Characterization of a Rhizobium meliloti proline dehydrogenase mutant altered in nodulation efficiency and competitiveness on alfalfa roots.

Rhizobium meliloti strain GRM8 is able to transform ornithine into proline by means of an ornithine cyclodeaminase and, therefore, has the ability to use either of these amino acids as its sole carbon and nitrogen source. By Tn5 insertion mutagenesis we obtained a GRM8 mutant derivative strain (LM1) unable to catabolize either ornithine or proline. DNA hybridization studies showed that the LM1 mutant carries a single Tn5 insertion within a chromosomally located gene that, as deduced from a partial nucleotide sequence, encodes a proline dehydrogenase (ProDH). Enzymatic assays confirmed the lack of ProDH activity in cell extracts of strain LM1 and revealed that production of this enzyme is inducible in the parental strain by proline and ornithine. Ultrastructural nodule microscopy analysis, acetylene reduction assays, and dry-weight determinations of nodulated alfalfa plants showed no obvious defect in the nitrogen fixation process of the ProDH- mutant LM1. However, nodulation tests and competition assays demonstrated that in R. meliloti ProDH is required for nodulation efficiency and competitiveness on alfalfa roots.