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J C Hinton

Publications and source records attributed to J C Hinton.

At least 19 recordsLinked to original sources
PubMed2006

Butyrate specifically down-regulates salmonella pathogenicity island 1 gene expression.

Invasion of intestinal epithelial cells by Salmonella enterica is decreased after exposure to butyric acid. To understand the molecular mechanisms of this phenomenon, a comparative transcriptomic analysis of Salmonella enterica serovar Enteritidis and Salmonella enterica serovar Typhimurium grown in medium supplemented with butyrate was performed. We found that butyrate down-regulated the expression of 19 genes common to both serovars by a factor of twofold or more, and 17 of these genes localized to the Salmonella pathogenicity island 1 (SPI1). These included the SPI1 regulatory genes hilD and invF. Of the remaining two genes, ampH has 91% homology to an Escherichia coli penicillin-binding protein and sopE2 encodes a type III-secreted effector protein associated with invasion but located at a separate site on the chromosome from SPI1.

Animals↗
PubMed2001

Structural characterization of the N-terminal oligomerization domain of the bacterial chromatin-structuring protein, H-NS.

The H-NS protein plays a key role in condensing DNA and modulating gene expression in bacterial nucleoids. The mechanism by which this is achieved is dependent, at least in part, on the oligomerization of the protein. H-NS consists of two distinct domains; the N-terminal domain responsible for protein oligomerization, and the C-terminal DNA binding domain, which are separated by a flexible linker region. We present a multidimensional NMR study of the amino-terminal 64 residues of H-NS (denoted H-NS1-64) from Salmonella typhimurium, which constitute the oligomerization domain. This domain exists as a homotrimer, which is predicted to be self-associated through a coiled-coil configuration. NMR spectra show an equivalent magnetic environment for each monomer indicating that the polypeptide chains are arranged in parallel with complete 3-fold symmetry. Despite the limited resonance dispersion, an almost complete backbone assignment for 1H(N), 1H(alpha), 15N, 13CO and 13C(alpha) NMR resonances was obtained using a suite of triple resonance experiments applied to uniformly 15N-, 13C/15N- and 2H/13C/15N-labelled H-NS1-64 samples. The secondary structure of H-NS1-64 has been identified on the basis of the analysis of 1H(alpha), 13C(alpha), 13Cbeta and 13CO chemical shifts, NH/solvent exchange rates, intra-chain H(N)-H(N) and medium-range nuclear Overhauser enhancements (NOEs). Within the context of the homotrimer, each H-NS1-64 protomer consists of three alpha-helices spanning residues 2-8, 12-20 and 22-53, respectively. A topological model is presented for the symmetric H-NS1-64 trimer based upon the combined analysis of the helical elements and the pattern of backbone amide group 15N nuclear relaxation rates within the context of axially asymmetric diffusion tensor. In this model, the longest of the three helices (helix 3, residues 22-53) forms a coiled-coil interface with the other chains in the homotrimer. The two shorter N-terminal helices fold back onto the outer surface of the coiled-coil core and potentially act to stabilise this configuration.

Bacterial Proteins↗
PubMed2001

The nucleoid-associated protein StpA binds curved DNA, has a greater DNA-binding affinity than H-NS and is present in significant levels in hns mutants.

The StpA protein is closely related to H-NS, the well-characterised global regulator of gene expression which is a major component of eubacterial chromatin. Despite sharing a very high degree of sequence identify and having biochemical properties in common with H-NS, the physiological function of StpA remains unknown. We show that StpA exhibits similar DNA-binding activities to H-NS. Although both display a strong preference for binding to curved DNA, StpA binds DNA with a four-fold higher affinity than H-NS, with K(d)s of 0.7 microM and 2.8 microM, respectively. It has previously been reported that expression of stpA is derepressed in an hns mutant. We have quantified the amount of StpA protein produced under this condition and find it to be only one-tenth the level of H-NS protein in wild-type cells. Our findings explain why the presence of StpA does not compensate for the lack of H-NS in an hns mutant, and why the characteristic pleiotropic hns mutant phenotype is observed.

