Abstract

Studies were undertaken to clone and characterise a two-component regulatory system from a clinical isolate (204) of the human and animal pathogen Burkholderia pseudomallei. A number of genomic libraries were constructed in E. coli host-vector systems and screened for the presence of a two-component system using oligonucleotide probes based on nucleotide sequence homology. Fragments of genomic DNA were cloned and sequenced and found to possess two open reading frames (ORFs) that overlap with a single nucleotide and are believed to encode a novel two-component regulatory system. A possible promoter region was identified upstream of the two ORFs, mrgR and mrgS, which read in the same direction and may represent an operon. The deduced translation of mrgR reveals a protein, MrgR, which possesses conserved motifs that are consistent with the phosphorylation domains and DNA-binding helix-turn-helix structure of a family of response regulatory proteins. The deduced translation of mrgS reveals that the MrgS protein possesses all the invariant amino acids that characterise other sensor regulatory proteins. Southern hybridisation studies showed that the mrgRS locus was present in 19 isolates of B. pseudomallei from a wide geographical derivation, but not in any closely related bacterial species, including Burkholderia thailandensis. The expression of the two genes was verified using antibodies developed to synthetic peptides based on sequences from the C- and N-terminal regions of MrgR and MrgS, respectively. The specificity of the antibodies was confirmed in Western blotting studies in which almost all of mrgR and the proximal quarter of mrgS were translationally fused with malE (MBP-MrgR and MBP-MrgS) and expressed in E. coli K12. The antibodies were used to probe Western blots of cellular and extracellular extracts of different isolates of B. pseudomallei and identified multiple bands in whole-cell lysates. The sizes of two of these bands were 24 kDa and 115 kDa, which may represent the unprocessed forms of MrgR and MrgS, respectively. It was proposed that the other bands represented either isoforms or degradation products of the full-length proteins. The recognition of all bands was abolished following pre-incubation of the antibodies with the immunising peptide but remained unaffected if an irrelevant peptide, was used for this purpose. Western blot analysis demonstrated that serum antibodies from a patient with acute melioidosis recognised MBP-MrgR but not MBP-MrgS suggesting a possible role for MrgR in the disease process. The expression of mrgR and mrgS was found to be constitutive in B. pseudomallei that had been cultured using different combinations of temperature, pH and NaCl suggesting that the genes perform a number of biological functions. There is some evidence that at 42°C the processing of MrgR and MrgS may be altered and the possible mechanisms for this are discussed. B. pseudomallei grew better at 42°C and pH 5 and less well at 25°C and pH 8 and this was influenced by NaCl concentration partly reflecting the environmental distribution and intracellular nature of the pathogen. Environmental and clinical isolates of B. pseudomallei differed in the pH optimum for growth at 42°C. The DNA flanking the mrgRS locus in isolate 204 was cloned, sequenced, and seven ORFs were identified including a transcriptional regulatory gene similar to bvgR of Bordetella pertussis. Southern blot analysis using three different DNA probes revealed restriction fragment length polymorphisms (RFLPs) in the region downstream of mrgRS. Two distinct RFLP patterns were identified among 16 different isolates of B. pseudomallei. The potential effects of this variation on gene expression and protein function await further investigation.

Document Type

Thesis

Publication Date

2001

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