PubMed Health⌕ Search

Biomedical subjects

Julian M Ketley

Publications and source records attributed to Julian M Ketley.

11 recordsLinked to original sources
PubMed2006

Heme utilization in Campylobacter jejuni.

A putative iron- and Fur-regulated hemin uptake gene cluster, composed of the transport genes chuABCD and a putative heme oxygenase gene (Cj1613c), has been identified in Campylobacter jejuni NCTC 11168. Mutation of chuA or Cj1613c leads to an inability to grow in the presence of hemin or hemoglobin as a sole source of iron. Mutation of chuB, -C, or -D only partially attenuates growth where hemin is the sole iron source, suggesting that an additional inner membrane (IM) ABC (ATP-binding cassette) transport system(s) for heme is present in C. jejuni. Genotyping experiments revealed that Cj1613c is highly conserved in 32 clinical isolates. One strain did not possess chuC, though it was still capable of using hemin/hemoglobin as a sole iron source, supporting the hypothesis that additional IM transport genes are present. In two other strains, sequence variations within the gene cluster were apparent and may account for an observed negative heme utilization phenotype. Analysis of promoter activity within the Cj1613c-chuA intergenic spacer region revealed chuABCD and Cj1613c are expressed from separate iron-repressed promoters and that this region also specifically binds purified recombinant Fur(Cj) in gel retardation studies. Absorbance spectroscopy of purified recombinant His(6)-Cj1613c revealed a 1:1 heme:His(6)-Cj1613c binding ratio. The complex was oxidatively degraded in the presence of ascorbic acid as the electron donor, indicating that the Cj1613c gene product functions as a heme oxygenase. In conclusion, we confirm the involvement of Cj1613c and ChuABCD in heme/hemoglobin utilization in C. jejuni.

Bacterial Proteins↗
PubMed2005

The Campylobacter jejuni glycome.

Microbial cell surface glycans in the form of glycolipids and glycoproteins frequently play important roles in cell-cell interaction and host immune responses. Given the likely importance of these surface structures in the survival and pathogenesis of Campylobacter jejuni, a concerted effort has been made to characterise these determinants genetically and structurally since the genome was sequenced in 2000. We review the considerable progress made in characterising the Campylobacter glycome including the lipooligosaccharide (LOS), the capsule and O- and N-linked protein glycosylation systems, and speculate on the roles played by glycan surface structures in the life-cycle of C. jejuni.

Animals↗
PubMed2005

Campylobacter jejuni gene expression in response to iron limitation and the role of Fur.

Campylobacter jejuni is a zoonotic pathogen and the most common cause of bacterial foodborne diarrhoeal illness worldwide. To establish intestinal colonization prior to either a commensal or pathogenic interaction with the host, C. jejuni will encounter iron-limited niches where there is likely to be intense competition from the host and normal microbiota for iron. To gain a better understanding of iron homeostasis and the role of ferric uptake regulator (Fur) in iron acquisition in C. jejuni, a proteomic and transcriptome analysis of wild-type and fur mutant strains in iron-rich and iron-limited growth conditions was carried out. All of the proposed iron-transport systems for haemin, ferric iron and enterochelin, as well as the putative iron-transport genes p19, Cj1658, Cj0177, Cj0178 and cfrA, were expressed at higher levels in the wild-type strain under iron limitation and in the fur mutant in iron-rich conditions, suggesting that they were regulated by Fur. Genes encoding a previously uncharacterized ABC transport system (Cj1660-Cj1663) also appeared to be Fur regulated, supporting a role for these genes in iron uptake. Several promoters containing consensus Fur boxes that were identified in a previous bioinformatics search appeared not to be regulated by iron or Fur, indicating that the Fur box consensus needs experimental refinement. Binding of purified Fur to the promoters upstream of the p19, CfrA and CeuB operons was verified using an electrophoretic mobility shift assay (EMSA). These results also implicated Fur as having a role in the regulation of several genes, including fumarate hydratase, that showed decreased expression in response to iron limitation. The known PerR promoters were also derepressed in the C. jejuni Fur mutant, suggesting that they might be co-regulated in response to iron and peroxide stress. These results provide new insights into the effects of iron on metabolism and oxidative stress response as well as the regulatory role of Fur.

