1887

Abstract

mutants of Gram-negative and some Gram-positive bacteria have an obligate requirement for diaminopimelic acid (DAP), an essential constituent of the cell wall of these organisms. In environments deprived of DAP, for example mammalian tissues, they will undergo lysis. This was previously exploited to develop vaccine strains of and cloning vectors containing as an selectable marker. As a first step for development of such systems for , the gene from wild-type strain PAO1 was cloned by a combined approach of PCR amplification from chromosomal DNA, construction of mini-libraries and by complementation of an mutant. The nucleotide sequence of a 2433 bp fragment was determined. This fragment contained the C-terminal 47 nucleotides of , encoding 3-isopropylmalate dehydrogenase; , encoding aspartate-β-semialdehyde dehydrogenase (Asd); and , whose product showed similarity to the Asd proteins from spp. By subcloning, was localized to a 1.24 kb DNA fragment which in an T7 expression system strongly expressed a 40000 Da protein. The amino acid sequence was deduced from the DNA sequence. A comparison of the Asd proteins from and revealed greater than 63% identity, demonstrating the conserved nature of Asd in Gram-negative bacteria, and defined the active-site-containing consensus sequence GGNCTVXMLMXXXLGLF as a possible signature motif. Chromosomal δ mutants were isolated. They were auxotrophic for DAP, lysine, methionine and threonine, and lysed in the absence of DAP. Genetic analyses indicated that probably is naturally frame-shifted and does not contribute to the Asd phenotype. By PFGE, the gene was mapped to between coordinates 1.89 and 2.15 Mbp, or 37-40 min, on the 5.9 Mbp chromosome.

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1997-03-01
2021-08-01
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