Submitted by Jean Medeiros (jeanletras@uepb.edu.br) on 2016-08-29T12:34:04Z No. of bitstreams: 1 PDF - Dalila Ribeiro Rodrigues.pdf: 1923244 bytes, checksum: f45088c1a8a811273f1ac7c37c1e3aec (MD5) / Approved for entry into archive by Secta BC (secta.csu.bc@uepb.edu.br) on 2016-08-29T17:02:09Z (GMT) No. of bitstreams: 1 PDF - Dalila Ribeiro Rodrigues.pdf: 1923244 bytes, checksum: f45088c1a8a811273f1ac7c37c1e3aec (MD5) / Made available in DSpace on 2016-08-29T17:02:09Z (GMT). No. of bitstreams: 1 PDF - Dalila Ribeiro Rodrigues.pdf: 1923244 bytes, checksum: f45088c1a8a811273f1ac7c37c1e3aec (MD5) Previous issue date: 2016-04-01 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The use of legumes inoculated with competitive and efficient nitrogen-fixing bacteria is a strategy to enhance the practices in the regeneration of degraded areas. In addition to encouraging the establishment of plants, the use of efficient strains reduces the cost of deployment and maintenance of agricultural systems, eliminating the use of nitrogenous fertilizers. But for the success of these practices is necessary to know the diversity of bacteria in the legumes nodules and evaluate their proper efficiency. The objective of this study was to evaluate the diversity and efficiency of bacterial isolates from three native species nodules Caatinga, grown in the State of Pernambuco soils deposited in the Agricultural Interest Collection of Microorganisms of Embrapa Semi -Arid. After certification purity of the stock of bacteria, all 308 isolates passed through a selection of amplicons nodC and nifH genes using a duplex-PCR approach. Among the bacteria evaluated, positive amplification were obtained for 18 isolated from mulungu, 40 to jurema-preta and 44 to angico. All these isolates were evaluated for NaCl tolerance and high temperatures "in vitro", ability to metabolize different carbon sources and enzymatic activity (only bacteria from mulungu), their “in vitro” plant growth promotion mechanisms by means of the production of indole acetic aci d (IAA) and calcium phosphate solubilization and finally tested in the greenhouse with the original 2 host plants, to determine the symbiotic efficiency of the isolates. Isolates of mulungu showed variability for enzyme activity and metabolism of carbon sources. Five isolated mulungu and jurema-preta and 6 isolates from angico were able to produce IAA in L-tryptophan supplemented medium, reaching rates above those achieved by the reference strain. As the potential of calcium phosphate solubilization for mulungu isolates, only one bacteria was able to achieve high solubilization equal to the reference strain, seven and two isolates from jurema-preta and angico, solubilized calcium phosphate above the reference strains value. For each species there bacteria were able to show tolerance to high temperatures and salinity. Isolates of angico and jurema were submitted to ARDRA using three endonucleases: MspI, HinfI and HhaI. The genetic diversity evaluation indicated that to angico a large cluster with around 40% of similarity was formed. To jurema-preta a major cluster with 65% of similarity among the isolates were achieved. In addition to high diversity of bacteria, patterns regarding its geographical occurrence were also observed. Bacteria from mulungu had the sequences of the 16S rRNA gene partially determinated. Among the 13 bacteria with good sequences, six belong to Rhizobium, six Bradyrhizobium and one belongs to Burkholderia. The Efficiency of tests, 22 isolates of mimosa and 10 mulungu were able to renodulate their original host strains and A27 from Angico and M14 and M31 can be highlighted to mulungu. / A utilização de leguminosas inoculadas com bactérias fixadoras de nitrogênio nativas eficientes e competitivas representa uma estratégia para potencializar as práticas na regeneração de áreas degradadas. Além de favorecer o estabelecimento das plantas, a utilização de estirpes eficientes reduz os custos da implantação e manutenção dos sistemas agrícolas, dispensando a utilização de fertilizantes químicos nitrogenados. Mas, para o sucesso destas práticas é preciso conhecer a diversidade das bactérias presentes nos nódulos de tais espécies de leguminosas e sua devida eficiência. O objetivo deste trabalho foi avaliar a diversidade e eficiência de bactérias oriundas de solos do Estado de Pernambuco e isoladas de nódulos de três espécies leguminosas arbóreas nativas da Caatinga, depositadas na Coleção de Microorganismos de Interesse Agrícola da Embrapa Semiárido. Após certificação da pureza do estoque de bactérias, os isolados passaram por