The fluorescent dihydroxyquinoline chromophore of the pyoverdine siderophore in Pseudomonas is a condensation product of D-tyrosine and L-2,4-diaminobutyrate. Both pvdH and asd (encoding aspartate -semialdehyde dehydrogenase) knockout mutants of Pseudomonas aeruginosa PAO1 were unable to synthesize pyoverdine under iron-limiting conditions in the absence of L-2,4-diaminobutyrate in the culture media. The pvdH gene was subcloned, and the gene product was hyperexpressed and purified from P. aeruginosa PAO1. PvdH was found to catalyze an aminotransferase reaction, interconverting aspartate -semialdehyde and L-2,4-diaminobutyrate. Steady-state kinetic analysis with a novel coupled assay established that the enzyme adopts a ping-pong kinetic mechanism and has the highest specificity for ␣-ketoglutarate. The specificity of the enzyme toward the smaller keto acid pyruvate is 41-fold lower. The enzyme has negligible activity toward other keto acids tested. Homologues of PvdH were present in the genomes of other Pseudomonas spp. These homologues were found in the DNA loci of the corresponding genomes that contain other pyoverdine synthesis genes. This suggests that there is a general mechanism of L-2,4-diaminobutyrate synthesis in Pseudomonas strains that produce the pyoverdine siderophore.
We have shown that it is feasible to isolate pure cff DNA from routinely discarded AF supernatant to perform QF-PCR and microarray analyses, providing timely and informative results even for problematic grossly bloody and otherwise compromised AF samples or culture failures.
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