Ian Roberts
University of Leicester
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Molecular Microbiology | 1990
Annabel Smith; Graham J. Boulnois; Ian Roberts
The complete nucleotide sequence has been determined of a region of the Escherichia coli K5 antigen gene cluster postulated to encode functions for the translocation of capsular polysaccharide across the inner membrane. This revealed two genes, designated kpsM and kpsT, organized in a single transcriptional unit. Analysis of the predicted amino acid sequence of the KpsM and KpsT proteins indicates that they may function as dual components in a polysaccharide export system analogous to the periplasmic binding protein‐dependent transport systems of Gram‐negative bacteria. We propose that the KpsT protein acts as an energizer, coupling ATP hydrolysis to the transport process mediated by the KpsM protein. Extensive sequence homology between the KpsM and KpsT proteins and the products of the bexB and bexA genes present in the capsulation (cap) locus of Haemophilus influenzae, indicates that a common mechanism for the export of polysaccharide across the inner membrane may exist in these two microorganisms.
Molecular Microbiology | 1995
Chantal Petit; Gordon P. Rigg; Carlo Pazzani; Annabel Smith; Veit Sieberth; Mark P. Stevens; Graham J. Boulnois; Klaus Jann; Ian Roberts
The nucleotide sequence of region 2 of the Escherichia coli K5 capsule gene cluster has been determined. This region, essential for the biosynthesis of the K5 poly‐saccharide, contained four genes, termed kfiA‐D. The G+C ratio was 33.4%, which was lower than the typical G+C ratio for E. coli and that of the flanking regions 1 and 3 in the K5 capsule gene cluster. Three major RNA transcripts were detected within region 2 by Northern blotting and three promoters located by transcript mapping. Promoter activity was confirmed by promoter‐probe analysis. The predicted amino acid sequence of KfiC had homology to a number of glycosyl transferase enzymes and overexpression of the kfiC gene resulted in increased K5 transferase activity. The predicted amino acid sequence of KfiD had homology to a number of NAD‐dependent dehydrogenase enzymes and was demonstrated to be a UDP‐glucose dehydrogenase that catalyses the formation of UDP‐glucuronic acid from UDP‐glucose.
Molecular Genetics and Genomics | 1987
Graham J. Boulnois; Ian Roberts; Rachel Hodge; Kim R. Hardy; Klaus Jann; Kenneth N. Timmis
SummaryTransposon and deletion analysis of the cloned K1 capsule biosynthesis genes of Escherichia coli revealed that approximately 17 kb of DNA, split into three functional regions, is required for capsule production. One block (region 1) is required for translocation of polysaccharide to the cell surface and mutations in this region result in the intracellular appearance of polymer indistinguishable on immunoelectrophoresis to that found on the surface of K1 encapsulated bacteria. This material was released from the cell by osmotic shock indicating that the polysaccharide was probably present in the periplasmic space. Insertions in a second block (region 2) completely abolished polymer production and this second region is believed to encode the enzymes for the biosynthesis and polymerisation of the K1 antigen. Addition of exogenous N-acetylneuraminic acid to one insertion mutant in this region restored its ability to express surface polymer as judged by K1 phage sensitivity. This insertion probably defines genes involved in biosynthesis of N-acetylneuraminic acid. Insertions in a third block (region 3) result in the intracellular appearance of polysaccharide with a very low electrophoretic mobility. The presence of the cloned K1 capsule biosynthesis genes on a multicopy plasmid in an E. coli K-12 strain did not increase the yields of capsular polysaccharide produced compared to the K1+ isolate from which the genes were cloned.
web science | 1997
C.E. Jones; Gilbert Shama; David R. Jones; Ian Roberts; Peter W. Andrew
Studies were undertaken to explain the ability of Listeria monocytogenes to grow at low temperatures in a chemostat. It was found that when grown in continuous culture at a dilution rate of 0·02 h−1L. monocytogenes had a lower proportion of anteiso‐17:0, and a higher proportion of anteiso‐15:0, and smaller chain fatty acids when grown at 10°C compared to 30°C. A previously unreported glycolipid was only seen after growth at low temperature. Growth temperature had no effect on the rate of glucose uptake.
web science | 1994
Stephen V. Gordon; T. Parish; Ian Roberts; Peter W. Andrew
This paper describes the construction of pSG10, the first mycobacterial promoter probe shuttle vector to use the structural gene of a bacterial luciferase as a reporter gene. To examine the utility of using bacterial luciferase to measure gene expression in mycobacteria, the authors have used this vector to monitor the induction of the acetamidase gene promoter of Mycobacterium smegmatis. Luciferase proved to be a rapid, sensitive and easily assayable reporter of changes in gene activity in response to environment in mycobacteria.
web science | 1990
Janet E. Alexander; Peter W. Andrew; Dorothy Jones; Ian Roberts
Electroporation was used to facilitate transformation of Listeria species with plasmid DNA. Optimal conditions for transformation of L. monocytogenes were a field strength of 8·5 kV/cm, 200 Ohms resistance, 25 μF capacitor with a time constant of 5 ms. With these conditions, 3·9 × 106 transformants/pg DNA were obtained. Under the same conditions, L. innocua and L. iuanouii exhibited a frequency of transformation similar to that of L. monocytogenes but a somewhat lower level was obtained with L. seeligeri.
Letters in Applied Microbiology | 1992
A. Wallace; G. Rigg; S.C. Hymanf; R. James; Ian Roberts
An IgM monoclonal antibody specified for the thiol‐activated proteinase of the oral pathogen Porphyromonas gingivalis W83 was generated. The antibody reacted with a single protein of approximate molecular mass 43 kDa in outer membrane preparations of P. gingivalis. Immuno‐electron microscopy using the monoclonal antibody and immunogold labelling confirmed the cell surface location of the thiol‐activated proteinase. The monoclonal antibody failed to detect any proteins in Western blot analysis of other closely related oral bacteria. The specificity of this monoclonal antibody to the thiol‐activated proteinase of P. gingivalis should allow its use as a diagnostic tool for the rapid enumeration of P. gingivalis in clinical samples.
Journal of Bacteriology | 1988
Ian Roberts; R Mountford; Rachel Hodge; K Jann; Graham J. Boulnois
Journal of Bacteriology | 1986
Ian Roberts; R Mountford; N High; D Bitter-Suermann; Klaus Jann; Kenneth N. Timmis; Graham J. Boulnois
Journal of Bacteriology | 1993
Carlo Pazzani; C Rosenow; Graham J. Boulnois; D Bronner; K Jann; Ian Roberts