Root nodule bacteria require access to adequate concentrations of mineral nutrients for metabolic processes to enable their survival and growth as free-living soil saprophytes, and in their symbiotic relationship with legumes. Essential nutrients, with a direct requirement in metabolism of rhizobia are carbon, hydrogen, oxygen, nitrogen, phosphorus, sulfur, potassium, calcium, magnesium, iron, manganese, copper, zinc, molybdenum, nickel, cobalt and selenium. Boron does not seem to be required by rhizobia, but is essential for the establishment of effective legume symbioses. Nutrient constraints can affect both free-living and symbiotic forms of root nodule bacteria, but whether they do is a function of a complex series of events and interactions. Important physiological characteristics of rhizobia involved in, or affected by, their mineral nutrition include nutrient uptake, growth rate, gene regulation, nutrient storage, survival, genetic exchange and the viable non-culturable state. There is considerable variation between genera, species and strains of rhizobia in their response to nutrient deficiency. The effects of nutrient deficiencies on free-living rhizobia in the soil are poorly understood. Competition between strains of rhizobia for limiting phosphorus and iron in the rhizosphere may affect their ability to nodulate legumes. Processes in the development of some legume symbioses specifically require calcium, cobalt, copper, iron, potassium, molybdenum, nickel, phosphorus, selenium, zinc and boron. Limitations of phosphorus, calcium, iron and molybdenum in particular, can reduce legume productivity by affecting nodule development and function. The effects of nutrient deficiencies on rhizobia–legume signalling are not understood. The supply of essential inorganic nutrients to bacteroids in relation to nutrient partitioning in nodule tissues and nutrient transport to the symbiosome may affect effectiveness of nitrogen fixation. An integration of molecular approaches with more traditional biochemical, physiological and field-based studies is needed to improve understanding of the agricultural importance of rhizobia response to nutrient stress.

Summary• Medicago truncatula (barrel medic) A17 is currently being sequenced as a model legume, complementing the sequenced root nodule bacterial strain Sinorhizobium meliloti 1021 (Sm1021). In this study, the effectiveness of the Sm1021-M. truncatula symbiosis at fixing N 2 was evaluated.• N 2 fixation effectiveness was examined with eight Medicago species and three accessions of M. truncatula with Sm1021 and two other Sinorhizobium strains. Plant shoot dry weights, plant nitrogen content and nodule distribution, morphology and number were analysed.• Compared with nitrogen-fed controls, Sm1021 was ineffective or partially effective on all hosts tested (excluding M. sativa), as measured by reduced dry weights and shoot N content. Against an effective strain, Sm1021 on M. truncatula accessions produced more nodules, which were small, pale, more widely distributed on the root system and with fewer infected cells.• The Sm1021-M. truncatula symbiosis is poorly matched for N 2 fixation and the strain could possess broader N 2 fixation deficiencies. A possible origin for this reduction in effectiveness is discussed. An alternative sequenced strain, effective at N 2 fixation on M. truncatula A17, is Sinorhizobium medicae WSM419.New Phytologist (2008) 179: 62-66

The multi-billion dollar asset attributed to symbiotic nitrogen fixation is often threatened by the nodulation of legumes by rhizobia that are ineffective or poorly effective in N(2) fixation. This study investigated the development of rhizobial diversity for the pasture legume Biserrula pelecinus L., 6 years after its introduction, and inoculation with Mesorhizobium ciceri bv. biserrulae strain WSM1271, to Western Australia. Molecular fingerprinting of 88 nodule isolates indicated seven were distinctive. Two of these were ineffective while five were poorly effective in N(2) fixation on B. pelecinus. Three novel isolates had wider host ranges for nodulation than WSM1271, and four had distinct carbon utilization patterns. Novel isolates were identified as Mesorhizobium sp. using 16S rRNA, dnaK and GSII phylogenies. In a second study, a large number of nodules were collected from commercially grown B. pelecinus from a broader geographical area. These plants were originally inoculated with M. c bv. biserrulae WSM1497 5-6 years prior to isolation of strains for this study. Nearly 50% of isolates from these nodules had distinct molecular fingerprints. At two sites diverse strains dominated nodule occupancy indicating recently evolved strains are highly competitive. All isolates tested were less effective and six were ineffective in N(2) fixation. Twelve randomly selected diverse isolates clustered together, based on dnaK sequences, within Mesorhizobium and distantly to M. c bv. biserrulae. All 12 had identical sequences for the symbiosis island insertion region with WSM1497. This study shows the rapid evolution of competitive, yet suboptimal strains for N(2) fixation on B. pelecinus following the lateral transfer of a symbiosis island from inoculants to other soil bacteria.

Integrative and conjugative elements (ICEs) are ubiquitous mobile genetic elements present as "genomic islands" within bacterial chromosomes. Symbiosis islands are ICEs that convert nonsymbiotic mesorhizobia into symbionts of legumes. Here we report the discovery of symbiosis ICEs that exist as three separate chromosomal regions when integrated in their hosts, but through recombination assemble as a single circular ICE for conjugative transfer. Whole-genome comparisons revealed exconjugants derived from nonsymbiotic mesorhizobia received three separate chromosomal regions from the donor Mesorhizobium ciceri WSM1271. The three regions were each bordered by two nonhomologous integrase attachment (att) sites, which together comprised three homologous pairs of attL and attR sites. Sequential recombination between each attL and attR pair produced corresponding attP and attB sites and joined the three fragments to produce a single circular ICE, ICEMcSym 1271 . A plasmid carrying the three attP sites was used to recreate the process of tripartite ICE integration and to confirm the role of integrase genes intS, intM, and intG in this process. Nine additional tripartite ICEs were identified in diverse mesorhizobia and transfer was demonstrated for three of them. The transfer of tripartite ICEs to nonsymbiotic mesorhizobia explains the evolution of competitive but suboptimal N 2 -fixing strains found in Western Australian soils. The unheralded existence of tripartite ICEs raises the possibility that multipartite elements reside in other organisms, but have been overlooked because of their unusual biology. These discoveries reveal mechanisms by which integrases dramatically manipulate bacterial genomes to allow cotransfer of disparate chromosomal regions.integrative and conjugative elements | integrase | recombination | conjugation | symbiosis H orizontal gene transfer plays an instrumental role in prokaryotic evolution because it facilitates the rapid acquisition of complex phenotypic traits required for pathogenicity, symbiosis, metabolism, fitness, and antimicrobial resistance (1-8). Integrative and conjugative elements (ICEs) are an abundant class of conjugative elements in bacteria, but they are also the most recently recognized and least characterized (8,9). An ICE integrates site-specifically within the chromosome of its host and is flanked by a direct repeat sequence that demarcates the insertion site. Before transfer, site-specific recombination between the flanking sequences results in excision and circularization of the ICE and restoration of the host chromosome. A single-stranded DNA copy of the circularized ICE is then formed via rollingcircle replication and transferred to recipient cells via an ICEencoded conjugative type IV secretion system. Following delivery of the ICE to the recipient cell, the second DNA strand of the ICE is synthesized and the circularized ICE integrates sitespecifically into the recipient genome (10).Symbiosis islands are the largest documented ICEs and their transfer converts nonsymb...

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