Animals↗
PubMed2005

The Campylobacter jejuni response regulator, CbrR, modulates sodium deoxycholate resistance and chicken colonization.

Two-component regulatory systems play a major role in the physiological response of bacteria to environmental stimuli. Such systems are composed of a sensor histidine kinase and a response regulator whose ultimate function is to affect the expression of target genes. Response regulator mutants of Campylobacter jejuni strain F38011 were screened for sensitivity to sodium deoxycholate. A mutation in Cj0643, which encodes a response regulator with no obvious cognate histidine kinase, resulted in an absence of growth on plates containing a subinhibitory concentration of sodium deoxcholate (1%, wt/vol). In broth cultures containing 0.05% (wt/vol) sodium deoxycholate, growth of the mutant was significantly inhibited compared to growth of the C. jejuni F38011 wild-type strain. Complementation of the C. jejuni cbrR mutant in trans restored growth in both broth and plate cultures supplemented with sodium deoxycholate. Based on the phenotype displayed by its mutation, we designated the gene corresponding to Cj0643 as cbrR (Campylobacter bile resistance regulator). While the MICs of a variety of bile salts and other detergents for the C. jejuni cbrR mutant were lower, no difference was noted in its sensitivity to antibiotics or osmolarity. Finally, chicken colonization studies demonstrated that the C. jejuni cbrR mutant had a reduced ability to colonize compared to the wild-type strain. These data support previous findings that bile resistance contributes to colonization of chickens and establish that the response regulator, CbrR, modulates resistance to bile salts in C. jejuni.

Amino Acid Sequence↗
PubMed2002

The role of iron in Campylobacter gene regulation, metabolism and oxidative stress defense.

Enteric Campylobacter species cause gastrointestinal diseases in humans. Like almost all organisms, campylobacters have an absolute requirement for iron, but are faced with variable availability of iron in the environment and host tissues. Campylobacters have developed mechanisms to scavenge sufficient iron for metabolism and growth. However, iron also participates in the formation of reactive oxygen species, and this forces pathogens to maintain intracellular iron homeostasis and to cope with oxidative stresses. The presence of two separate, but possibly overlapping iron-responsive regulatory systems, which regulate iron acquisition and oxidative stress defense, and the presence of genes encoding multiple iron acquisition and detoxification systems in Campylobacter indicate the central role that iron plays in Campylobacter gene regulation and virulence.

Bacterial Proteins↗
PubMed2002

Mutational and transcriptional analysis of the Campylobacter jejuni flagellar biosynthesis gene flhB.

A Campylobacter jejuni gene encoding a homologue of the flagellar biosynthesis gene flhB was identified downstream of the peroxide stress defence gene ahpC. Insertional mutagenesis of the flhB gene rendered C. jejuni non-motile, with most cells aflagellate, although a small number expressed truncated flagella. The absence of FlhB also appeared to affect cell shape, as the majority of cells were straight rather than curved rods. Transcription of the flagellin gene flaA was significantly reduced in the C. jejuni flhB mutants, which also did not express significant amounts of flagellin proteins, indicating that FlhB is an essential protein for subsequent expression of flagellar genes. The transcription start site of the flhB gene, as determined by primer extension, was located 91 bp upstream of the flhB start codon, but no recognizable promoter sequence could be identified immediately upstream of this transcription start site. Transcriptional flhB::lacZ reporter gene fusions confirmed that the flhB gene has its own promoter region, is expressed at very low levels and is transcribed independently of ahpC, and that its transcription is not regulated by iron or growth phase.

Bacterial Proteins↗
PubMed2002

Characterization of the Campylobacter jejuni heptosyltransferase II gene, waaF, provides genetic evidence that extracellular polysaccharide is lipid A core independent.

