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ACTIVATION OF A CRYPTIC D-SERINE DEAMINASE (DSD) GENE FROM PSEUDOMONAS CEPACIA 17616
Montgomery, S. O. AND T. G. Lessie. ACTIVATION OF A CRYPTIC D-SERINE DEAMINASE (DSD) GENE FROM PSEUDOMONAS CEPACIA 17616. Presented at Presented at the 1995 Annual Meeting of the American Society for Microbiology, Washington, DC, May 21 - 25, 1995.
D-serine inhibits growth of P. cepacia 17616; however, resistant mutants able to express an ordinarily cryptic D-serine deaminase (dsd) gene were isolated readily. The resistant strains formed high levels of a D-serine deaminase active on D-threonine as well as D-serine. IS elements have been shown to play an important role in the activation of gene expression in P. cepacia. To examine if D-serine resistance was due to IS-element-dependent dsd gene expression, we cloned and determined nucleotide sequence of a DNA fragment containing this gene. Southern hybridization experiments using the cloned DNA as a probe failed to reveal any genomic rearrangements consistent with insertional activation. We presume that the majority of mutations are due to small alterations at sites upstream of the dsd gene. We have designed PCR primers suitable for amplification of this region and are attempting to define regulatory sequences important for dsd gene expression. When a representative Dsd+ mutant was plated to medium containing mannitol and D-serine, colonies appeared within two days. In contrast, Dsd+ derivatives of the wild-type appeared only after 3-4 days. Addition of L-lysine to selection plates decreased the number of Dsd+ colonies more then ten-fold. L-homoserine caused a corresponding increase in the number of Dsd+ colonies. Our results suggest that the majority of dsd mutations occur post-exponentially and that adaptation to D-serine resistance can be modulated by members of the aspartate family of amino acids.
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