Molecular genetic analysis of the region containing the essential Pseudomonas aeruginosa asd gene encoding aspartate-beta-semialdehyde dehydrogenase.
asd 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 Salmonella typhimurium and cloning vectors containing asd as an in vivo selectable marker. As a first step for development of such systems for Pseudomonas aeruginosa, the asd 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 Escherichia coli delta asd mutant. The nucleotide sequence of a 2433 bp Smal-Nsil fragment was determined. This fragment contained the C-terminal 47 nucleotides of leuB, encoding 3-isopropylmalate dehydrogenase; asd, encoding aspartate-beta-semialdehyde dehydrogenase (Asd); and orfA, whose product showed similarity to the Asd proteins from Vibrio spp. By subcloning, asd was localized to a 1.24 kb DNA fragment which in an E. coli T7 expression system strongly expressed a 40,000 Da protein. The amino acid sequence was deduced from the DNA sequence. A comparison of the Asd proteins from P. aeruginosa, E. coli and Haemophilus influenzae 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 delta asd mutants were isolated. They were auxotrophic for DAP, lysine, methionine and threonine, and lysed in the absence of DAP. Genetic analyses indicated that orfA probably is naturally frame-shifted and does not contribute to the Asd phenotype. By PFGE, the asd gene was mapped to between coordinates 1.89 and 2.15 Mbp, or 37-40 min, on the 5.9 Mbp P. aeruginosa chromosome.
Hoang
Tung T
TT
Department of Microbiology & Infectious Diseases, University of Calgary Health Sciences Center,Calgary, Alberta,Canada T2N 4N1.|Department of Microbiology, Colorado State University,Fort Collins, CO 80523,USA.
Williams
Scott
S
Department of Microbiology & Infectious Diseases, University of Calgary Health Sciences Center,Calgary, Alberta,Canada T2N 4N1.
Schweizer
Herbert P
HP
Canadian Bacterial Diseases Network, University of Guelph,Guelph, Ontario,Canada N1G 2W1.|Department of Microbiology, Colorado State University,Fort Collins, CO 80523,USA.
Lam
Joseph S
JS
Department of Microbiology, University of Guelph,Guelph, Ontario,Canada N1G 2W1.|Canadian Bacterial Diseases Network, University of Guelph,Guelph, Ontario,Canada N1G 2W1.
D000595:Amino Acid Sequence; D001223:Aspartate-Semialdehyde Dehydrogenase / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D011550:Pseudomonas aeruginosa / Q000235:genetics*; D016415:Sequence Alignment; D017421:Sequence Analysis
D001223:Aspartate-Semialdehyde Dehydrogenase