O'Callaghan, Kenneth J.; Stone, Philip J.; Hu, Xiaojia; Griffiths, D. Wynne; Davey, Michael R.; Cocking, Edward C.
Plants of Brassica napus were assessed quantitatively for their susceptibility to lateral root crack colonization by Azorhizobium caulinodans ORS571(pXLGD4) (a rhizobial strain carrying the lacZ reporter gene) and for the concentration of glucosinolates in their roots by high-pressure liquid chromatography (HPLC). High- and low-glucosinolate-seed (HGS and LGS) varieties exhibited a relatively low and high percentage of colonized lateral roots, respectively. HPLC showed that roots of HGS plants contained a higher concentration of glucosinolates than roots of LGS plants. One LGS variety showing fewer colonized lateral roots than other LGS varieties contained a higher concentration of glucosinolates than other LGS plants. Inoculated HGS plants treated with the flavonoid naringenin showed significantly more colonization than untreated HGS plants. This increase was not mediated by a naringenin-induced lowering of the glucosinolate content of HGS plant roots, nor did naringenin induce bacterial resistance to glucosinolates or increase the growth of bacteria. The erucic acid content of seed did not appear to influence colonization by azorhizobia. Frequently, leaf assays are used to study glucosinolates and plant defense; this study provides data on glucosinolates and bacterial colonization in roots and describes a bacterial reporter gene assay tailored easily to the study of ecologically important phytochemicals that influence bacterial colonization. These data also form a basis for future assessments of the benefits to oilseed rape plants of interaction with plant growth-promoting bacteria, especially diazotrophic bacteria potentially able to extend the benefits of nitrogen fixation to nonlegumes. PMID:10788398
Full Text Available Cyanuric acid hydrolase (CAH catalyzes the hydrolytic ring-opening of cyanuric acid (2,4,6-trihydroxy-1,3,5-triazine, an intermediate in s-triazine bacterial degradation and a by-product from disinfection with trichloroisocyanuric acid. In the present study, an X-ray crystal structure of the CAH-barbituric acid inhibitor complex from Azorhizobium caulinodans ORS 571 has been determined at 2.7 Å resolution. The CAH protein fold consists of three structurally homologous domains forming a β-barrel-like structure with external α-helices that result in a three-fold symmetry, a dominant feature of the structure and active site that mirrors the three-fold symmetrical shape of the substrate cyanuric acid. The active site structure of CAH is similar to that of the recently determined AtzD with three pairs of active site Ser-Lys dyads. In order to determine the role of each Ser-Lys dyad in catalysis, a mutational study using a highly sensitive, enzyme-coupled assay was conducted. The 10⁹-fold loss of activity by the S226A mutant was at least ten times lower than that of the S79A and S333A mutants. In addition, bioinformatics analysis revealed the Ser226/Lys156 dyad as the only absolutely conserved dyad in the CAH/barbiturase family. These data suggest that Lys156 activates the Ser226 nucleophile which can then attack the substrate carbonyl. Our combination of structural, mutational, and bioinformatics analyses differentiates this study and provides experimental data for mechanistic insights into this unique protein family.
Full Text Available Azorhizobium caulinodans ORS571 is a motile soil bacterium that interacts symbiotically with legume host Sesbania rostrata, forming nitrogen-fixing root and stem nodules. Bacterial chemotaxis plays an important role in establishing this symbiotic relationship. To determine the contribution of chemotaxis to symbiosis in A. caulinodans ORS571-S. rostrata, we characterized the function of TlpA1 (transducer-like protein in A. caulinodans, a chemoreceptor predicted by SMART (Simple Modular Architecture Research Tool, containing two N-terminal transmembrane regions. The tlpA1 gene is located immediately upstream of the unique che gene cluster and is transcriptionally co-oriented. We found that a ΔtlpA1 mutant is severely impaired for chemotaxis to various organic acids, glycerol and proline. Furthermore, biofilm forming ability of the strain carrying the mutation is reduced under certain growth conditions. Interestingly, competitive colonization ability on S. rostrata root surfaces is impaired in the ΔtlpA1 mutant, suggesting that chemotaxis of the A. caulinodans ORS571 contributes to root colonization. We also found that TlpA1 promotes competitive nodulation not only on roots but also on stems of S. rostrata. Taken together, our data strongly suggest that TlpA1 is a transmembrane chemoreceptor involved in A. caulinodans-S. rostrata symbiosis.
