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Mesorhizobia are soil bacteria that establish nitrogen-fixing symbioses with various legumes. Novel symbiotic mesorhizobia frequently evolve following horizontal transfer of symbiosis-gene-carrying integrative and conjugative elements (ICESyms) to indigenous mesorhizobia in soils. Evolved symbionts exhibit a wide range in symbiotic effectiveness, with some fixing nitrogen poorly or not at all. Little is known about the genetic diversity and symbiotic potential of indigenous soil mesorhizobia prior to ICESym acquisition. Here we sequenced genomes of 144 Mesorhizobium spp. strains cultured directly from cultivated and uncultivated Australian soils. Of these, 126 lacked symbiosis genes. The only isolated symbiotic strains were either exotic strains used previously as legume inoculants, or indigenous mesorhizobia that had acquired exotic ICESyms. No native symbiotic strains were identified. Indigenous nonsymbiotic strains formed 22 genospecies with phylogenomic diversity overlapping the diversity of internationally isolated symbiotic Mesorhizobium spp. The genomes of indigenous mesorhizobia exhibited no evidence of prior involvement in nitrogen-fixing symbiosis, yet their core genomes were similar to symbiotic strains and they generally lacked genes for synthesis of biotin, nicotinate and thiamine. Genomes of nonsymbiotic mesorhizobia harboured similar mobile elements to those of symbiotic mesorhizobia, including ICESym-like elements carrying aforementioned vitamin-synthesis genes but lacking symbiosis genes. Diverse indigenous isolates receiving ICESyms through horizontal gene transfer formed effective symbioses with Lotus and Biserrula legumes, indicating most nonsymbiotic mesorhizobia have an innate capacity for nitrogen-fixing symbiosis following ICESym acquisition. Non-fixing ICESym-harbouring strains were isolated spo-radically within species alongside effective symbionts, indicating chromosomal lineage does not predict symbiotic potential. Our observations sugges

Rhizobia induce nodule formation on legume roots and differentiate into bacteroids, which catabolize plant-derived dicarboxylates to reduce atmospheric N-2 into ammonia. Despite the agricultural importance of this symbiosis, the mechanisms that govern carbon and nitrogen allocation in bacteroids and promote ammonia secretion to the plant are largely unknown. Using a metabolic model derived from genome-scale datasets, we show that carbon polymer synthesis and alanine secretion by bacteroids facilitate redox balance in microaerobic nodules. Catabolism of dicarboxylates induces not only a higher oxygen demand but also a higher NADH/NAD(+) ratio than sugars. Modeling and C-13 metabolic flux analysis indicate that oxygen limitation restricts the decarboxylating arm of the tricarboxylic acid cycle, which limits ammonia assimilation into glutamate. By tightly controlling oxygen supply and providing dicarboxylates as the energy and electron source donors for N-2 fixation, legumes promote ammonia secretion by bacteroids. This is a defining feature of rhizobium-legume symbioses., QC 20220922

Rhizobia are soil bacteria capable of forming N2-fixing symbioses with legumes, with highly effective strains often selected in agriculture as inoculants to maximize symbiotic N2 fixation. When rhizobia in the genus Mesorhizobium have been introduced with exotic legumes into farming systems, horizontal transfer of symbiosis Integrative and Conjugative Elements (ICEs) from the inoculant strain to soil bacteria has resulted in the evolution of ineffective N2-fixing rhizobia that are competitive for nodulation with the target legume. In Australia, Cicer arietinum (chickpea) has been inoculated since the 1970's with Mesorhizobium ciceri sv. ciceri CC1192, a highly effective strain from Israel. Although the full genome sequence of this organism is available, little is known about the mobility of its symbiosis genes and the diversity of cultivated C. arietinum-nodulating organisms. Here, we show the CC1192 genome harbors a 419-kb symbiosis ICE (ICEMcSym1192) and a 648-kb repABC-type plasmid pMC1192 carrying putative fix genes. We sequenced the genomes of 11 C. arietinum nodule isolates from a field site exclusively inoculated with CC1192 and showed they were diverse unrelated Mesorhizobium carrying ICEMcSym1192, indicating they had acquired the ICE by environmental transfer. No exconjugants harboured pMc1192 and the plasmid was not essential for N2 fixation in CC1192. Laboratory conjugation experiments confirmed ICEMcSym1192 is mobile, integrating site-specifically within the 3' end of one of the four ser-tRNA genes in the R7ANS recipient genome. Strikingly, all ICEMcSym1192 exconjugants were as efficient at fixing N2 with C. arietinum as CC1192, demonstrating ICE transfer does not necessarily yield ineffective microsymbionts as previously observed.Importance Symbiotic N2 fixation is a key component of sustainable agriculture and in many parts of the world legumes are inoculated with highly efficient strains of rhizobia to maximise fixed N2 inputs into farming systems. Sy

