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Identification and characterization of a cryptic D-serine deaminase (DSD) gene from Burkholderia cepacia

by Montgomery, Stacy O

Abstract (Summary)
Isolates of Burkholderia cepacia differed in their ability to utilize D-serine as a carbon and energy source. Strain 17616 is one of the strains which ordinarily fails to utilize D-serine. D-serine also inhibited the growth of this strain on alternative carbon sources such as mannitol. D-serine resistant (Dsd$\sp+$) mutants of strain 17616 were isolated which formed high constitutive levels of a D-serine deaminase not present in the wild type. The majority of such mutants also utilized D-serine as sole carbon source. A 5.5-kb fragment of B. cepacia DNA containing the dsd gene was cloned into the DsdA$\sp-$ strain E. coli AC6082. Introduction of recombinant plasmids carrying the dsd gene into strains 17616 or AC6082 resulted in high levels of constitutive D-serine deaminase activity as well as expression of a new peptide with the predicted size (45 kDa) of the the dsd gene product. Efforts were made to define the mechanism of activation of the cryptic dsd gene in strain 17616. Comparison of PCR products of the region upstream of the dsd gene in the wild type and Dsd$\sp+$ strains indicated that dsd gene expression was not a consequence of insertion of tranposable-gene-activating elements upstream of the cryptic gene as had been observed for activation of foreign bla and lac genes in this strain. Furthermore, no other alterations were detected in this upstream region that would account for dsd gene activation. Analysis the region downstream of the dsd gene revealed the presence of a marR-like repressor gene and adjacent multi-drug resistance transporter gene. The orientation of the latter two genes was opposite to that of the dsd gene. We were interested in the possibility that the marR (multiple antibiotic resistance/multiple adaptive response) protein might control dsd gene expression. To explore this possibility, I compared the nucleotide sequences of the marR gene from the representative Dsd$\sp+$ strain 249-50 with the corresponding wild type gene. The wild type and mutant sequences were identical.
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Advisor:

School:University of Massachusetts Amherst

School Location:USA - Massachusetts

Source Type:Master's Thesis

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Date of Publication:01/01/1998

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