Jan Jacek Slaski
Full Text Available A protocol was developed to monitor persistence and spread of a genetically modified strain of the growth promoting diazotroph Azorhizobium caulinodans used to inoculate field grown wheat. The protocol was used to identify an endogluconase (egl overproducing strain of A. caulinodans isolated from inoculated soils. A detection limit of 2500 cfu g-1 and 108_1010cfu g-1 of soil was determined using the BIOLOG® fingerprinting method and PCR technique, respectively. Since none of the tested samples were positive for the bacteria or the Egl 1 gene construct, it was concluded that A. caulinodans ORS 571 pGV910-C1 did not persist or spread in any of the tested field locations. This result may be due to low soil temperatures and competition of indigenous microorganisms: environmental factors that were not favorable for the diazotroph to thrive in the test locations. The application of different inoculation methodologies, as well as the study of other rhizobial genera for the inoculation of wheat in further experiments, is strongly recommended. Key words: detection, diazotroph, endogluconase, ORS 571, wheat
Liu, Xiaolin; Liu, Wei; Sun, Yu; Xia, Chunlei; Elmerich, Claudine; Xie, Zhihong
Chemotaxis can provide bacteria with competitive advantages for survival in complex environments. The CheZ chemotaxis protein is a phosphatase, affecting the flagellar motor in Escherichia coli by dephosphorylating the response regulator phosphorylated CheY protein (CheY∼P) responsible for clockwise rotation. A cheZ gene has been found in Azorhizobium caulinodans ORS571, in contrast to other rhizobial species studied so far. The CheZ protein in strain ORS571 has a conserved motif similar to that corresponding to the phosphatase active site in E. coli The construction of a cheZ deletion mutant strain and of cheZ mutant strains carrying a mutation in residues of the putative phosphatase active site showed that strain ORS571 participates in chemotaxis and motility, causing a hyperreversal behavior. In addition, the properties of the cheZ deletion mutant revealed that ORS571 CheZ is involved in other physiological processes, since it displayed increased flocculation, biofilm formation, exopolysaccharide (EPS) production, and host root colonization. In particular, it was observed that the expression of several exp genes, involved in EPS synthesis, was upregulated in the cheZ mutant compared to that in the wild type, suggesting that CheZ negatively controls exp gene expression through an unknown mechanism. It is proposed that CheZ influences the Azorhizobium -plant association by negatively regulating early colonization via the regulation of EPS production. This report established that CheZ in A. caulinodans plays roles in chemotaxis and the symbiotic association with the host plant. IMPORTANCE Chemotaxis allows bacteria to swim toward plant roots and is beneficial to the establishment of various plant-microbe associations. The level of CheY phosphorylation (CheY∼P) is central to the chemotaxis signal transduction. The mechanism of the signal termination of CheY∼P remains poorly characterized among Alphaproteobacteria , except for Sinorhizobium meliloti , which
Full Text Available R bodies are insoluble large polymers consisting of small proteins encoded by reb genes and are coiled into cylindrical structures in bacterial cells. They were first discovered in Caedibacter species, which are obligate endosymbionts of paramecia. Caedibacter confers a killer trait on the host paramecia. R-body-producing symbionts are released from their host paramecia and kill symbiont-free paramecia after ingestion. The roles of R bodies have not been explained in bacteria other than Caedibacter. Azorhizobium caulinodans ORS571, a microsymbiont of the legume Sesbania rostrata, carries a reb operon containing four reb genes that are regulated by the repressor PraR. Herein, deletion of the praR gene resulted in R-body formation and death of host plant cells. The rebR gene in the reb operon encodes an activator. Three PraR binding sites and a RebR binding site are present in the promoter region of the reb operon. Expression analyses using strains with mutations within the PraR binding site and/or the RebR binding site revealed that PraR and RebR directly control the expression of the reb operon and that PraR dominantly represses reb expression. Furthermore, we found that the reb operon is highly expressed at low temperatures and that 2-oxoglutarate induces the expression of the reb operon by inhibiting PraR binding to the reb promoter. We conclude that R bodies are toxic not only in paramecium symbiosis but also in relationships between other bacteria and eukaryotic cells and that R-body formation is controlled by environmental factors.
