Proline Catabolism by Pseudomonas putida : Cloning, Characterization, and Expression of the put Genes in the Presence of Root Exudates

Abstract

Pseudomonas putida KT2442 is a root-colonizing strain which can use proline, one of the major components in root exudates, as its sole carbon and nitrogen source. A P. putida mutant unable to grow with proline as the sole carbon and nitrogen source was isolated after random mini-Tn5-Km mutagenesis. The mini-Tn5 insertion was located at the putA gene, which is adjacent to and divergent from the putP gene. The putA gene codes for a protein of 1,315 amino acid residues which is homologous to the PutA protein of Escherichia coli, Salmonella enterica serovar Typhimurium, Rhodobacter capsulatus, and several Rhizobium strains. The central part of P. putida PutA showed homology to the proline dehydrogenase of Saccharomyces cerevisiae and Drosophila melanogaster, whereas the C-terminal end was homologous to the pyrroline-5-carboxylate dehydrogenase of S. cerevisiae and a number of aldehyde dehydrogenases. This suggests that in P. putida, both enzymatic steps for proline conversion to glutamic acid are catalyzed by a single polypeptide. The putP gene was homologous to the putP genes 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 a P. putida putA-deficient background, expression of both putA and putP genes was maximal and proline independent. Corn root exudates collected during 7 days also strongly induced the P. putida put genes, as determined by using fusions of the put promoters to lacZ. The induction ratio for the putA promoter (about 20-fold) was 6-fold higher than the induction ratio for the putP promoter.

Expression, Purification and Characterization of the Proline Dehydrogenase Domain of PutA from Pseudomonas putida POS-F84

“…From this feature, it can be inferred that like many other ProDHs [6], the P. putida ProDH was a form of mesophilic enzymes. The obtained data were in good agreement with those previously ProDHs from P. aeruginosa [6], P. putida [15] and P. fluorescence [3]. The effect of various pH values on the enzymatic reaction of ProDH was evaluated in the pH range from 3.0 to 12.0 at 30°C.…”

Selection of hyperadherent mutants in Pseudomonas putida biofilms

“…The gene disrupted in ibi-815 corresponded to putA, encoding proline dehydrogenase. This enzyme catalyses the two-step conversion of proline into glutamic acid, and has a regulatory role in its own expression, as well as in that of the proline transporter putP; it also plays a role in redox signalling (Vílchez et al, 2000;Fernández-Piñar et al, 2008).…”

“…The gene disrupted in ibi-815 corresponded to putA, encoding proline dehydrogenase. This enzyme catalyses the two-step conversion of proline into glutamic acid, and has a regulatory role in its own expression, as well as in that of the proline transporter putP; it also plays a role in redox signalling (Vílchez et al, 2000; Fernández-Piñar et al, 2008).In the remaining four mutants, arbitrary PCR and DNA sequencing placed the insertion of mini-Tn5 in the same position in locus PP_0116. This gene encodes a 235 aa protein highly conserved in all Pseudomonas species sequenced so far.…”

Identification of a Chemoreceptor for Tricarboxylic Acid Cycle Intermediates

“…However, most of these MCPs are of unknown function. To compensate for nutritional shortages, many soil microorganisms are able to use TCA cycle intermediates present in plant root exudates or cell debris for growth (4,5). This, combined with the fact that TCA cycle intermediates are abundantly present in plant tissue and root exudates (4, 6) might explain why many bacteria show a chemotactic response toward TCA cycle intermediates (7)(8)(9)(10)(11)(12)(13).…”