Campylobacter jejuni produces both lipooligosaccharide (LOS) and a higher-molecular-weight polysaccharide that is believed to form a capsule. The role of these surface polysaccharides in C. jejuni-mediated enteric disease is unclear; however, epitopes associated with the LOS are linked to the development of neurological complications. In Escherichia coli and Salmonella enterica serovar Typhimurium the waaF gene encodes a heptosyltransferase, which catalyzes the transfer of the second L-glycero-D-manno-heptose residue to the core oligosaccharide moiety of lipopolysaccharide (LPS), and mutation of waaF results in a truncated core oligosaccharide. In this report we confirm experimentally that C. jejuni gene Cj1148 encodes the heptosyltransferase II enzyme, WaaF. The Campylobacter waaF gene complements an S. enterica serovar Typhimurium waaF mutation and restores the ability to produce full-sized lipopolysaccharide. To examine the role of WaaF in C. jejuni, waaF mutants were constructed in strains NCTC 11168 and NCTC 11828. Loss of heptosyltransferase activity resulted in the production of a truncated core oligosaccharide, failure to bind specific ligands, and loss of serum reactive GM(1), asialo-GM(1), and GM(2) ganglioside epitopes. The mutation of waaF did not affect the higher-molecular-weight polysaccharide supporting the production of a LOS-independent capsular polysaccharide by C. jejuni. The exact structural basis for the truncation of the core oligosaccharide was verified by comparative chemical analysis. The NCTC 11168 core oligosaccharide differs from that known for HS:2 strain CCUG 10936 in possessing an extra terminal disaccharide of galactose-beta(1,3) N-acetylgalactosamine. In comparison, the waaF mutant possessed a truncated molecule consistent with that observed with waaF mutants in other bacterial species.

Campylobacter jejuni↗
PubMed1999

A histotoxin produced by Salmonella.

Salmonella Typhimurium strain GM3, known to be histotoxic for explants of terminal rabbit ileum in vitro, produces similar lesions in vitro when sterile filtrates, obtained from live organisms after interaction with gut explants in vitro, are used and when rabbit ligated ileal loops are challenged with live organisms. Epithelial damage occurs rapidly, within 2 h of adding organisms or sterile filtrates. This evidence is construed in terms of a secreted salmonella histotoxin that causes epithelial damage, detaching enterocytes which rapidly degenerate into spheroid cells devoid of microvilli. Typhimurium strain GM3 invades ileal mucosa and bacteria are found in the subepithelial tissues. After 12 h, bacteria were seen to be expelled from infected villi in a manner similar to that seen in non-histotoxic infection with Typhimurium strain TML.

Animals↗
PubMed1999

Cloning, mutation and distribution of a putative lipopolysaccharide biosynthesis locus in Campylobacter jejuni.

A region encoding ORFs with homology to known lipopolysaccharide (LPS) biosynthesis genes was isolated from two strains of Campylobacter jejuni. One of the strains produces LPS, but the second strain is reported to produce only lipooligosaccharide (LOS) and therefore lacks the O-chain. The two strains shared six predicted ORFs, but an additional ORF, orfE, of unknown function was identified in the LOS-producing strain. Mutation of the shared wbeE (rfbE) homologue (orfF) or deletion of five of the seven genes reduced core reactivity with specific antiserum without affecting O-chain production. Mutation of either the capD homologue (orfG) or the unique orfE had no detectable effect on LOS or LPS production. The presence or absence of orfE in 36 isolates of C. jejuni did not correlate with LOS/LPS phenotype or serotype. However, after insertion of orfE into a LPS-producing orfE-negative strain the O-chain ladder was no longer detectable on Western blots. We were not able to disrupt the wbaP (rfbP) homologue (orfC) in C jejuni.

Campylobacter jejuni↗
PubMed1998

An improved physical and genetic map of Campylobacter jejuni NCTC 11168 (UA580).

Campylobacter jejuni is recognized as the major cause of food-borne gastrointestinal disease in the developed world. To facilitate the molecular genetic analysis of this pathogen, an approximately 18-fold redundant Tropist3 cosmid library was constructed from C. jejuni NCTC 11168 genomic DNA. The cosmid library was partially ordered by hybridization to 15 pulsed-field electrophoresis (PFGE) restriction fragments. This analysis confirmed the estimated size of the genome to be 1730 kb, but suggested discrepancies in some regions of the published physical map. The precise locations of two of the three rRNA gene clusters were mapped using a combination of restriction fingerprinting, sample sequencing and riboprobing. Additionally, 15 further genes were located on the revised map. A more detailed physical and genetic map of C. jejuni NCTC 11168 is presented.

Campylobacter jejuni↗