Regulation and Characterization of the dadRAX Locus for d-Amino Acid Catabolism in Pseudomonas aeruginosa PAO1▿

摘要

d-Amino acids are essential components for bacterial peptidoglycan, and these natural compounds are also involved in cell wall remodeling and biofilm disassembling. In Pseudomonas aeruginosa, the dadAX operon, encoding the d-amino acid dehydrogenase DadA and the amino acid racemase DadX, is essential for d- and l-Ala catabolism, and its expression requires a transcriptional regulator, DadR. In this study, purified recombinant DadA alone was sufficient to demonstrate the proposed enzymatic activity with very broad substrate specificity; it utilizes all d-amino acids tested as substrates except d-Glu and d-Gln. DadA also showed comparable kcat and Km values on d-Ala and several d-amino acids. dadRAX knockout mutants were constructed and subjected to analysis of their growth phenotypes on amino acids. The results revealed that utilization of l-Ala, l-Trp, d-Ala, and a specific set of d-amino acids as sole nitrogen sources was abolished in the dadA mutant and/or severely hampered in the dadR mutant while growth yield on d-amino acids was surprisingly improved in the dadX mutant. The dadA promoter was induced by several l-amino acids, most strongly by Ala, and only by d-Ala among all tested d-amino acids. Enhanced growth of the dadX mutant on d-amino acids is consistent with the finding that the dadA promoter was constitutively induced in the dadX mutant, where exogenous d-Ala but not l-Ala reduced the expression. Binding of DadR to the dadA regulatory region was demonstrated by electromobility shift assays, and the presence of l-Ala but not d-Ala increased affinity by 3-fold. The presence of multiple DadR-DNA complexes in the dadA regulatory region was demonstrated in vitro, and the formation of these nucleoprotein complexes exerted a complicated impact on promoter activation in vivo. In summary, the results from this study clearly demonstrate DadA to be the enzyme solely responsible for the proposed d-amino acid dehydrogenase activity of broad substrate specificity and the physiological functions of DadRAX in catabolism of several d-amino acids and support l-Ala as the signal molecule for induction of the dadAX genes through DadR binding to several putative operator sites.