uma seleção através da amplificação de fragmentos dos genes nodC e nifH utilizando a abordagem de duplex PCR. Apresentaram amplificação positiva 18 isolados de mulungu, 40 de jurema-preta e 44 de angico, esses isolados foram avaliados quanto a tolerância de NaCl e altas temperaturas “in vitro”, habilidade de metabolizar diferentes fontes de carbono e atividade enzimática (apenas isolados de mulungu), sua capacidade de promoção do crescimento vegetal, através da produção do Ácido indol-acético (AIA) e solubilização de fosfato de cálcio e, por fim, foram testados em casa de vegetação com as plantas hospedeiras originais, para determinação da eficiência simbiótica. Os isolados do mulungu mostraram variabilidade para atividade enzimática e para metabolização das fontes de carbono. Cinco isolados de mulungu e jurema-preta e 6 isolados de angico foram capazes de produzir AIA, na presença do L-triptofano, acima da estirpe de referência. Quanto ao potencial de solubilização de fosfato de cálcio apenas um isolado de mulungu apresentou valor igual a estirpe de referência, sete isolados de jurema-preta e dois de angico, solubilizaram fosfato de cálcio acima do valor da referência. Para cada espécie existem representantes potenciais na tolerância de altas temperaturas e altos teores salinos. Os isolados de angico e jurema-preta com características promíscuas foram submetidos ao ARDRA, utilizando três enzimas de restrição MspI, HinfI e HhaI, gerando para o angico um grande grupo com aproximadamente 40% de similaridade e para juremapreta um principal grupo com 65% de similaridade entre os isolados, constatando então a presença da diversidade dentre os isolados. Nos testes de autenticidade, 22 isolados de angico e 10 de mulungu foram capazes de renodular a estirpe hospedeira e o isolado A27, é eficiente na fixação biológica de nitrogênio, quando comparado ao controle nitrogenado. As bactérias do mulungu que se destacaram tiveram o gene 16S do rRNA parcialmente sequenciado para determinar o posicionamento taxonômico.

Nous nous sommes intéressés à la compréhension des mécanismes régissant la symbiose Rhizobium-Acacia dans les conditions de stress salin. Les lipopolysaccharides jouent un rôle important dans les étapes de cette symbiose. Le but était de mettre en évidence les modifications pariétales de la bactérie en réponse au stress salin par l'étude de la structure des lipopolysaccharides des souches isolées du désert marocain tolérant NaCl 7%. Ainsi, une nouvelle méthode d'hydrolyse des lipopolysaccharides sensible, non destructive et compatible avec la spectrométrie de masse a été développée. En présence de stress salin, nous avons montré que la membrane externe devenait plus hydrophobe en augmentant l'acylation de la région lipidique ainsi qu'en réduisant la présence des molécules de LPSs à longues chaînes de sucres.Des essais d'évaluation de l'efficience et de l'infectivité des Rhizobia étudiés ont été mis en œuvre pour déterminer l'impact de ces modifications des LPSs sur la symbiose sous stress salin.

Most members of the Rhizobiaceae possess single copies of the poly-3-hydroxybutyrate biosynthesis genes, phbA, phbB and phbC. Analysis of the genome sequence of Bradyrhizobium japonicum reveals the presence of five homologues of the PHB synthase gene phbC as well as two homologues of the biosynthesis operon, phbAB. The presence of multiple, seemingly redundant homologues may suggest a functional importance. Each B. japonicum phbC gene was cloned and used to complement the pleiotropic phenotype of a Sinorhizobium meliloti phbC mutant; this mutant is unable to synthesize PHB, grow on certain PHB cycle intermediates and forms non-mucoid colonies on yeast mannitol medium. Two of the five putative B. japonicum phbC genes were found to complement the S. meliloti phbC mutant phenotype on D-3-hydroxybutyrate although none of them could fully complement the phenotype on acetoacetate. Both complementing genes were also able to restore PHB accumulation and formation of mucoid colonies on yeast mannitol agar to phbC mutants. In-frame deletions were constructed in three of the five phbC open reading frames in B. japonicum, as well as in both phbAB operons, by allelic replacement. One of the phbC mutants was unable to synthesize PHB under free-living conditions; one of the two phbAB operons was shown to be necessary and sufficient for PHB production under free-living conditions. These mutants also demonstrated an exopolysaccharide phenotype that was comparable to S meliloti PHB synthesis mutants. These strains were non-mucoid when grown under PHB-inducing conditions