Phenotypic and molecular diversity of Lupinus mariae-josephi endosymbiotic bacteria.

Abstract

Lupinus mariae-josephi is a new Lupinus species recently described in a Southeastern area of Spain (Valencia) in soils of singularly high pH and active lime content. Bacteria from L. mariaejosephi have been isolated and correspond to extra-slow growing bacteria symbiotically and phylogenetically distant to endosymbiotic strains nodulating other Lupinus sp. native of the Iberian Peninsula and adapted to growth in acid soils. Cross-inoculation experiments revealed that the L. mariae-josephi endosymbiotic bacteria are unable to nodulate or efficiently fix nitrogen with well-known Lupinus spp. Their species affiliation was examined by a multilocus sequence analysis of four housekeeping genes (16S rDNA, glnII, recA, atpD) and the symbiotic nodC gene. Single and concatenated phylogenetic analyses of these genes consistently revealed that L. mariae-josephi endosymbiotic bacteria belong to a clade, within the Bradyrhizobium genus, highly differentiated from the Bradyrhizobium clade that includes currently named Bradyrhizobium species as well as the endosymbiotic bacteria from Lupinus species tested in this study. Within this new clade the L. mariae-josephi bacteria nested in several subgroups that may correspond to novel sister species. The phylogenetic analysis based on the nodC gene showed that L. mariae-josephi endosymbiotic bacteria define a novel branch in the nodC Bradyrhizobium tree and likely have a common unique ancestor for the symbiotic genes with nodule isolates from Retama spp. At this moment two draft genome sequences belonging to a Bradyrhizobium isolated from L. angustifolius (ISLU101) and to a Bradyrhizobium isolated from L. mariae-josephi (LmjC) have been obtained. The first analysis showed that both genomes correspond to very large chromosomes (>8000 genes) with a high number of unique proteins. nod genes organization are highly conserved among ISLU101, LmjC and B. japonicum USDA110. LmjC presents a single cluster with nod genes from diverse origins. ISLU101 fix genes are found in a single cluster homologous to that of USDA110. LmjC possess a complete copy of fix genes homologous to that from USDA110 and an incomplete one similar to S. meliloti. LmjC possess TypeIII and TypeIV secretion systems. ISLU101 has a Type IV homologous to that of photosynthetic Bradyrhizobium BTAi1, and two copies of Type VI, one homologous to that of USDA110 and the other to R. leguminosarum. Preliminary data indicate that ISLU101 contains a potential second replicon (~100 genes) with high homology to Bradyrhizobium BTai1 plasmid sequence