Ling, Jun; Wang, Hui; Wu, Ping; Li, Tao; Tang, Yu; Naseer, Nawar; Zheng, Huiming; Masson-Boivin, Catherine; Zhong, Zengtao; Zhu, Jun
Horizontal gene transfer (HGT) of genomic islands is a driving force of bacterial evolution. Many pathogens and symbionts use this mechanism to spread mobile genetic elements that carry genes important for interaction with their eukaryotic hosts. However, the role of the host in this process remains unclear. Here, we show that plant compounds inducing the nodulation process in the rhizobium-legume mutualistic symbiosis also enhance the transfer of symbiosis islands. We demonstrate that the symbiosis island of the Sesbania rostrata symbiont, Azorhizobium caulinodans, is an 87.6-kb integrative and conjugative element (ICE Ac ) that is able to excise, form a circular DNA, and conjugatively transfer to a specific site of gly-tRNA gene of other rhizobial genera, expanding their host range. The HGT frequency was significantly increased in the rhizosphere. An ICE Ac -located LysR-family transcriptional regulatory protein AhaR triggered the HGT process in response to plant flavonoids that induce the expression of nodulation genes through another LysR-type protein, NodD. Our study suggests that rhizobia may sense rhizosphere environments and transfer their symbiosis gene contents to other genera of rhizobia, thereby broadening rhizobial host-range specificity.
Biological nitrogen fixation is the microbial process by which atmospheric dinitrogen (N 2 ) is reduced to ammonia. In all microbes studied, dinitrogen reduction is catalyzed by a highly conserved enzyme complex, called nitrogenase.
Pronk, A.F.; Boogerd, F C; Stoof, C.; Oltmann, L F; Stouthamer, A.H.; van Verseveld, H W
The determination of the in situ reduction levels of cytochromes b and c in growing bacteria is achieved by coupling a chemostat with a dual wavelength spectrophotometer. Visible light absorption spectra of cytochromes present in bacterial cells actively growing in a chemostat at a specific growth
Full Text Available Estudos foram realizados no Departamento de Ciência do Solo da Universidade Federal de Lavras (MG, no período de novembro/1999 a janeiro/2000, com o objetivo de avaliar a sobrevivência de estirpe e isolados de rizóbio em solo contaminado com metais pesados e verificar a relação entre tolerância do rizóbio a metais pesados em meio de cultura e sua sobrevivência em solo contaminado. Foram utilizados os dois microrganismos mais tolerantes [BR-4406 (estirpe recomendada para Enterolobium spp. e UFLA-01-457 (isolado de solo contaminado, ambos pertencentes ao gênero Bradyrhizobium ] e os dois mais sensíveis (UFLA-01-486 e UFLA-01-510, isolados de solo contaminado, pertencentes ao gênero Azorhizobium , todos selecionados de um grupo de 60estirpes/isolados em estudos prévios deste laboratório, em meio de cultura suplementado com metais pesados.Empregaram-se misturas de um Latossolo