Horizontal gene transfer among nitrogen-fixing bacteria in the guts of termite Coptotermes gestroi

Aims@#Arthropods guts, such as termite harbor diverse microorganisms including those that are capable of fixing atmospheric nitrogen (N2). Nitrogen-fixing bacteria can help termite to overcome their shortage of dietary N by providing fixed N2. Nitrogenase enzyme is responsible for this trait and encoded by nif genes which are highly conserved and are primarily used in the identification of N2-fixing microorganisms. Here, we characterized N2-fixing bacteria isolated from the hindguts of termite Coptotermes gestroi.@*Methodology and results@#A total of 46 bacterial isolates were obtained after a primary screening based on their ability to grow on Burk’s media. Subsequently, the nifH gene from two of these isolates, namely S7 and S20, were successfully amplified and sequenced. Molecular phylogenetic analysis of 16S rRNA gene sequence revealed that isolate S7 is closely related to Ralstonia pickettii ATCC27511 (99.34% similarity, 1059 bp), whereas isolate S20 is closely related to Microbacterium sp. NCCP-451 (LC488936) (99.06% similarity, 948 bp). Besides that, the recA gene of isolate S7 is closely related to Ralstonia pickettii 12D (CP001644) (100% similarity, 442 bp) and the type strain of Ralstonia pickettii (ATCC 27511) (NZ KN050646) (98.97% similarity, 438 bp). Meanwhile, nifH gene of isolate S7 showed highest similarity to the uncultured bacterium NR1606 (AF035490) (99.93% similarity, 277 bp). Moreover, the nifH gene of isolate S20 is clearly separated from Azoarcus sp. and distantly related to Microbacterium sp. The incongruence between the partial 16S rRNA and nifH gene sequences could indicate the possibility of horizontal transfer of nif genes.@*Conclusion, significance and impact of study@#The phylogenetic incongruence between housekeeping genes (16S rRNA and RecA) and nifH gene in these bacteria provides new insight on potential horizontal gene transfer (HGT) activity taking place in bacterial communities particularly in the guts of arthropods. The finding of this study on potential HGT can also aid in the prediction of origins and evolution of gene transfer among bacteria.

Photo-fermentational hydrogen production of Rhodobacter sp. KKU-PS1 isolated from an UASB reactor

Background In this study, the detection of nifH and nifD by a polymerase chain reaction assay was used to screen the potential photosynthetic bacteria capable of producing hydrogen from five different environmental sources. Efficiency of photo-hydrogen production is highly dependent on the culture conditions. Initial pH, temperature and illumination intensity were optimized for maximal hydrogen production using response surface methodology with central composite design. Results Rhodobacter sp. KKU-PS1 (GenBank Accession No. KC478552) was isolated from the methane fermentation broth of an UASB reactor. Malic acid was the favored carbon source while Na-glutamate was the best nitrogen source. The optimum conditions for simultaneously maximizing the cumulative hydrogen production (Hmax) and hydrogen production rate (Rm) from malic acid were an initial of pH 7.0, a temperature of 25.6°C, and an illumination intensity of 2500 lx. Hmax and Rm levels of 1264 ml H2/l and 6.8 ml H2/L-h were obtained, respectively. The optimum initial pH and temperature were further used to optimize the illumination intensity for hydrogen production. An illumination intensity of 7500 lx gave the highest values of Hmax (1339 ml H2/l) and Rm (12.0 ml H2/L-h) with a hydrogen yield and substrate conversion efficiency of 3.88 mol H2/mol malate and 64.7%, respectively. Conclusions KKU-PS1 can produce hydrogen from at least 8 types of organic acids. By optimizing pH and temperature, a maximal hydrogen production by this strain was obtained. Additionally, by optimizing the light intensity, Rm was increased by approximately two fold and the lag phase of hydrogen production was shortened.

Regulation of nitrogen metabolism in salt tolerant and salt sensitive Frankia strains.