Bacterial Proteins↗
PubMed2001

It's easy to build your own microarrayer!

DNA microarrays are becoming the tool of choice for microbial gene-expression profiling and genotypic analysis. The construction of a gridding robot for the 'in-house' production of microarrays is a choice worth considering, and offers distinct advantages over other options in terms of cost effectiveness and scale. Having built our own robot, we want to dispel some of the myths that might be associated with such a project, as well as provide practical advice for potential builders in the UK and Europe.

Biotechnology↗
PubMed2001

Role of the nucleoid-associated protein Fis in the regulation of virulence properties of enteropathogenic Escherichia coli.

Virulence gene expression in enteropathogenic Escherichia coli (EPEC) is governed by a combination of environmental factors and virulence regulators. These factors control the expression of the bundle-forming pili (BFP), intimin, the type III secretion apparatus and the secreted proteins EspA, EspB, EspD and Tir. Expression of the bfp genes occurs for a short period in early exponential phase during growth in tissue culture medium. The nucleoid-associated regulator protein, Fis, is also expressed transiently during this period. To determine whether Fis was responsible for the growth phase-dependent expression of bfp, fis was deleted from the EPEC strain E2348/69S. Paradoxically, the Delta fis mutant retained the ability to colonize HEp-2 cells in a characteristic localized adherence pattern, and Fis was found negatively to regulate the expression of BFP. However, the Delta fis mutant failed to induce the accretion of filamentous actin, which is associated with attaching and effacing lesions. Using a combination of Western blotting and a novel multiplex primer extension assay (MPEA), we showed that, although the expression of intimin and Tir was not affected, transcription of the LEE4 operon encoding espADB and the virulence activator, Ler, were found to be Fis dependent.

Adhesins, Bacterial↗
PubMed2001

Virulence gene regulation in Salmonella enterica.

In order to infect a host, a microbe must be equipped with special properties known as virulence factors. Bacterial virulence factors are required to facilitate colonization, to survive under host defenses, and to permit multiplication inside the host. However, the possession of genes encoding virulence factors does not guarantee effective infection. There is considerable evidence that tight regulation of a given virulence factor is as important as the possession of the virulence factors themselves. Thus, an understanding of the regulation of virulence expression is fundamental to our comprehension of any infection process and can identify potential targets for disease prevention and therapy. We have summarized the lessons learned from experimental salmonellosis in terms of virulence regulation and hope to illustrate the differing requirements for gene and virulence expression.

Adaptation, Physiological↗
PubMed2000

Measurement of bacterial gene expression in vivo.

The complexities of bacterial gene expression during mammalian infection cannot be addressed by in vitro experiments. We know that the infected host represents a complex and dynamic environment, which is modified during the infection process, presenting a variety of stimuli to which the pathogen must respond if it is to be successful. This response involves hundreds of ivi (in vivo-induced) genes which have recently been identified in animal and cell culture models using a variety of technologies including in vivo expression technology, differential fluorescence induction, subtractive hybridization and differential display. Proteomic analysis is beginning to be used to identify IVI proteins, and has benefited from the availability of genome sequences for increasing numbers of bacterial pathogens. The patterns of bacterial gene expression during infection remain to be investigated. Are ivi genes expressed in an organ-specific or cell-type-specific fashion? New approaches are required to answer these questions. The uses of the immunologically based in vivo antigen technology system, in situ PCR and DNA microarray analysis are considered. This review considers existing methods for examining bacterial gene expression in vivo, and describes emerging approaches that should further our understanding in the future.

Animals↗
PubMed2000

Oligomerization of the chromatin-structuring protein H-NS.