Vermelho-Escuro (LE que continham 0, 15 e 45% (v/v de um Latossolo Vermelho-Amarelo plíntico contaminado com Zn, Cd, Pb e Cu. As misturas de solo contaminado foram inoculadas com 20mL de cultura em YM na fase log das estirpes mencionadas, as quais foram testadas separadamente com três repetições. A avaliação do número de células viáveis no solo, realizada aos 0, 7, 14, 21 e 28dias de incubação, pelo método das diluições sucessivas e inoculação em placas com meio YMA, revelou comportamento diferenciado entre os organismos estudados. O número médio de células que sobreviveram ao final de 28 dias de incubação foi de (em UFCg-1de solo: 10(10,36, 10(10,29 e 10(9,70, para Bradyrhizobium, e 10(9,36, 10(7,54 e 0, para Azorhizobium em misturas de 0, 15 e 45% de solo contaminado, respectivamente. Portanto, houve maior sobrevivência de Bradyrhizobium do que de Azorhizobium , indicando maior tolerância a metais pesados do primeiro gênero.Como Bradyrhizobium foi também mais tolerante "in vitro", os resultados indicam haver relação entre o
I. C. B. Trannin
Full Text Available Com o objetivo de avaliar a tolerância a metais pesados "in vitro" de estirpes inoculantes (I, isolados de solos contaminados com metais (ISC e de solos não contaminados (ISNC de Bradyrhizobium, simbiontes de Enterolobium contortisiliquum (tamboril e de Acacia mangium (acácia e de Azorhizobium, simbiontes de Sesbania virgata (sesbânia, foram realizados dois experimentos. No primeiro, dez estirpes e, ou, isolados para cada espécie vegetal foram testados em meio YMA modificado por adição de tampões biológicos (HEPES e MES, suplementado com diferentes concentrações de Cu, Cd e Zn. Cobre e Cd foram testados em concentrações de 0 a 40 mg L-1, para ambos os gêneros, enquanto Zn variou de 0 a 1.000 mg L-1, para Bradyrhizobium, e de 0 a 500 mg L-1, para Azorhizobium. O crescimento de rizóbio nas diferentes concentrações de metais foi avaliado com atribuição de valores para os padrões observados (0 a 5. Os ISC de ambos os gêneros foram mais tolerantes, mas Bradyrhizobium tolerou Zn (800 mg L-1 até duas vezes e Cu (40 mg L-1 até oito vezes mais que Azorhizobium. No segundo experimento, estirpes e isolados tolerantes (T, sensíveis (S e de tolerância média (TM a metais selecionados em meio YMA modificado foram estudados em soluções aquosas com diferentes concentrações de Cu (0 a 0,01 mg L-1, Cd e Zn (0 a 1,0 mg L-1. A avaliação do número de células viáveis em soluções de metais foi feita por contagem das unidades formadoras de colônia em 0, 24, 48, 72 e 96 h de incubação, pelo método das diluições sucessivas e inoculação em YMA. Embora as soluções de metais tenham sido mais discriminatórias quanto a tolerância a metais que o meio YMA, estes dois métodos mostraram que: (a Azorhizobium foi mais sensível que Bradyrhizobium, (b os ISC de ambos os gêneros foram mais tolerantes do que os ISNC e (c a ordem de toxidez dos metais foi Cu > Cd > Zn.
