Identification of a novel Rhizobium meliloti nodulation efficiency nfe gene homolog of Agrobacterium ornithine cyclodeaminase

Abstract

The nfe genes located on the large plasmid pRmeGR4b are involved in the nodulation efficiency and competitiveness of Rhizobium meliloti GR4 on alfalfa roots. One hundred twenty-eight base-pairs downstream of nfe2 gene we found an open reading frame designated ORFC, 970 bp long and potentially coding for a 320 amino acid long protein. The amino acid sequence of the putatively encoded ORFC product shows similarity with ornithine cyclodeaminase (OCD) of Agrobacterium tumefaciens an unusual enzyme that converts ornithine into proline. The gene product of ORFC was identified as a 37-kDa protein by in vitro-coupled transcription-translation and in vivo by the T7 RNA polymerase/promoter system. DNA hybridization studies showed that strain GR4 carries a single copy of the ocd-like gene. No homologous sequences to GR4 ORFC DNA were found in other R. meliloti strains or Rhizobium spp. assayed. Furthermore, a GR4 derivative mutant obtained by plasmid disruption of ORFC showed an impaired nodulation efficiency as compared to that of the wild-type strain GR4. Thus, the former locus should be considered a novel nfe gene. We propose to rename the nfe genes, nfe1, 2 and ORFC as nfeA, B, and D, respectively.