H-NS is a major component of the bacterial nucleoid, involved in condensing and packaging DNA and modulating gene expression. The mechanism by which this is achieved remains unclear. Genetic data show that the biological properties of H-NS are influenced by its oligomerization properties. We have applied a variety of biophysical techniques to study the structural basis of oligomerization of the H-NS protein from Salmonella typhimurium. The N-terminal 89 amino acids are responsible for oligomerization. The first 64 residues form a trimer dominated by an alpha-helix, likely to be in coiled-coil conformation. Extending this polypeptide to 89 amino acids generated higher order, heterodisperse oligomers. Similarly, in the full-length protein no single, defined oligomeric state is adopted. The C-terminal 48 residues do not participate in oligomerization and form a monomeric, DNA-binding domain. These N- and C-terminal domains are joined via a flexible linker which enables them to function independently within the context of the full-length protein. This novel mode of oligomerization may account for the unusual binding properties of H-NS.

Amino Acid Sequence↗
PubMed1997

DNA binding is not sufficient for H-NS-mediated repression of proU expression.

H-NS is a major component of bacterial chromatin and influences the expression of many genes. H-NS has been shown to exhibit a binding preference for certain AT-rich curved DNA elements in vitro. In this study we have addressed the factors that determine the specificity of H-NS action in vitro and in vivo. In bandshift studies, H-NS showed a slight binding preference for all curved sequences tested whether GC-based or AT-based; the specific architecture of the curve also influenced H-NS binding. In filter retention assays little difference in affinity could be detected for any sequence tested, including the downstream regulatory element (DRE) a downstream curved DNA element required for H-NS to repress transcription of the Salmonella typhimurium proU operon in vivo. A Kd of 1-2 microM was estimated for binding of H-NS to each of these sequences. In vivo, the distance between the proU promoter and the DRE, their relative orientations on the face of the DNA helix, and translation of the DRE had no major effect on proU regulation. None of the synthetic curved sequences tested could functionally replace the DRE in vivo. These data show that differential binding to curved DNA cannot account for the specificity of H-NS action in vivo. Furthermore, binding of H-NS to DNA per se is insufficient to repress the proU promoter. Thus, the DRE does not simply act as an H-NS binding site but must have a more specific role in mediating H-NS regulation of proU transcription.

Amino Acid Transport Systems↗
PubMed1997

The Escherichia coli genome sequence: the end of an era or the start of the FUN?

Our dream of determining the entire Escherichia coli K12 genome sequence has been realized. This calls for new approaches for the analysis of gene expression and function in biology's best-understood organism. Comparison of the E. coli genome sequence with others will provide important taxonomic insights and have implications for the study of bacterial virulence. Approximately 20% of E. coli genes have been designated FUN genes, because they have no known function or homologies to sequence databases. FUN genes promise to have an exciting impact on bacterial research. The post-genome era requires novel strategies that address gene regulation at the level of the entire cell. These strategies need to supersede the reductionist approach to genetic analysis. Only then will the genome sequence lead us to an understanding of how a bacterial cell really works.

Escherichia coli↗
PubMed1994

The chromatin-associated protein H-NS alters DNA topology in vitro.

H-NS is one of the two most abundant proteins in the bacterial nucleoid and influences the expression of a number of genes. We have studied the interaction of H-NS with DNA; purified H-NS was demonstrated to constrain negative DNA supercoils in vitro. This provides support for the hypothesis that H-NS influences transcription via changes in DNA topology, and is evidence of a structural role for H-NS in bacterial chromatin. The effects of H-NS on topology were only observed at sub-saturating concentrations of the protein. In addition, a preferred binding site on DNA was identified by DNase I footprinting at sub-saturating H-NS concentrations. This site corresponded to a curved sequence element which we previously showed, by in vivo studies, to be a site at which H-NS influences transcription of the proU operon. When present in saturating concentrations, H-NS did not constrain supercoils and bound to DNA in a sequence-independent fashion, covering all DNA molecules from end to end, suggesting that H-NS may form distinct complexes with DNA at different H-NS:DNA ratios. The data presented here provide direct support for the hypothesis that H-NS acts at specific sites to influence DNA topology and, hence, transcription.

Bacterial Proteins↗
PubMed1994

The chromatin-associated protein H-NS.