Ng, Gordon; Tom, Curtis G S; Park, Angela S; Zenad, Lounis; Ludwig, Robert A
Nitrogen (N(2)) fixation also yields hydrogen (H(2)) at 1:1 stoichiometric amounts. In aerobic diazotrophic (able to grow on N(2) as sole N-source) bacteria, orthodox respiratory hupSL-encoded hydrogenase activity, associated with the cell membrane but facing the periplasm (exo-hydrogenase), has nevertheless been presumed responsible for recycling such endogenous hydrogen. As shown here, for Azorhizobium caulinodans diazotrophic cultures open to the atmosphere, exo-hydrogenase activity is of no consequence to hydrogen recycling. In a bioinformatic analysis, a novel seven-gene A. caulinodans hyq cluster encoding an integral-membrane, group-4, Ni,Fe-hydrogenase with homology to respiratory complex I (NADH: quinone dehydrogenase) was identified. By analogy, Hyq hydrogenase is also integral to the cell membrane, but its active site faces the cytoplasm (endo-hydrogenase). An A. caulinodans in-frame hyq operon deletion mutant, constructed by "crossover PCR", showed markedly decreased growth rates in diazotrophic cultures; normal growth was restored with added ammonium--as expected of an H(2)-recycling mutant phenotype. Using A. caulinodans hyq merodiploid strains expressing beta-glucuronidase as promoter-reporter, the hyq operon proved strongly and specifically induced in diazotrophic culture; as well, hyq operon induction required the NIFA transcriptional activator. Therefore, the hyq operon is constituent of the nif regulon. Representative of aerobic N(2)-fixing and H(2)-recycling alpha-proteobacteria, A. caulinodans possesses two respiratory Ni,Fe-hydrogenases: HupSL exo-hydrogenase activity drives exogenous H(2) respiration, and Hyq endo-hydrogenase activity recycles endogenous H(2), specifically that produced by N(2) fixation. To benefit human civilization, H(2) has generated considerable interest as potential renewable energy source as its makings are ubiquitous and its combustion yields no greenhouse gases. As such, the reversible, group-4 Ni,Fe-hydrogenases, such
Full Text Available 2e-79 AM494938_8( AM494938 |pid:none) Leishmania braziliensis chromosome... 300 2...7( CP001154 |pid:none) Laribacter hongkongensis HLHK9, ... 182 7e-44 CP001322_2533( CP001322 |pid:none) Desulfatibacillum alkeni...tckkmgiktva ihsdvdknakhvnmadeaicvgpaptsesylnidaiveaikmtgaqavhpgygflsensr fvkelenigvtfigpgsyamhalgdkie...84 |pid:none) Azorhizobium caulinodans ORS 57... 249 4e-64 AY810800_1( AY810800 |pid:none) Schistosoma japoni...e-57 CP000529_2359( CP000529 |pid:none) Polaromonas naphthalenivorans C... 226 4e-57 CP000821_1453( CP000821 |pid:none) Shewan
Yao Yunyin; Zhen Ming; Chang Xizhong
The results in pot experiments showed that the treating of 2,4-D and Azorhizobium caulinodans (2,4-D+A) could induce para-nodule formation on wheat roots. Plants treated grew normally. The plant height and dry weight are significantly higher than reference plants which are treated with 2,4-D+azorhizobium sterilized (2,4-D+AS). The nitrogenase activity is detected by ARA method. The N yield of most treated plants, especially in root systems, is higher than reference group that is measured by Kjeldahl method. The atom % 15 N excess in leaf and stem of treated plants measured by 15 N isotope dilution method is lower than that of reference group. Through four years experiments, it shows that para-nodules of wheat treated with 2,4-D+A could fix N 2 from air, but the ability of nitrogen fixation is lower and unstable. Although the nodulation efficiency could reach 100%, not each para-nodule induced can present activity of dinitrogen fixation. The amount of N fixed is 0.05∼18.1 mg/pot (0.01∼3.87 mg/plant). The net %Ndfa is 2.32%∼18.07%. The free-living N 2 fixing activity of azorhizobium is detected by 15 N isotope dilution method. The calculation of %Ndfa of nodulated wheat accurately is also discussed
