Functional Genomics Enables Identification of Genes of the Arginine Transaminase Pathway in Pseudomonas aeruginosa

Abstract

ABSTRACT Arginine utilization in Pseudomonas aeruginosa with multiple catabolic pathways represents one of the best examples of the metabolic versatility of this organism. To identify genes involved in arginine catabolism, we have employed DNA microarrays to analyze the transcriptional profiles of this organism in response to l -arginine. While most of the genes involved in arginine uptake, regulation, and metabolism have been identified as members of the ArgR (arginine-responsive regulatory protein) regulon in our previous study, they did not include any genes of the arginine dehydrogenase (ADH) pathway. In this study, 18 putative transcriptional units of 38 genes, including the two known genes of the ADH pathway, kauB and gbuA , were found to be inducible by exogenous l -arginine in the absence of ArgR. To identify the missing genes that encode enzymes for the initial steps of the ADH pathway, the potential physiological functions of those candidate genes in arginine utilization were studied by growth phenotype analysis of knockout mutants. Expression of these genes was induced by l -arginine in an aruF mutant strain devoid of a functional arginine succinyltransferase pathway, the major route of arginine utilization. Disruption of dadA , a putative catabolic alanine dehydrogenase-encoding gene, in the aruF mutant produced no growth on l -arginine, suggesting the involvement of l -alanine in arginine catabolism. This hypothesis was further supported by the detection of an l -arginine-inducible arginine:pyruvate transaminase activity in the aruF mutant. Knockout of aruH and aruI , which encode an arginine:pyruvate transaminase and a 2-ketoarginine decarboxylase in an operon, also abolished the ability of the aruF mutant to grow on l -arginine. The results of high-performance liquid chromatography analysis demonstrated consumption of 2-ketoarginine and suggested that generation of 4-guanidinobutyraldehyde occurred in the aruF mutant but not in the aruF aruI mutant. These results led us to propose the arginine transaminase pathway that removes the α-amino group of l -arginine via transamination instead of oxidative deamination by dehydrogenase or oxidase as originally proposed. In the same genetic locus, we also identified a two-component system, AruRS, for the regulation of arginine-responsive induction of the arginine transaminase pathway. This work depicted a wider network of arginine metabolism than we previously recognized.