Nodulation and nitrogen fixation genes of Mesorhizobium loti are encoded on the chromosome of the bacterium. Nevertheless, there is strong evidence that these genes can be transferred from an inoculant strain to nonsymbiotic mesorhizobia in the field environment. Here we report that the chromosomal symbiotic element of M. loti strain ICMP3153 is transmissible in laboratory matings to at least three genomic species of nonsymbiotic mesorhizobia. The element is 500 kb in size, integrates into a phe-tRNA gene, and encodes an integrase of the phage P4 family just within its left end. The entire phe-tRNA gene is reconstructed at the left end of the element upon integration, whereas the 3 17 nucleotides of the tRNA gene are present as a direct repeat at the right end. We termed the element a symbiosis island on the basis of its many similarities to pathogenicity islands. It may represent a class of genetic element that contributes to microbial evolution by acquisition.The considerable impact of horizontal gene transfer on microbial evolution has become apparent from recent molecular analyses (1-4). For example, many bacterial pathogens contain clusters of virulence genes not present in closely related nonpathogenic strains or species. These gene clusters may be located on transmissible phage or plasmids, but are often found as so-called pathogenicity islands on the chromosome (5-8). The acquisition of a pathogenicity island is likely to have been a key step in the evolution of the pathogen. For example, the SPI-1 pathogenicity island was probably acquired very early in the evolution of Salmonella and enabled that genus to invade epithelial cells (9, 10). Pathogenicity islands range up to 190 kb in size, and most are found adjacent to or integrated within tRNA genes or flanked by insertion sequences. tRNA genes are targets for integration of plasmids and phage in a wide variety of bacteria, and pathogenicity islands in Dichelobacter nodosus and Vibrio cholerae are linked to phagederived integrase genes, suggesting that pathogenicity islands may have been acquired by a phage-mediated process (11-13). However, transmissibility of pathogenicity islands has not been demonstrated and their evolutionary origin remains unknown.We are working with Mesorhizobium loti, a species previously named Rhizobium loti, that is able to form nodules on several Lotus species. Mesorhizobium was recently described as a new genus of root nodule bacteria phylogenetically distinct from other root nodule bacteria of the genera Rhizobium and Bradyrhizobium on the basis of 16S rRNA gene sequence and other properties (14). M. loti differs from Rhizobium species and some other Mesorhizobium species in that its symbiotic information is chromosome rather than plasmid encoded (15-17). Nevertheless, we observed that genetically diverse symbiotic mesorhizobia, isolated from nodules off a stand of Lotus corniculatus established with a single inoculant strain, M. loti strain ICMP3153, in an area devoid of naturalized rhizobia able to nodulate the pl...

Bacterial infection of interior tissues of legume root nodules is controlled at the epidermal cell layer and is closely coordinated with progressing organ development. Using spontaneous nodulating Lotus japonicus plant mutants to uncouple nodule organogenesis from infection, we have determined the role of 16 genes in these two developmental processes. We show that host-encoded mechanisms control three alternative entry processes operating in the epidermis, the root cortex and at the single cell level. Single cell infection did not involve the formation of trans-cellular infection threads and was independent of host Nod-factor receptors and bacterial Nod-factor signals. In contrast, Nod-factor perception was required for epidermal root hair infection threads, whereas primary signal transduction genes preceding the secondary Ca2+ oscillations have an indirect role. We provide support for the origin of rhizobial infection through direct intercellular epidermal invasion and subsequent evolution of crack entry and root hair invasions observed in most extant legumes.

Surface polysaccharides are important for bacterial interactions with multicellular organisms, and some are virulence factors in pathogens. In the legume-rhizobium symbiosis, bacterial exopolysaccharides (EPS) are essential for the development of infected root nodules. We have identified a gene in Lotus japonicus, Epr3, encoding a receptor-like kinase that controls this infection. We show that epr3 mutants are defective in perception of purified EPS, and that EPR3 binds EPS directly and distinguishes compatible and incompatible EPS in bacterial competition studies. Expression of Epr3 in epidermal cells within the susceptible root zone shows that the protein is involved in bacterial entry, while rhizobial and plant mutant studies suggest that Epr3 regulates bacterial passage through the plant's epidermal cell layer. Finally, we show that Epr3 expression is inducible and dependent on host perception of bacterial nodulation (Nod) factors. Plant-bacterial compatibility and bacterial access to legume roots is thus regulated by a two-stage mechanism involving sequential receptor-mediated recognition of Nod factor and EPS signals.

Rhizobia were isolated from nodules off a stand ofLotus corniculatus established with a single inoculant strain, ICMP3153, 7 years earlier in an area devoid of naturalized Rhizobium loti. The isolates showed diversity in growth rate, Spe I fingerprint of genomic DNA, and hybridization pattern to genomic DNA probes. The 19%o of isolates that grew at the same rate as strain ICMP3153 were the only isolates that had the same fingerprint as strain ICMP3153. Sequencing of part of the 16S rRNA gene of several diverse isolates confirmed that they were not derived from the inoculant strain. Nevertheless, all non-ICMP3153 strains gave EcoRI and Spe I hybridization patterns identical to ICMP3153 when hybridized to nodulation gene cosmids. Hybridization of digests generated by the very rare cutting enzyme Swa I revealed that the symbiotic DNA region (at least 105 kb) was chromosomally integrated in the strains. The results suggest that the diverse strains arose by transfer of chromosomal symbiotic genes from ICMP3153 to nonsymbiotic rhizobia in the environment.

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