization and which is lost following Pro115 mutation. Linker deletion or substitution with KorB linker abolishes H‐NS oligomerization. A model describing H‐NS dimerization and tetramerization based on all available data and suggesting the existence in the tetramer of a bundle of four α‐helices, each contributed by an H‐NS monomer, is presented.