and, in contrast to wild-type B. japonicum, formed a compact pellet upon centrifugation. Interestingly, none of the mutants exhibited carbon-utilization phenotypes similar to those exhibited by S. meliloti PHB mutants. Wild-type B. japonicum accumulates PHB during symbiosis, and plants inoculated with the phbC mutants demonstrate a reproducible reduction in shoot dry mass. Analysis of bacteroid PHB accumulation in the mutant strains suggests that the phbAB operons of B. japonicum are differently regulated relative to growth under free-living conditions; mutants of the second phbAB operon demonstrated a significant reduction in PHB accumulation during symbiosis. These data suggest that the first phbAB operon is required for PHB synthesis only under free-living conditions, but is able to partially substitute for the second operon during symbiosis. Deletion of both phbAB operons completely abolished PHB synthesis in bacteroids. Analysis of the upstream regions of these genes suggest the existence of putative RpoN binding sites, perhaps indicating a potential mode of regulation and highlighting the metabolic complexity that is characteristic of the Rhizobiaceae. PHB metabolism in S. meliloti has been studied in considerable detail with two notable exceptions. No reports of the construction of either a β-ketothiolase (phbA) or a PHB depolymerase (phaZ ) mutant have ever been documented. The phaZ gene, encoding the first enzyme of the catabolic half of the PHB cycle in S. meliloti, was identified and a phaZ mutant strain was generated by insertion mutagenesis. The phaZ mutant demonstrates a Fix+ symbiotic phenotype and, unlike other PHB cycle mutants, does not demonstrate reduced rhizosphere competitiveness. Bacteroids of this strain were shown to accumulate PHB, demonstrating for the first time that S. meliloti is able to synthesize and accumulate PHB during symbiosis. Interestingly, there is no significant difference in shoot dry mass of plants inoculated with the phaZ mutant, suggesting that PHB accumulation does not occur at the expense of nitrogen fixation. The phaZ mutant strain was also used to demonstrate roles for PhaZ in the control of PHB accumulation and exopolysaccharide production. When grown on high-carbon media, this mutant demonstrates a mucoid phenotype characteristic of exopolysaccharide production. Subsequent analyses of a phoA::exoF fusion confirmed elevated transcription levels in the phaZ mutant background. In contrast, mutants of the PHB biosynthesis gene, phbC, have a characteristically dry phenotype and demonstrate reduced exoF transcriptional activity. The phaZ mutant also demonstrates a significant increase in PHB accumulation relative to the wild-type strain. Previous work on phasin mutants in S. meliloti demonstrated that they lack the ability to synthesize PHB. Transduction of the phaZ lesion into the phasin mutant background was used to construct a phaZ-phasin mutant strain. Analysis of the PHB biosynthesis capacity of this strain showed that the lack of PHB synthesis exhibited by S. meliloti phasin mutants is due to loss of PHB biosynthesis activity and not due to an inherent instability in the PHB granules themselves. A recent study suggested that some bacteria may possess an alternate pathway for acetate assimilation that would bypass the need for the glyoxylate cycle in organisms that do not possess the enzyme, isocitrate lyase. In these organisms, acetate is assimilated through the ethylmalonyl-CoA pathway, which has significant overlap with the anabolic half of the PHB cycle, including reliance on the PHB intermediate 3-hydroxybutyryl-CoA. The observation that phbB and phbC mutants of S. meliloti are unable to grow well on acetoacetate -- coupled with previously unexplained data that show a class of mutants (designated bhbA-D) are able to grow on acetate, but not on hydroxybutyrate or acetoacetate -- made it tempting to speculate that an ethylmalonyl-CoA-like pathway might be present in S. meliloti, and that this pathway might overlap with the PHB cycle at the point of 3-hydroxybutyryl-CoA. An in-frame mutation of phbA was constructed by cross-over PCR and allelic replacement. This mutant exhibited a complete abolition of growth on acetoacetate, suggesting that PhbA represents the only exit point for carbon from the PHB cycle and that an alternative ethylmalonyl-CoA-like pathway is not present in this organism. During symbiosis, rhizobial cells are dependent on the provision of carbon from the host plant in order to fuel cellular metabolism. This carbon is transported into the bacteroids via the dicarboxylate transport