Functional analysis of the genes encoding diaminopropionate ammonia lyase in Escherichia coli and Salmonella enterica serovar Typhimurium.
Diaminopropionate ammonia lyase (DAPAL) is a pyridoxal-5'phosphate (PLP)-dependent enzyme that catalyzes the conversion of diaminopropionate (DAP) to pyruvate and ammonia and plays an important role in cell metabolism. We have investigated the role of the ygeX gene of Escherichia coli K-12 and its ortholog, STM1002, in Salmonella enterica serovar Typhimurium LT2, presumed to encode DAPAL, in the growth kinetics of the bacteria. While Salmonella Typhimurium LT2 could grow on dl-DAP as a sole carbon source, the wild-type E. coli K-12 strain exhibited only marginal growth on dl-DAP, suggesting that DAPAL is functional in S. Typhimurium. The expression of ygeX in E. coli was low as detected by reverse transcriptase PCR (RT-PCR), consistent with the poor growth of E. coli on dl-DAP. Strains of S. Typhimurium and E. coli with STM1002 and ygeX, respectively, deleted showed loss of growth on dl-DAP, confirming that STM1002 (ygeX) is the locus encoding DAPAL. Interestingly, the presence of dl-DAP caused a growth inhibition of the wild-type E. coli strain as well as the knockout strains of S. Typhimurium and E. coli in minimal glucose/glycerol medium. Inhibition by dl-DAP was rescued by transforming the strains with plasmids containing the STM1002 (ygeX) gene encoding DAPAL or supplementing the medium with Casamino Acids. Growth restoration studies using media lacking specific amino acid supplements suggested that growth inhibition by dl-DAP in the absence of DAPAL is associated with auxotrophy related to the inhibition of the enzymes involved in the biosynthetic pathways of pyruvate and aspartate and the amino acids derived from them.
Department of Biochemistry, Indian Institute of Science, Bangalore, India.
J N
JN
Kalyani
Nagaraju
N
Ramachandra
Aashiq H
AH
Kachroo
S
S
Mahadevan
H S
HS
Savithri
D000642:Ammonia-Lyases / Q000235:genetics*; D001224:Aspartic Acid / Q000378:metabolism; D002244:Carbon / Q000378:metabolism; D003470:Culture Media / Q000737:chemistry; D048168:Escherichia coli K12 / Q000201:enzymology* / Q000235:genetics* / Q000254:growth & development; D017353:Gene Deletion; D020869:Gene Expression Profiling; D005816:Genetic Complementation Test; D019289:Pyruvic Acid / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012486:Salmonella typhimurium / Q000201:enzymology* / Q000235:genetics* / Q000254:growth & development; D015091:beta-Alanine / Q000031:analogs & derivatives / Q000378:metabolism
D003470:Culture Media; D015091:beta-Alanine; D001224:Aspartic Acid; D002244:Carbon; D019289:Pyruvic Acid; C011757:2,3-diaminopropionic acid; D000642:Ammonia-Lyases; C023147:diaminopropionate ammonia-lyase