Legumes play an integral role in increasing agricultural productivity, particularly in low input agricultural systems in Australia, due to their ability to form symbiotic interactions with a group of soil bacteria called rhizobia. However, in medium-to-low rainfall areas of southern Australia, there is a lack of suitable annual pasture legumes, which is limiting agricultural productivity and profitability in these farming systems. Scorpiurus muricatus is an annual legume from the Mediterranean which possesses high nutritive value and palatability for livestock, is high yielding, capable of self-seeding and is well-adapted to hot and dry summers. As such, S. muricatus is currently being evaluated as a new pasture legume for southern Australia. Crucial to the success of introducing this legume will be the availability of a highly effective rhizobial inoculant strain. This thesis therefore sought to characterise the phylogeny, free-living and symbiotic phenotype of a range of bacteria isolated from Scorpiurus spp. A total of 19 strains were investigated, with 16s rRNA sequencing demonstrating that 18 of these strains belonged to the genus Mesorhizobium, with the remaining strain (WSM1184) most closely related to Agrobacterium tumefaciens. Analysis of nifH and nodC symbiosis genes further showed that the characterised Mesorhizobium strains generally shared highly similar sequences for these loci, indicating a comparatively high degree of genetic similarity. In particular, WSM1343 (isolated from Scorpiurus sulcatus growing in Morocco) and WSM1386 (isolated from S. sulcatus in Manjimup, Western Australia) were shown to share highly similar symbiosis genes, but divergent 16S rRNA genes, suggesting the possibility that these strains may contain symbiosis genes on mobile Integrative and Conjugative Elements (ICEs). While the temperature tolerance and apparent optimum growth temperature of the test strains of 28°C was consistent with that commonly reported for Mesorhizobium s

Bacterial integrative & conjugative elements (ICEs) are chromosomally-integrated DNA islands that excise to form circular molecules capable of horizontal self-transmission via conjugation (cell-to-cell contact). Symbiosis ICEs, such as ICEMlSymR7A of Mesorhizobium loti, are a group of ICEs that carry genes enabling rhizobial bacteria to engage in N2-fixing symbioses with leguminous plants. Transfer of symbiosis ICEs can convert non-symbiotic rhizobia into legume symbionts in a single evolutionary step. In this thesis, a novel form of “tripartite” ICE (ICE3) is reported that exists as three entirely separated regions of DNA residing in the chromosomes of genetically diverse N2-fixing Mesorhizobium spp. These ICE3 regions did not excise independently, rather through multiple recombinations with the host chromosome they formed a single contiguous region of DNA prior to excision and conjugative transfer. Upon integration into a recipient chromosome, the ICE3 recombined the recipient chromosome to disassemble into the tripartite form. These recombination reactions were catalysed by three Integrase proteins IntG, IntM, and IntS, acting on three associated integrase attachment sites. The “excisive” recombination reactions (i.e. assembly and excision) were stimulated by three recombination directionality factors RdfG, RdfM, and RdfS. Expression of ICE3 transfer and conjugation genes were found to be induced by quorum-sensing. Quorum-sensing activated expression of rdfS, and in turn RdfS stimulated transcription of both rdfG and rdfM. Therefore, RdfS acts as a “master controller” of ICE3 assembly and excision. A model for ICE3 recombination and transfer is presented in this thesis. The conservation of gene content between symbiosis ICE and ICE3 indicated that these elements share a common evolutionary history. However, the persistence of ICE3 structure in diverse mesorhizobia is perplexing due to its seemingly unnecessary complexity. Bioinformatic comparisons of ICE and ICE3