protein, DctA. Most rhizobia possess single copies of the transporter gene dctA and its corresponding two-component regulatory system dctBD. The completed genome sequence of B. japonicum suggests that it possesses seven copies of dctA. Complementation of Sinorhizobium meliloti dct mutants using the cosmid bank of B. japonicum USDA110 led to the identification a dctA locus and a dctBD operon. Interestingly, the B. japonicum dctABD system carried on the complementing cosmid was not able to complement the symbiotic deficiency of S. meliloti strains carrying individual mutations in either dctA, dctB, or dctD suggesting that the B. japonicum dctBD is unable to recognize either DctB/DctD or the DctB/DctD-independent regulatory elements in S. meliloti. All seven B. japonicum dctA ORFs were cloned and an analysis of their capacity to complement the free-living phenotype of a S. meliloti dctA mutant demonstrated that they all possess some capacity for dicarboxylate transport. Mutants of all seven B. japonicum dctA ORFs were constructed and an analysis of their free-living phenotypes suggested that significant functional redundancy exists in B. japonicum DctA function. Given the large number of potential dctA genes in the genome, coupled with an apparent lack of dctBD regulators, it is tempting to speculate that different DctA isoforms may be used during free-living and symbiotic growth and may be subject to different regulatory mechanisms than those of better-studied systems. A comprehensive analysis of desiccation tolerance and ion sensitivity in S. meliloti was conducted. The results of these analyses suggest that genetic elements on both pSymA and pSymB may play a significant role in enhancing cell survival under conditions of osmotic stress. The S. meliloti expR+ strains SmUW3 and SmUW6 were both shown to exhibit considerably higher desiccation tolerance than Rm1021, suggesting a role for enhanced exopolysaccharide production in facilitating survival under adverse conditions. Furthermore, scanning electron microscopy of inoculated seeds suggests that S. meliloti cells initiate biofilm formation upon application to the surface of seeds. This finding has implications for the analysis of OSS and the development of desiccation assays and may explain some of the variability that is characteristic of desiccation studies.

Exopolysaccharides are essential for the establishment of the symbiosis between Sinorhizobium meliloti and Medicago sativa (alfalfa). The ExoS/ChvI two-component regulatory system is known as a regulator of succinoglycan production but the genes that are directly regulated by ChvI have not been determined. Difficulty isolating exoS and chvI null mutants has prompted the suggestion that these genes are essential for S. meliloti viability. We have successfully isolated exoS and chvI null mutants using a merodiploid facilitated strategy. We present evidence that the S. meliloti ExoS/ChvI two-component regulatory system is essential for symbiosis with alfalfa. Phenotypic analyses of exoS and chvI null mutant strains demonstrate that ExoS/ChvI controls both succinoglycan and galactoglucan production and is required for growth on over 21 different carbon sources. These new findings suggest that the ExoS/ChvI regulatory targets might not be the exo genes that are specific for succinoglycan biosynthesis but rather genes that have common influence on both succinoglycan and galactoglucan production. To obtain further insight into the nature of the ChvI regulon, we obtained a purified His•Tag-ChvI and used it to perform modified electrophoretic mobility shift assays. These assays were done using genomic DNA and were followed by cloning of DNA fragments having the highest affinity for ChvI. Sequencing of these fragments revealed that ChvI has a diverse regulon, it affects transcription of genes encoding enzymes that are involved in different pathways. Transcriptional gene fusion assays confirmed that ChvI is important for the activation of the transcription of the msbA2 operon, as well as repression of the transcription of the rhizobactin 1021 operon and genes SMc00262-61. ChvI-regulation of genes that are part of the connected thiamine and histidine biosynthesis pathways suggest that ChvI could act in a concerted manner to avoid limitation of important intermediates in these pathways. This study presents for the first time genes directly regulated by ChvI and this includes none of the exo genes. This work opens new avenues in the understanding of the global regulatory role of the symbiotically important ExoS/ChvI two-component regulatory system.

