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B R Clarke

Publications and source records attributed to B R Clarke.

12 recordsLinked to original sources
PubMed1992

Molecular cloning of the rfb region of Klebsiella pneumoniae serotype O1:K20: the rfb gene cluster is responsible for synthesis of the D-galactan I O polysaccharide.

Previous chemical analyses identified two structurally distinct O polysaccharides in the lipopolysaccharide of Klebsiella pneumoniae serotype O1:K20 (C. Whitfield, J. C. Richards, M. B. Perry, B. R. Clarke, and L. L. MacLean, J. Bacteriol. 173:1420-1431, 1991). The polysaccharides were designated D-galactan I and D-galactan II; both are homopolymers of galactose. To begin investigation of the synthesis and expression of these O polysaccharides, we have cloned a 7.3-kb region of the chromosome of K. pneumoniae O1:K20, containing the his-linked rfbkpO1 (O-antigen biosynthesis) gene cluster. In Escherichia coli K-12 and Salmonella typhimurium, rfbkpO1 directed the synthesis of D-galactan I but not D-galactan II. The cloned rfbkpO1 genes did not complement a mutation affecting D-galactan II synthesis in K. pneumoniae CWK37, suggesting that another (unlinked) locus is also required for D-galactan II expression. However, plasmids carrying rfbkpO1 did complement a mutation in K. pneumoniae CWK43 which eliminated expression of both D-galactan I and D-galactan II, indicating that at least one function is common to synthesis of both polymers. Synthesis of D-galactan I was dependent on chromosomal galE and rfe genes. Hybridization experiments indicated that the rfbkpO1 sequences from different serotype O1 Klebsiella isolates showed some restriction fragment length polymorphism.

Cloning, Molecular↗
PubMed1991

Expression of two structurally distinct D-galactan O antigens in the lipopolysaccharide of Klebsiella pneumoniae serotype O1.

The lipopolysaccharide (LPS) molecule is an important virulence determinant in Klebsiella pneumoniae. Studies on the serotype O1 LPS were initiated to determine the basis for antigenic heterogeneity previously observed in the O1 side chain polysaccharides and to resolve apparent ambiguities in the reported polysaccharide structure. Detailed chemical analysis, involving methylation and 1H- and 13C-nuclear magnetic resonance studies, demonstrated that the O-side chain polysaccharides of serotype O1 LPS contained a mixture of two structurally distinct D-galactan polymers. The repeating unit structures of these two polymers were identified as [----3)-beta-D-Galf-(1----3)-alpha-D-Galp-(1----] (D-galactan I) and [----3)-alpha-D-Galp-(1----3)-beta-D-Galp-(1----] (D-Galactan II). D-Galactan I polysaccharides were heterogeneous in size and were detected throughout the sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) profile of O1 LPS. In contrast, D-galactan II was confined to the higher-molecular-weight region. The structures of the two D-galactans were not influenced by simultaneous synthesis of a capsular K antigen. Apparently, neither of the D-galactans constitutes a common antigen widespread in Klebsiella spp. as determined by immunochemical analysis. Examination of the LPSs in mutants indicated that expression of D-galactan I can occur independently of D-galactan II. Transconjugants of Escherichia coli K-12 strains carrying the his region of K. pneumoniae were constructed by chromosome mobilization with RP4::mini-Mu. In these transconjugants, the O antigen encoded by the his-linked rfb locus was determined to be D-galactan I, suggesting that genes involved in the expression of D-galactan II are not closely linked to the rfb cluster.

Antigens, Bacterial↗
PubMed1990

Use of oligonucleotide probes to identify members of two-component regulatory systems in Xanthomonas campestris pathovar campestris.

Two-component regulatory systems comprising a sensor and a regulator protein, both with highly conserved amino acid domains, and commonly genetically linked, have been described in a range of bacterial species and are involved in sensing environmental stimuli. We used two oligonucleotide probes matching the postulated coding regions for domains of sensor and regulator proteins respectively in Xanthomonas campestris pathovar campestris (Xcc) to identify possible two-component regulatory systems in Xcc. Two different fragments of Xcc DNA with homology to both of these probes were cloned. The DNA sequence of part of one of these fragments encompassed a potential open reading frame (ORF), the predicted amino acid sequence of which had extensive homology with regulator proteins of two-component regulatory systems. Analysis of the predicted amino acid sequence for the 3' end of an adjacent ORF revealed a very high level of homology with the C-terminal end of sensor proteins. Strains of Xcc with Tn5-induced mutations in the regulator gene were affected in extracellular polysaccharide production, and also in resistance to salt and chloramphenicol. No effects of mutation in the second clone were observed.