H-NS is a major component of chromatin in enteric bacteria. H-NS plays a structural role in organising the chromosome, and influences DNA rearrangements as well as the expression of many genes. The biochemical and functional characteristics of H-NS are distinct from those of 'typical' DNA-binding proteins and much remains to be learned about the mechanism(s) by which H-NS acts. In this article we review our current understanding of the role of H-NS, and describe possible models by which H-NS might influence DNA structure and gene expression.

Amino Acid Sequence↗
PubMed1993

A pleiotropic reduced virulence (Rvi-) mutant of Erwinia carotovora subspecies atroseptica is defective in flagella assembly proteins that are conserved in plant and animal bacterial pathogens.

Erwinia carotovora subsp. atroseptica was mutagenized and assayed for virulence in planta. Those mutants which exhibited reduced virulence (Rvi-) were assayed for growth rate, auxotrophy and extracellular enzyme secretion and seven mutants were found to be wild type for all of these phenotypes. When screened for other phenotypes, two were found to be non-motile. One mutant was complemented for motility by a heterologous gene library. A 2.7kb XmaIII-ClaI complementing fragment was sequenced and the gene products were found to have similarity to flagella biosynthesis gene products from several bacteria. Further similarity was found to a pathogenicity protein from the plant pathogen Xanthomonas campestris pv. glycines and to the Spa pathogenicity proteins of the human pathogen Shigella flexneri, which are involved in the surface presentation of antigens. These studies highlight the emergence of common themes in the molecular strategies employed by both plant and animal bacterial pathogens for the targeting of proteins involved in the elaboration of disease.

Amino Acid Sequence↗
PubMed1992

The chromatin-associated protein H-NS interacts with curved DNA to influence DNA topology and gene expression.

H-NS is an abundant structural component of bacterial chromatin and influences many cellular processes, including recombination, transposition, and transcription. We have studied the mechanism of action of H-NS at the osmotically regulated proU promoter. The interaction of H-NS with a curved DNA element located downstream of the proU promoter is required for normal regulation of expression. Heterologous curved sequences can replace the regulatory role of the proU curve. Hence, the luxAB and lacZ reporter genes, which differ in the presence or absence of a curve, can indicate very different patterns of transcription. H-NS interacts preferentially with these curved DNA elements in vitro. Furthermore, in vivo the interaction of H-NS with curved DNA participates in the control of plasmid linking number. The data suggest that H-NS-dependent changes in DNA topology play a role in the osmoregulation of proU expression.

Bacterial Proteins↗
PubMed1992

Expression and mutational analysis of the nucleoid-associated protein H-NS of Salmonella typhimurium.

The H-NS (H1) protein is a major component of bacterial chromatin. Mutations in the hns (osmZ) gene encoding H-NS are highly pleiotropic, affecting the expression of many unrelated genes in an allele-specific manner. H-NS expression was found not to vary with growth phase or growth medium osmolarity. Additionally, 10 independent hns mutations were isolated and characterized. Five of these mutations were the result of an IS10 insertion, each generating a truncated polypeptide. The other five mutations were the same specific deletion of one amino acid, delta Ala46. The various hns mutations exhibited different phenotypes and influenced DNA topology to variable extents. Implications for the mechanism by which H-NS influences gene expression are discussed.

Bacterial Outer Membrane Proteins↗
PubMed1990

Histone-like protein H1 (H-NS), DNA supercoiling, and gene expression in bacteria.

Changes in DNA supercoiling in response to environmental signals such as osmolarity, temperature, or anaerobicity appear to play an underlying role in the regulation of gene expression in bacteria. Extensive genetic analyses have implicated the osmZ gene in this regulatory process: osmZ mutations are highly pleiotropic and alter the topology of cellular DNA. We have shown that the product of the osmZ gene is the "histone-like" protein H1 (H-NS). Protein H1 is one of the most abundant components of bacterial chromatin and binds to DNA in a relatively nonspecific fashion. These data imply a regulatory role for one of the major components of bacterial chromatin and provide support for the notion that changes in DNA topology and/or chromatin structure play a role in regulating gene expression.

Bacterial Proteins↗