Most members of the Rhizobiaceae possess single copies of the poly-3-hydroxybutyrate biosynthesis genes, phbA, phbB and phbC. Analysis of the genome sequence of Bradyrhizobium japonicum reveals the presence of five homologues of the PHB synthase gene phbC as well as two homologues of the biosynthesis operon, phbAB. The presence of multiple, seemingly redundant homologues may suggest a functional importance. Each B. japonicum phbC gene was cloned and used to complement the pleiotropic phenotype of a Sinorhizobium meliloti phbC mutant; this mutant is unable to synthesize PHB, grow on certain PHB cycle intermediates and forms non-mucoid colonies on yeast mannitol medium. Two of the five putative B. japonicum phbC genes were found to complement the S. meliloti phbC mutant phenotype on D-3-hydroxybutyrate although none of them could fully complement the phenotype on acetoacetate. Both complementing genes were also able to restore PHB accumulation and formation of mucoid colonies on yeast mannitol agar to phbC mutants. In-frame deletions were constructed in three of the five phbC open reading frames in B. japonicum, as well as in both phbAB operons, by allelic replacement. One of the phbC mutants was unable to synthesize PHB under free-living conditions; one of the two phbAB operons was shown to be necessary and sufficient for PHB production under free-living conditions. These mutants also demonstrated an exopolysaccharide phenotype that was comparable to S meliloti PHB synthesis mutants. These strains were non-mucoid when grown under PHB-inducing conditions and, in contrast to wild-type B. japonicum, formed a compact pellet upon centrifugation. Interestingly, none of the mutants exhibited carbon-utilization phenotypes similar to those exhibited by S. meliloti PHB mutants. Wild-type B. japonicum accumulates PHB during symbiosis, and plants inoculated with the phbC mutants demonstrate a reproducible reduction in shoot dry mass. Analysis of bacteroid PHB accumulation in the mutant strains suggests that the phbAB operons of B. japonicum are differently regulated relative to growth under free-living conditions; mutants of the second phbAB operon demonstrated a significant reduction in PHB accumulation during symbiosis. These data suggest that the first phbAB operon is required for PHB synthesis only under free-living conditions, but is able to partially substitute for the second operon during symbiosis. Deletion of both phbAB operons completely abolished PHB synthesis in bacteroids. Analysis of the upstream regions of these genes suggest the existence of putative RpoN binding sites, perhaps indicating a potential mode of regulation and highlighting the metabolic complexity that is characteristic of the Rhizobiaceae. PHB metabolism in S. meliloti has been studied in considerable detail with two notable exceptions. No reports of the construction of either a β-ketothiolase (phbA) or a PHB depolymerase (phaZ ) mutant have ever been documented. The phaZ gene, encoding the first enzyme of the catabolic half of the PHB cycle in S. meliloti, was identified and a phaZ mutant strain was generated by insertion mutagenesis. The phaZ mutant demonstrates a Fix+ symbiotic phenotype and, unlike other PHB cycle mutants, does not demonstrate reduced rhizosphere competitiveness. Bacteroids of this strain were shown to accumulate PHB, demonstrating for the first time that S. meliloti is able to synthesize and accumulate PHB during symbiosis. Interestingly, there is no significant difference in shoot dry mass of plants inoculated with the phaZ mutant, suggesting that PHB accumulation does not occur at the expense of nitrogen fixation. The phaZ mutant strain was also used to demonstrate roles for PhaZ in the control of PHB accumulation and exopolysaccharide production. When grown on high-carbon media, this mutant demonstrates a mucoid phenotype characteristic of exopolysaccharide production. Subsequent analyses of a phoA::exoF fusion confirmed elevated transcription levels in the phaZ mutant background. In contrast, mutants of the PHB biosynthesis gene, phbC, have a characteristically dry phenotype and demonstrate reduced exoF transcriptional activity. The phaZ mutant also demonstrates a significant increase in PHB accumulation relative to the wild-type strain. Previous work on phasin mutants in S. meliloti demonstrated that they lack the ability to synthesize PHB. Transduction of the phaZ lesion into the phasin mutant background was used to construct a phaZ-phasin mutant strain. Analysis of the PHB biosynthesis capacity of this strain showed that the lack of PHB synthesis exhibited by S. meliloti phasin mutants is due to loss of PHB biosynthesis activity and not due to an inherent instability in the PHB granules themselves. A recent study suggested that some bacteria may possess an alternate pathway for acetate assimilation that would bypass the need for the glyoxylate cycle in organisms that do not possess the enzyme, isocitrate lyase. In these organisms, acetate is assimilated through the ethylmalonyl-CoA pathway, which has significant overlap with the anabolic half of the PHB cycle, including reliance on the PHB intermediate 3-hydroxybutyryl-CoA. The observation that phbB and phbC mutants of S. meliloti are unable to grow well on acetoacetate -- coupled with previously unexplained data that show a class of mutants (designated bhbA-D) are able to