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Regulation of the dauBAR Operon and Characterization of D-Amino Acid Dehydrogenase DauA in Arginine and Lysine Catabolism of Pseudomonas aeruginosa PAO1

¹ Laboratory of Pharmacology, Institute of Medicinal Biotechnology, Chinese Academy of Medical Science;
² Dept. of Biology, Georgia State University

A unique D-to-L racemization of arginine by coupled arginine dehydrogenases DauA and DauB encoded by the dauBAR operon was recently reported as pre-requisite for D-arginine utilization as sole source of carbon and nitrogen through L-arginine catabolic pathways in P. aeruginosa. Here in this study, enzymatic properties of the catabolic FAD-dependent D-amino acid dehydrogenase DauA and the physiological functions of the dauBAR operon were further characterized with other D-amino acids. These results establish DauA as D-amino acid dehydrogenase of broad substrate specificity, with D-Arg and D-Lys as the two most effective substrates based on the kinetic parameters. In addition, expression of dauBAR is specifically induced by exogenous D-Arg and D-Lys, and mutations in dauBAR operon affect utilization of these two amino acids only. The function of DauR as repressor in control of the dauBAR operon was demonstrated by dauB promoter activity measurements in vivo and mobility shift assays with purified His-tagged protein in vitro. The potential effect of 2-ketoarginine (2-KA) derived from D-Arg deamination by DauA as signal molecule of dauBAR induction was first revealed from mutation analysis and further supported by its in vitro effect on alleviation of DauR-DNA interactions. Through sequence analysis, putative DauR operators were identified and confirmed by mutation analysis. Induction of the dauBAR operon to the maximal level was found to require the L-arginine responsive regulator ArgR, as supported by the loss of inductive effect by L-arg on dauBAR expression in the argR mutant and binding of purified ArgR to the dauB regulatory region in vitro. In summary, this study established that optimal induction of the dauBAR operon requires relief of DauR repression by 2-KA and activation of ArgR by L-Arg as a result of D-Arg racemization by the encoded DauA and DauB.

³ E-mail: biocdl{at}langate.gsu.edu