Amino Acid Sequence↗
PubMed1990

A multipurpose broad host range cloning vector and its use to characterise an extracellular protease gene of Xanthomonas campestris pathovar campestris.

A multipurpose broad host range plasmid, pIJ3200, was constructed by inserting the polylinker-containing 445 bp PvuII fragment of Bluescript M13 into the EcoRI site of the cosmid pLAFR1. Using this vector a protease gene of Xanthomonas campestris pathovar campestris, previously cloned in the recombinant plasmid pIJ3070, was located by deletion to a 2.2 kb DNA region. Sequencing of the protease gene revealed an open reading frame encoding a 580 amino acid polypeptide with molecular weight of 57,000. The deduced amino acid sequence showed strong homology with the subtilisin family of serine proteases. This, together with its sensitivity to inhibition by phenylmethylsulphonyl fluoride, suggests that the enzyme belongs to this family of proteases.

Amino Acid Sequence↗
PubMed1990

Extracellular proteases from Xanthomonas campestris pv. campestris, the black rot pathogen.

Two proteases (PRT1 and PRT2) were fractionated from culture supernatants of wild-type Xanthomonas campestris pv. campestris by cation-exchange chromatography on SP-5PW. Inhibitor experiments showed that PRT 1 was a serine protease which required calcium ions for activity or stability or both and that PRT 2 was a zinc-requiring metalloprotease. PRT 1 and PRT 2 showed different patterns of degradation of beta-casein. The two proteases comprised almost all of the extracellular proteolytic activity of the wild type. A protease-deficient mutant which lacked both PRT 1 and PRT 2 showed considerable loss of virulence in pathogenicity tests when bacteria were introduced into mature turnip leaves through cut vein endings. This suggests that PRT 1 and PRT 2 have a role in black rot pathogenesis.

Bacterial Proteins↗
PubMed1979

Children who wear individual hearing aids in British Columbia, Canada.

Data collected on the use of hearing aids by 680 school-aged hearing-impaired subjects are examined and aid use is related to gender, age, hearing loss, age at onset, aetiology, age at diagnosis, educational setting, method of communication, primary language in the home, hearing status of parents and additional handicaps. The likelihood of a student wearing an aid is significantly related to certain educational settings, the degree of hearing loss, age and to a lesser extent, his method of communication.

Adolescent↗
PubMed1978

Hearing impairment in children of low birthweight.

Audiometer evaluations were carried out on a population of 204 low birth weight (LBW) children and 123 controls. In the LBW group, 6 children (3.3%) had a bilateral loss and 5 (2.5%) had a unilateral sensorineural high-frequency hearing loss. No case of sensorineural hearing loss was found among the controls. There were 13 (6.4%) cases of conductive loss among the LBW sample, compared with 3 (2.4%) among the controls. Correlation coefficients showed a relationship between sensorineural impairment and: bilirubin level, incubator time, antibiotic history, and neurological status. Subsequent multiple classification analyses showed that, while incubator time and bilirubin level are each significant predictors of sensorineural loss, this is not the case with antibiotics or neurological status. Neurological status was closely associated with the syndrome of LBW, high bilirubin level, extended incubator time and sensorineural loss. However, no significant relationship could be found between neurological impairment and these predictors nor can it be regarded as useful in predicting hearing loss in this population.

Brain Diseases↗
PubMed1975

B.R. Clarke.

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History of Medicine↗
PubMedDate not supplied

Identification and DNA sequence of a pathogenicity gene of Xanthomonas campestris pv. campestris.

A region of Xanthomonas campestris pv. campestris DNA containing at least two pathogenicity genes was identified. Mutants in one gene were clearly reduced in pathogenicity while mutants in the other were only moderately reduced. Both classes of mutants were prototrophic and motile, and had wild-type levels of extracellular enzymes and extracellular polysaccharide. They also grew in vitro and in planta at the same rate as the wild type. Experiments involving one of the clear pathogenicity mutants indicated that the recovery of mutant cells from turnip seedlings 24 hr after inoculation was lower than for the wild type. This may be due to cell death as a result of action by some preformed or induced plant factor. From DNA sequencing an open reading frame was identified that encompassed the site of the mutations giving a clear reduction in pathogenicity. The predicted protein sequence had no homology with other proteins in the computer data base.

Amino Acid Sequence↗