grow on acetate, but not on hydroxybutyrate or acetoacetate -- made it tempting to speculate that an ethylmalonyl-CoA-like pathway might be present in S. meliloti, and that this pathway might overlap with the PHB cycle at the point of 3-hydroxybutyryl-CoA. An in-frame mutation of phbA was constructed by cross-over PCR and allelic replacement. This mutant exhibited a complete abolition of growth on acetoacetate, suggesting that PhbA represents the only exit point for carbon from the PHB cycle and that an alternative ethylmalonyl-CoA-like pathway is not present in this organism. During symbiosis, rhizobial cells are dependent on the provision of carbon from the host plant in order to fuel cellular metabolism. This carbon is transported into the bacteroids via the dicarboxylate transport protein, DctA. Most rhizobia possess single copies of the transporter gene dctA and its corresponding two-component regulatory system dctBD. The completed genome sequence of B. japonicum suggests that it possesses seven copies of dctA. Complementation of Sinorhizobium meliloti dct mutants using the cosmid bank of B. japonicum USDA110 led to the identification a dctA locus and a dctBD operon. Interestingly, the B. japonicum dctABD system carried on the complementing cosmid was not able to complement the symbiotic deficiency of S. meliloti strains carrying individual mutations in either dctA, dctB, or dctD suggesting that the B. japonicum dctBD is unable to recognize either DctB/DctD or the DctB/DctD-independent regulatory elements in S. meliloti. All seven B. japonicum dctA ORFs were cloned and an analysis of their capacity to complement the free-living phenotype of a S. meliloti dctA mutant demonstrated that they all possess some capacity for dicarboxylate transport. Mutants of all seven B. japonicum dctA ORFs were constructed and an analysis of their free-living phenotypes suggested that significant functional redundancy exists in B. japonicum DctA function. Given the large number of potential dctA genes in the genome, coupled with an apparent lack of dctBD regulators, it is tempting to speculate that different DctA isoforms may be used during free-living and symbiotic growth and may be subject to different regulatory mechanisms than those of better-studied systems. A comprehensive analysis of desiccation tolerance and ion sensitivity in S. meliloti was conducted. The results of these analyses suggest that genetic elements on both pSymA and pSymB may play a significant role in enhancing cell survival under conditions of osmotic stress. The S. meliloti expR+ strains SmUW3 and SmUW6 were both shown to exhibit considerably higher desiccation tolerance than Rm1021, suggesting a role for enhanced exopolysaccharide production in facilitating survival under adverse conditions. Furthermore, scanning electron microscopy of inoculated seeds suggests that S. meliloti cells initiate biofilm formation upon application to the surface of seeds. This finding has implications for the analysis of OSS and the development of desiccation assays and may explain some of the variability that is characteristic of desiccation studies.

Exopolysaccharides are essential for the establishment of the symbiosis between Sinorhizobium meliloti and Medicago sativa (alfalfa). The ExoS/ChvI two-component regulatory system is known as a regulator of succinoglycan production but the genes that are directly regulated by ChvI have not been determined. Difficulty isolating exoS and chvI null mutants has prompted the suggestion that these genes are essential for S. meliloti viability. We have successfully isolated exoS and chvI null mutants using a merodiploid facilitated strategy. We present evidence that the S. meliloti ExoS/ChvI two-component regulatory system is essential for symbiosis with alfalfa. Phenotypic analyses of exoS and chvI null mutant strains demonstrate that ExoS/ChvI controls both succinoglycan and galactoglucan production and is required for growth on over 21 different carbon sources. These new findings suggest that the ExoS/ChvI regulatory targets might not be the exo genes that are specific for succinoglycan biosynthesis but rather genes that have common influence on both succinoglycan and galactoglucan production. To obtain further insight into the nature of the ChvI regulon, we obtained a purified His•Tag-ChvI and used it to perform modified electrophoretic mobility shift assays. These assays were done using genomic DNA and were followed by cloning of DNA fragments having the highest affinity for ChvI. Sequencing of these fragments revealed that ChvI has a diverse regulon, it affects transcription of genes encoding enzymes that are involved in different pathways. Transcriptional gene fusion assays confirmed that ChvI is important for the activation of the transcription of the msbA2 operon, as well as repression of the transcription of the rhizobactin 1021 operon and genes SMc00262-61. ChvI-regulation of genes that are part of the connected thiamine and histidine biosynthesis pathways suggest that ChvI could act in a concerted manner to avoid limitation of important intermediates in these pathways. This study presents for the first time genes directly regulated by ChvI and this includes none of the exo genes. This work opens new avenues in the understanding of the global regulatory role of the symbiotically important ExoS/ChvI two-component regulatory system.