Full Text Available BACKGROUND: Nitrogen (N(2 fixation also yields hydrogen (H(2 at 1:1 stoichiometric amounts. In aerobic diazotrophic (able to grow on N(2 as sole N-source bacteria, orthodox respiratory hupSL-encoded hydrogenase activity, associated with the cell membrane but facing the periplasm (exo-hydrogenase, has nevertheless been presumed responsible for recycling such endogenous hydrogen. METHODS AND FINDINGS: As shown here, for Azorhizobium caulinodans diazotrophic cultures open to the atmosphere, exo-hydrogenase activity is of no consequence to hydrogen recycling. In a bioinformatic analysis, a novel seven-gene A. caulinodans hyq cluster encoding an integral-membrane, group-4, Ni,Fe-hydrogenase with homology to respiratory complex I (NADH: quinone dehydrogenase was identified. By analogy, Hyq hydrogenase is also integral to the cell membrane, but its active site faces the cytoplasm (endo-hydrogenase. An A. caulinodans in-frame hyq operon deletion mutant, constructed by "crossover PCR", showed markedly decreased growth rates in diazotrophic cultures; normal growth was restored with added ammonium--as expected of an H(2-recycling mutant phenotype. Using A. caulinodans hyq merodiploid strains expressing beta-glucuronidase as promoter-reporter, the hyq operon proved strongly and specifically induced in diazotrophic culture; as well, hyq operon induction required the NIFA transcriptional activator. Therefore, the hyq operon is constituent of the nif regulon. CONCLUSIONS: Representative of aerobic N(2-fixing and H(2-recycling alpha-proteobacteria, A. caulinodans possesses two respiratory Ni,Fe-hydrogenases: HupSL exo-hydrogenase activity drives exogenous H(2 respiration, and Hyq endo-hydrogenase activity recycles endogenous H(2, specifically that produced by N(2 fixation. To benefit human civilization, H(2 has generated considerable interest as potential renewable energy source as its makings are ubiquitous and its combustion yields no greenhouse gases. As
Cocking, Edward C.
Nitrogen-fixing bacteria are able to enter into roots from the rhizosphere, particularly at the base of emerging lateral roots, between epidermal cells and through root hairs. In the rhizosphere growing root hairs play an important role in symbiotic recognition in legume crops. Nodulated legumes in endosymbiosis with rhizobia are amongst the most prominent nitrogen-fixing systems in agriculture. The inoculation of non-legumes, especially cereals, with various non-rhizobial diazotrophic bacteria has been undertaken with the expectation that they would establish themselves intercellularly within the root system, fixing nitrogen endophytic ally and providing combined nitrogen for enhanced crop production. However, in most instances bacteria colonize only the surface of the roots and remain vulnerable to competition from other rhizosphere micro-organisms, even when the nitrogen-fixing bacteria are endophytic, benefits to the plant may result from better uptake of soil nutrients rather than from endophytic nitrogen fixation. Azorhizobium caulinodans is known to enter the root system of cereals, other nonlegume crops and Arabidopsis, by intercellular invasion between epidermal cells and to internally colonize the plant intercellularly, including the xylem. This raises the possibility that xylem colonization might provide a nonnodular niche for endosymbiotic nitrogen fixation in rice, wheat, maize, sorghum and other non-legume crops. A particularly interesting, naturally occurring, non-qodular xylem colonising endophytic diazotrophic interaction with evidence for endophytic nitrogen fixation is that of Gluconacetobacter diazotrophicus in sugarcane. Could this beneficial endophytic colonization of sugarcane by G. diazotrophicus be extended to other members of the Gramineae, including the major cereals, and to other major non-legume crops of the World? (author)
