Identification of Mesorhizobium loti Genes Relevant to Symbiosis by Using Signature-Tagged Mutants

Abstract

Signature-tagged mutagenesis was applied to Mesorhizobium loti, a nitrogen-fixing root-nodule symbiont of the leguminous plant Lotus japonicus. We arranged 1,887 non-redundant mutant strains of M. loti into 75 sets, each consisting of 24 to 26 strains with a different tag in each strain. These sets were each inoculated en masse onto L. japonicus plants. Comparative analysis of total DNA extracted from inoculants and resulting nodules based on quantitative PCR led to the selection of 69 strains as being reduced in relative abundance during nodulation. Plant assays conducted with individual strains confirmed that 3 were defective in nodulation (Nod − ) and that 10 were Nod + but defective in nitrogen fixation (Fix − ); in each case, the symbiosis deficiency could be attributed to the transposon insertion carried by that strain. Although the remaining 56 strains were Fix + , 33 of them showed significantly reduced competitiveness during nodulation. Among the mutants we identified are known genes that are diverse in predicted function as well as some genes of unknown function, which demonstrates the validity of this screening procedure for functional genomics in rhizobia.Key words: Mesorhizobium loti, signature-tagged mutagenesis, symbiosis, competitiveness, root nodule Rhizobia are a group of soil bacteria that establish a nitrogen-fixing symbiosis with leguminous plants. Mesorhizobium loti is an α-proteobacterial species of the group and a symbiotic partner of Lotus japonicus; this symbiotic pair is a well-documented model system for molecular genetic studies (20, 31). Some rhizobia, including M. loti, elicit root-hair curling and nodule organogenesis on roots of host plants at an early step during symbiosis. Such plant responses are triggered specifically by signaling compounds, called Nod factors, produced by compatible rhizobia. Rhizobia colonize curled root hairs and invade developing nodules via infection threads, which are formed by invagination of the root-hair cell membrane. The rhizobia are then released into the host nodule cells (33,42). These intracellular bacteria (bacteroids) reduce dinitrogen into ammonia by use of a carbon and energy source originating from photosynthates. Host plants are supplied with the fixed nitrogen and thus are able to grow under nitrogen-poor conditions. In a free-living state, rhizobia can metabolize a wide range of carbon and nitrogen sources. This metabolic versatility allows rhizobia to adapt to the nutritional complexity of the rhizosphere, which is affected by plant root exudates (14,15,25,36).Whole genome sequences have been determined for several rhizobial species; their genomes range in size from 5.4 Mb (Azorhizobium caulinodans), 6.7 Mb (Sinorhizobium meliloti) and 7.6 Mb (M. loti) to 8.5 Mb (Bradyrhizobium sp. BTAi1) and 9.1 Mb (Bradyrhizobium japonicum) (8,10,16,17,22). These large sizes can be explained by the need to accommodate the many genes responsible for symbiotic capacity and metabolic versatility. A number of genes have been assigned to functions require...

Genome Analysis Suggests that the Soil Oligotrophic Bacterium Agromonas oligotrophica (Bradyrhizobium oligotrophicum) Is a Nitrogen-Fixing Symbiont of Aeschynomene indica

“…A large-scale transposon mutagenesis study of strain ORS278 identified 29 ndv genes that, when mutated, resulted in severely impaired nodule development by ORS278 (7,9). Unexpectedly, the BiBlast comparison revealed that most of the ndv genes (27 of the 29 genes in ORS278), including purine biosynthesis genes (34), are conserved in the S58 genome (see Table S3 in the supplemental material).…”

Multidisciplinary approaches for studying rhizobium–legume symbioses

“…In further work, this library was used to show that ϳ2% of S. meliloti genes contribute to rhizosphere colonization ). Screening of a Mesorhizobium loti STM library on host plants identified 13 Nod − or Fix − mutants as well as 33 mutants impaired in symbiotic competitiveness (Borjigin et al 2011). Recently, a Sinorhizobium fredii STM library consisting of 25 500 mutants was reported, and an initial screen of 10% of these mutants identified four mutants impaired in symbiosis ).…”