This research investigated the rhizobia that are associated with New Zealand legume plants. Rhizobia are a diverse group of bacteria that live in symbiosis with legumes in root nodules. Rhizobia fix Nitrogen from the atmosphere and provide this nutrient to the plant. The objectives of this research were to: 1) Determine the identity of the rhizobial species nodulating the native legumes of New Zealand: Sophora (kowhai), Carmichaelia (NZ broom), and Clianthus (kakabeak); and the identity and origin of rhizobial species nodulating invasive exotic legumes in New Zealand: Ulex (gorse), Cytisus (broom), and Acacia (wattles). 2) Determine the specificity and nitrogen fixing capacity of both groups of rhizobia. 3) Investigate the possible exchange of transmissible symbiotic genetic elements. A polyphasic strategy was used to determine the identity of bacterial isolates. The 16S rRNA, atpD, recA, and glnII genes were PCR amplified and sequenced, then analysed by maximum likelihood and Bayesian methods. Phenotypic characters were also assessed by use of the Biolog and FAME techniques. Nodulation and fixation ability was assessed by inoculating legume seedlings with rhizobial strains, then determining nitrogenase activity after ten weeks by gas chromatography, and examining roots for nodules. A gene involved in symbiosis, nodA, was sequenced from rhizobial strains to determine if transmission between strains had occurred. The results of the experiments showed that the native legumes were predominately nodulated by diverse Mesorhizobium spp. that contain three different nodA genotypes (two of which are novel) that have transferred between rhizobial strains. The Mesorhizobium spp. showed little nodulation specificity and could nodulate an exotic legume Astragalus (milk vetch), but not the invasive weed legumes. Rhizobium leguminosarum was also found to nodulate native legumes, albeit ineffectively. The exotic invasive woody legumes of this study were nodulated by diverse Bradyrhizobium spp. that had nodA genotypes typical of Australian and European species. The origins of these bacteria can not be categorically determined. However the evidence is presented to suggest that nodulating Mesorhizobium spp. arrived with the ancestors of the native legumes, while Bradyrhizobium spp. nodulating Ulex and Cytisus arrived recently from Europe. Bradyrhizobium spp. nodulating Acacia may be recently introduced, possibly from Australia, although further work is required to confirm these hypotheses. / This study was supported by a grant from the Marsden Fund of the Royal Society of New Zealand, under contract 97-LAN-LFS-002, and a grant from the Non-Specific Output Fund of Landcare Research.

This research investigated the rhizobia that are associated with New Zealand legume plants. Rhizobia are a diverse group of bacteria that live in symbiosis with legumes in root nodules. Rhizobia fix Nitrogen from the atmosphere and provide this nutrient to the plant. The objectives of this research were to: 1) Determine the identity of the rhizobial species nodulating the native legumes of New Zealand: Sophora (kowhai), Carmichaelia (NZ broom), and Clianthus (kakabeak); and the identity and origin of rhizobial species nodulating invasive exotic legumes in New Zealand: Ulex (gorse), Cytisus (broom), and Acacia (wattles). 2) Determine the specificity and nitrogen fixing capacity of both groups of rhizobia. 3) Investigate the possible exchange of transmissible symbiotic genetic elements. A polyphasic strategy was used to determine the identity of bacterial isolates. The 16S rRNA, atpD, recA, and glnII genes were PCR amplified and sequenced, then analysed by maximum likelihood and Bayesian methods. Phenotypic characters were also assessed by use of the Biolog and FAME techniques. Nodulation and fixation ability was assessed by inoculating legume seedlings with rhizobial strains, then determining nitrogenase activity after ten weeks by gas chromatography, and examining roots for nodules. A gene involved in symbiosis, nodA, was sequenced from rhizobial strains to determine if transmission between strains had occurred. The results of the experiments showed that the native legumes were predominately nodulated by diverse Mesorhizobium spp. that contain three different nodA genotypes (two of which are novel) that have transferred between rhizobial strains. The Mesorhizobium spp. showed little nodulation specificity and could nodulate an exotic legume Astragalus (milk vetch), but not the invasive weed legumes. Rhizobium leguminosarum was also found to nodulate native legumes, albeit ineffectively. The exotic invasive woody legumes of this study were nodulated by diverse Bradyrhizobium spp. that had nodA genotypes typical of Australian and European species. The origins of these bacteria can not be categorically determined. However the evidence is presented to suggest that nodulating Mesorhizobium spp. arrived with the ancestors of the native legumes, while Bradyrhizobium spp. nodulating Ulex and Cytisus arrived recently from Europe. Bradyrhizobium spp. nodulating Acacia may be recently introduced, possibly from Australia, although further work is required to confirm these hypotheses. / This study was supported by a grant from the Marsden Fund of the Royal Society of New Zealand, under contract 97-LAN-LFS-002, and a grant from the Non-Specific Output Fund of Landcare Research.

