Proline Catabolism byPseudomonas putida: Cloning, Characterization, and Expression of theputGenes in the Presence of Root Exudates

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Abstract
Pseudomonas putidaKT2442 is a root-colonizing strain which can use proline, one of the major components in root exudates, as its sole carbon and nitrogen source. AP. putidamutant unable to grow with proline as the sole carbon and nitrogen source was isolated after random mini-Tn5–Km mutagenesis. The mini-Tn5insertion was located at theputAgene, which is adjacent to and divergent from theputPgene. TheputAgene codes for a protein of 1,315 amino acid residues which is homologous to the PutA protein ofEscherichia coli,Salmonella entericaserovar Typhimurium,Rhodobacter capsulatus, and severalRhizobiumstrains. The central part ofP. putidaPutA showed homology to the proline dehydrogenase ofSaccharomyces cerevisiaeandDrosophila melanogaster, whereas the C-terminal end was homologous to the pyrroline-5-carboxylate dehydrogenase ofS. cerevisiaeand a number of aldehyde dehydrogenases. This suggests that inP. putida, both enzymatic steps for proline conversion to glutamic acid are catalyzed by a single polypeptide. TheputPgene was homologous to theputPgenes of several prokaryotic microorganisms, and its gene product is an integral inner-membrane protein involved in the uptake of proline. The expression of both genes was induced by proline added in the culture medium and was regulated by PutA. In aP. putida putA-deficient background, expression of bothputAandputPgenes was maximal and proline independent. Corn root exudates collected during 7 days also strongly induced theP. putida putgenes, as determined by using fusions of theputpromoters to ′lacZ. The induction ratio for theputApromoter (about 20-fold) was 6-fold higher than the induction ratio for theputPpromoter.