The symbiosis between soil bacteria (rhizobia) and legumes is an integral component of sustainable agricultural practice, providing a source of bioavailable (or fixed) nitrogen to farming systems without supplementation with industrially synthesised fertiliser. Critical to the success of this association are rhizobial nodulation (nod) and nitrogen fixation (nif and fix) genes, that collectively allow for establishment and maintenance of nitrogen-fixing rhizobia-legume symbioses. In the Mesorhizobium genus symbiosis genes are chromosomally-encoded on Integrative and Conjugative Elements (ICEs). ICEs are a form of mobile genetic elements, capable of excising from the host chromosome through the action of the ICE-encoded integrase (IntS) and recombination and directionality factor (RdfS). The excised ICE can then transfer via conjugation into the recipient cell, where the ICE integrates into the host chromosome at specific attachment sites, usually adjacent to amino-acyl tRNA genes. Although recipients receive the complete symbiosis ICE from the donor strain and are subsequently able to nodulate a target legume, these exconjugants frequently fix nitrogen sub-optimally, indicating that a strong interaction between chromosomal and ICE-encoded genes exists. Understanding how different Mesorhizobium chromosomes and ICEs interact requires an array of ICE-devoid Mesorhizobium recipient strains. However currently, there is only such strain available, M. loti R7ANS, produced from the ICE-containing parent strain M. loti R7A. Furthermore, Mesorhizobium spp. may also harbour an accessory plasmid, which may have a role in nitrogen fixation. Recently, the genome of Mesorhizobium ciceri CC1192, the commercial inoculant for Cicer arietinum (chickpea) was reported. The genome of CC1192 consists of 6.29-Mb chromosome with a symbiosis ICE (ICEMcSym1192) of 419-kb, as well as a 648-kb accessory plasmid, pMc1192. Although the strain has been used as an inoculant for C. arietinum for more

Rhizobia are soil-dwelling bacteria capable of forming symbiotic associations with legumes, where they reduce atmospheric dinitrogen (N2) into ammonia (NH3), providing the host plant with a bioavailable nitrogen source. For rhizobia in the genus Mesorhizobium, genes essential to the establishment (nod) and maintenance of nitrogen fixing-legume symbioses (nif and fix) are chromosomally-encoded on mobile segments of DNA known as symbiosis integrative and conjugative elements (ICEs). Symbiosis ICEs are capable of excising from the host chromosome and transferring to a recipient ICE-free cell via bacterial conjugation. In the recipient cell, the invading ICE integrates into the recipient chromosome at conserved sites known as attachment sites. As well as encoding genes for excision, conjugation and integration, symbiosis ICEs often encode genes for vitamin biosynthesis, such as those for the synthesis of nicotinate, biotin and thiamine. Symbiosis ICEs can be monopartite or tripartite in nature and it appears that these two conformations of elements may be evolutionarily related, with the more complex tripartite ICE hypothesised to have evolved from two recombination reactions between three independent monopartite ancestral ICEs that co-existed in an ancestral bacterial cell. Testing this hypothesis for tripartite ICE evolution requires conjugation experiments between two strains that harbour different symbiosis ICEs, which has not previously been experimentally attempted. Symbiosis ICE transfer studies have been conducted between an ICE donor strain and an ICE-devoid vitamin auxotrophic recipient strain, with ICE transfer exconjugants selected on the basis of vitamin prototrophy. However, this method of selecting exconjugants is ineffective for conjugation experiments between two ICE-harbouring strains, as both usually harbour the operons for nicotinate, biotin and thiamine biosynthesis. Genetically-marking two structurally similar, yet phenotypically distinct monoparti

Phaseolus vulgaris (common bean) is an important food crop in Sub-Saharan Africa. Low soil nitrogen limits the productivity of P. vulgaris in Kenya and a greater exploitation of symbiotic nitrogen fixation, resulting from interactions with rhizobia, has the potential to improve yields. To enable the increased use of the symbiosis in Kenyan agriculture in the future, studies in this thesis examined the genetic diversity of rhizobia that nodulate P. vulgaris in the central and western parts of Kenya, their nitrogen-fixing capabilities, and their competitiveness against Rhizobium tropici CIAT 899, a leading commercial inoculant strain for P. vulgaris. Lastly, studies investigated the relative importance of the genotypes of resident soil rhizobia, soil rhizobial population densities, inocula densities, and levels of soil nitrogen, in determining nodule occupancy by R. tropici CIAT 899 inoculated onto P. vulgaris. Phylogenetic studies using 16S rRNA and recA genes indicated at least five species of Rhizobium viz., R. sophoriradicis, R. phaseoli, R. leucaenae, R. paranaense and R. etli nodulate P. vulgaris in Kenya. In addition to the five species, strains that likely belong to new species in the genus Rhizobium also widely nodulate P. vulgaris in Kenyan soils. In glasshouse studies, recovered strains were variably effective on Kenyan cultivars of P. vulgaris and 11 fixed as much nitrogen as R. tropici CIAT 899. From the 11, strains such as NAK 227, NAK 288, NAK 214 and NAK 157 were also highly competitive in liquid co-inoculation assays, carried out with the aid of gusA and celB marker genes, and are potential future inoculants for P. vulgaris in Kenya. The genotype of the rhizobia in the soil was found to be the primary determinant of the nodule occupancy achieved by the inoculant strain, a finding that conflicts previous reports that indicated nodule occupancy was mainly determined by soil rhizobial densities. The rhizobial genotypes varied in their rhizosphere compete