This research investigated the rhizobia that are associated with New Zealand legume plants. Rhizobia are a diverse group of bacteria that live in symbiosis with legumes in root nodules. Rhizobia fix Nitrogen from the atmosphere and provide this nutrient to the plant. The objectives of this research were to: 1) Determine the identity of the rhizobial species nodulating the native legumes of New Zealand: Sophora (kowhai), Carmichaelia (NZ broom), and Clianthus (kakabeak); and the identity and origin of rhizobial species nodulating invasive exotic legumes in New Zealand: Ulex (gorse), Cytisus (broom), and Acacia (wattles). 2) Determine the specificity and nitrogen fixing capacity of both groups of rhizobia. 3) Investigate the possible exchange of transmissible symbiotic genetic elements. A polyphasic strategy was used to determine the identity of bacterial isolates. The 16S rRNA, atpD, recA, and glnII genes were PCR amplified and sequenced, then analysed by maximum likelihood and Bayesian methods. Phenotypic characters were also assessed by use of the Biolog and FAME techniques. Nodulation and fixation ability was assessed by inoculating legume seedlings with rhizobial strains, then determining nitrogenase activity after ten weeks by gas chromatography, and examining roots for nodules. A gene involved in symbiosis, nodA, was sequenced from rhizobial strains to determine if transmission between strains had occurred. The results of the experiments showed that the native legumes were predominately nodulated by diverse Mesorhizobium spp. that contain three different nodA genotypes (two of which are novel) that have transferred between rhizobial strains. The Mesorhizobium spp. showed little nodulation specificity and could nodulate an exotic legume Astragalus (milk vetch), but not the invasive weed legumes. Rhizobium leguminosarum was also found to nodulate native legumes, albeit ineffectively. The exotic invasive woody legumes of this study were nodulated by diverse Bradyrhizobium spp. that had nodA genotypes typical of Australian and European species. The origins of these bacteria can not be categorically determined. However the evidence is presented to suggest that nodulating Mesorhizobium spp. arrived with the ancestors of the native legumes, while Bradyrhizobium spp. nodulating Ulex and Cytisus arrived recently from Europe. Bradyrhizobium spp. nodulating Acacia may be recently introduced, possibly from Australia, although further work is required to confirm these hypotheses. / This study was supported by a grant from the Marsden Fund of the Royal Society of New Zealand, under contract 97-LAN-LFS-002, and a grant from the Non-Specific Output Fund of Landcare Research.

This research investigated the rhizobia that are associated with New Zealand legume plants. Rhizobia are a diverse group of bacteria that live in symbiosis with legumes in root nodules. Rhizobia fix Nitrogen from the atmosphere and provide this nutrient to the plant. The objectives of this research were to: 1) Determine the identity of the rhizobial species nodulating the native legumes of New Zealand: Sophora (kowhai), Carmichaelia (NZ broom), and Clianthus (kakabeak); and the identity and origin of rhizobial species nodulating invasive exotic legumes in New Zealand: Ulex (gorse), Cytisus (broom), and Acacia (wattles). 2) Determine the specificity and nitrogen fixing capacity of both groups of rhizobia. 3) Investigate the possible exchange of transmissible symbiotic genetic elements. A polyphasic strategy was used to determine the identity of bacterial isolates. The 16S rRNA, atpD, recA, and glnII genes were PCR amplified and sequenced, then analysed by maximum likelihood and Bayesian methods. Phenotypic characters were also assessed by use of the Biolog and FAME techniques. Nodulation and fixation ability was assessed by inoculating legume seedlings with rhizobial strains, then determining nitrogenase activity after ten weeks by gas chromatography, and examining roots for nodules. A gene involved in symbiosis, nodA, was sequenced from rhizobial strains to determine if transmission between strains had occurred. The results of the experiments showed that the native legumes were predominately nodulated by diverse Mesorhizobium spp. that contain three different nodA genotypes (two of which are novel) that have transferred between rhizobial strains. The Mesorhizobium spp. showed little nodulation specificity and could nodulate an exotic legume Astragalus (milk vetch), but not the invasive weed legumes. Rhizobium leguminosarum was also found to nodulate native legumes, albeit ineffectively. The exotic invasive woody legumes of this study were nodulated by diverse Bradyrhizobium spp. that had nodA genotypes typical of Australian and European species. The origins of these bacteria can not be categorically determined. However the evidence is presented to suggest that nodulating Mesorhizobium spp. arrived with the ancestors of the native legumes, while Bradyrhizobium spp. nodulating Ulex and Cytisus arrived recently from Europe. Bradyrhizobium spp. nodulating Acacia may be recently introduced, possibly from Australia, although further work is required to confirm these hypotheses. / This study was supported by a grant from the Marsden Fund of the Royal Society of New Zealand, under contract 97-LAN-LFS-002, and a grant from the Non-Specific Output Fund of Landcare Research.