Full Text Available Sessenta estirpes/isolados dos gêneros Bradyrhizobium, Rhizobium, Sinorhizobium, Mesorhizobium e Azorhizobium, procedentes de diferentes locais (Mata Atlântica, Amazônia, culturas agrícolas e experimentos com metais pesados e de espécies hospedeiras pertencentes às subfamílias Papilionoideae, Mimosoideae e Caesalpinoideae, foram avaliadas quanto à tolerância a Zn, Cu e Cd em meio YMA modificado pela adição de tampões biológicos (HEPES e MES e suplementados com Cu (0 a 60 mg L-1, Cd (0 a 60 mg L-1 e Zn (0 a 1.000 mg L-1. Mediante padrões de crescimento atribuídos às culturas nas diferentes concentrações dos metais, avaliaram-se as concentrações máximas toleradas e as doses tóxicas destes metais para redução de crescimento em 25% (DT25 e 50% (DT50. Não houve influência da procedência na concentração máxima de metal tolerada. A ordem de sensibilidade aos metais, considerando-se as concentrações máximas toleradas, foi Azorhizobium > Rhizobium = Mesorhizobium = Sinorhizobium > Bradyrhizobium. A DT25 e a DT50 foram úteis para diferenciarem estirpes/isolados de um mesmo gênero, que atingiram a mesma concentração máxima tolerada a Zn, Cu e Cd. A ordem de toxicidade dos metais estudados foi Cu > Cd > Zn.Sixty strains/isolates of the genera Bradyrhizobium, Rhizobium, Sinorhizobium, Mesorhizobium and Azorhizobium, isolated from different hosts (legume subfamilies: Papilionoideae, Mimosoideae and Caesalpinoideae and location (Atlantic Forest, Amazon region, crop plantings and heavy metal experiments, were evaluated for Zn, Cu and Cd tolerance in YMA medium modified by the addition of biological buffers (HEPES and MES and supplemented with Cu (0 to 60 mg L-1, Cd (0 to 60 mg L-1, and Zn (0 to 1,000 mg L-1sulphates. Growth standards were applied to evaluate rhizobia cultures growth at different metal concentrations, allowing evaluation of highest tolerated concentrations of Zn, Cu, and Cd and the toxic doses
Banik, Avishek; Mukhopadhaya, Subhra Kanti; Dangar, Tushar Kanti
The diversity of endophytic and epiphytic diazotrophs in different parts of rice plants has specificity to the niche (i.e. leaf, stem and root) of different genotypes and nutrient availability of the organ. Inoculation of the indigenous, polyvalent diazotrophs can facilitate and sustain production of non-leguminous crops like rice. Therefore, N2-fixing plant growth promoting bacteria (PGPB) were isolated from different parts of three Indian cultivated [Oryza sativa L. var. Sabita (semi deep/deep water)/Swarna (rain fed shallow lowland)/Swarna-Sub1(submergence tolerant)] and a wild (O. eichingeri) rice genotypes which respond differentially to nitrogenous fertilizers. Thirty-five isolates from four rice genotypes were categorized based on acetylene reduction assay on nitrogenase activity, biochemical tests, BIOLOG and 16S rRNA gene sequencing. The bacteria produced 9.36-155.83 nmole C2H4 mg(-1) dry bacteria h(-1) and among them nitrogenase activity of 11 potent isolates was complemented by nifH-sequence analysis. Phylogenetic analysis based on 16S rDNA sequencing divided them into five groups (shared 95-100 % sequence homology with type strains) belonging to five classes-alpha (Ancylobacter, Azorhizobium, Azospirillum, Rhizobium, Bradyrhizobium, Sinorhizobium, Novosphingobium, spp.), beta (Burkholderia sp.), gamma (Acinetobacter, Aeromonas, Azotobacter, Enterobacter, Klebsiella, Pantoea, Pseudomonas, Stenotrophomonas spp.) Proteobacteria, Bacilli (Bacillus, Paenibacillus spp.) and Actinobacteria (Microbacterium sp.). Besides, all bacterial strains possessed the intrinsic PGP traits of like indole (0.44-7.4 µg ml(-1)), ammonia (0.18-6 mmol ml(-1)), nitrite (0.01-3.4 mol ml(-1)), and siderophore (from 0.16-0.57 μmol ml(-1)) production. Inoculation of rice (cv. Swarna) seedlings with selected isolates had a positive impact on plant growth parameters like shoot and root elongation which was correlated with in vitro PGP attributes. The results indicated that the