Biserrula pelecinus is a productive pasture legume with potential for replenishing soil fertility and providing quality livestock feed in Southern Australia. The experience with growing B. pelecinus in Australia suggests an opportunity to evaluate this legume in Ethiopia, due to its relevance to low-input farming systems such as those practiced in Ethiopia. However, the success of B. pelecinus is dependent upon using effective, competitive, and genetically stable inoculum strains of root nodule bacteria (mesorhizobia). Mesorhizobium strains isolated from the Mediterranean region were previously reported to be effective on B. pelecinus in Australian soils. Subsequently, it was discovered that these strains transferred genes required for symbiosis with B. pelecinus (contained on a “symbiosis island’ in the chromosome) to non-symbiotic soil bacteria. This transfer converted the recipient soil bacteria into symbionts that were less effective in N2-fixation than the original inoculant. This study investigated selection of effective, stable inoculum strains for use with B. pelecinus in Ethiopian soils. Genetically diverse and effective mesorhizobial strains of B. pelecinus were shown to be present in Ethiopian and Eritrean soils. These strains were shown to belong to the genus Mesorhizobium and carry highly mobile symbiosis islands, with a novel integration hotspot (ser-tRNA). In vitro, the transfer of the symbiosis island from these strains to a non-symbiotic recipient strain resulted in novel bacteria with a poorly effective phenotype, except for one highly effective strain. By deleting a relaxase gene, which is involved in the conjugative transfer of the symbiosis island, a more stable strain was created containing an immobile symbiosis island. The study highlights the presence of taxonomically and symbiotically distinct B. pelecinus-nodulating Mesorhizobium strains in East African soils. In these Mesorhizobium strains, the rate of symbiosis island transfer was as high

The reduction of atmospheric nitrogen (N2) to ammonia (N2 fixation) by bacteria (rhizobia) living symbiotically within legume root nodules contributes approximately 50% of the biosphere’s available nitrogen. This association provides the differentiated microsymbiont (the bacteroid) within the root nodule of the host legume a source of carbon in the form of malate. While studies suggest the oxidation of malate through the TCA cycle is essential for effective symbiotic N2 fixation in rhizobia, exactly how bacteroids balance their own biosynthetic requirements with the need to supply ATP and reductant to drive N2 fixation is currently unclear. However, this picture of bacteroid metabolism is limited to the detailed analysis of very few strains. Developing clearer explanations of how bacteroids power N2 fixation therefore requires analysis of a wider range of rhizobia, with particular focus on well-characterised strains with full genome sequences available that are also highly effective in N2 fixation on a legume host. Rhizobium tropici CIAT899 is a highly effective N2-fixing microsymbiont of the grain legume Phaseolus vulgaris (common bean). R. tropici CIAT899 is the commercial inoculant for P. vulgaris production in many parts of the world and the genome of this strain was recently fully sequenced. Despite its significant role in agriculture, very little is known about the metabolism of R. tropici CIAT899 in symbiotic and free-living conditions, making it an ideal strain for further investigations. The symbiotic and free-living metabolism of R. tropici CIAT899 was investigated using a combination of site-directed and random mutagenesis approaches. For the site directed approach, inactivation vectors pk19icdA and pJQ200SksucA were successfully created to target genes encoding the TCA cycle enzymes isocitrate dehydrogenase (icdA) and the E1 component of 2-oxogluarate dehydrogenase (sucA), respectively. However, isolation of icdA or sucA mutants was not achieved. In the