Effect of salinity (0, 50, 100, 250, 500 and 750 mM NaCl) was observed on some important physiological parameters of nitrogen metabolism such as nitrate uptake, intracellular and extracellular ammonium status and activities of nitrogenase, nitrate reductase, nitrite reductase and glutamine synthetase among Frankia strains differing in their salt tolerance capacity. Nitrogenase activity closely followed the growth pattern with regular decline on NaCl supplementation. All the other enzymes showed optimum activity at 100 mM and declined further. Co-regulation of the nitrate uptake system and sequential enzyme activities plays a crucial role in governing the nitrogen status of strains during salt stress. HsIi10 experiencing minimum decline in enzyme activities and best possible nitrogen regulation under NaCl replete condition showed adequate nutritional management. Among all the strains, HsIi10 proved to be salt tolerant on account of above features while the salt sensitive strain HsIi8 lacked the ability to regulate various steps of nitrogen metabolism during salinity, and thus Frankia strain HsIi10 can potentially serve as a potential biofertilizer in the saline soil.

Effect of UV-C on thylakoid arrangement, pigment content and nitrogenase activity in the cyanobacterium Microchaete sp.

The effect of UV-C radiation on thylakoid arrangement, chlorophyll-a and carotenoid content and nitrogenase activity of the cyanobacterium Microchaete sp. was studied. Chlorophyll-a and carotenoid content increased gradually up to 48 h of UV-C exposure but declined with longer exposures. Nitrogenase activity decreased moderately with 6 to 12 h exposure and decreased substantially afterwards. When cells exposed to UV-C for 12 to 24 h, grown under fluorescent light for 144 h, nitrogenase activity increased to levels greater than in the control cells. The exposure of UV-C treated cells to fluorescent light, however, did not result in recovery of pigment content. In Microchaete sp. cells treated with UV-C for 144 h, thylakoid membranes became dense, were aggregated into bundles, and were surrounded by spaces devoid of cytoplasm.

Caracterización fisiológica de la comunidad microbiana endófita de la caña de azúcar / Physiological characterization of sugarcane’s endophytic microbial community

El uso desmedido de fertilizantes químicos nitrogenados y pesticidas ha traído graves consecuencias ambientales, por lo que se ha prestado gran atención al estudio de la microbiota nativa de los cultivos y sus beneficios a la planta, incluyendo la caña de azúcar. Este trabajo se realizó con el objetivo de caracterizar la microbiota nativa de la caña de azúcar. Se utilizaron 5 cepas bacterianas y 50 aislados provenientes del interior de este cultivo. Se determinó la actividad nitrogenasa y la influencia de la fuente de carbono, nitrógeno y el pH en la misma, mediante cromatografía gaseosa. Se detectó la producción de ácido indolacético por Dot-Immunobinding y el método de Salkowski. Del total de cepas y aislados, 19 mostraron actividad nitrogenasa, con valores entre 100 y 5000 //g/mL, y 6, Gluconacetobacter diazotrophicus PAl-5, Gluconacetobacter diazotrophicus 1-05, Gluconacetobacter diazotrophicus 4-02, aislado 17, aislado 30 y aislado 305; además, tienen la capacidad de producir AIA (valores entre 1,7 y 2,5 JMg/mL). Se demostró que las fuentes nutricionales y el pH del medio de cultivo influyen sobre la actividad nitrogenasa de las cepas representativas de la comunidad endófita.

Excessive application of chemical nitrogen fertilisers and pesticides has badly affected the environment. This has led to great interest being shown in studying a crop’s native microbial community and its benefit for plants. This paper was thus aimed at characterising sugarcane’s endophytic microbial community. 5 sugar cane strains and 50 isolates were used. Gas chromatography was used for measuring nitrogenase activity and the influence of carbon and nitrogen sources and pH on cultures. Indol acetic (IAA) production was detected by Dot-Immunobinding and Salkowski’s method. These results show that 19 strains and isolates had nitrogenase activity, values ranging from 100 to 5000 /zg/mL; 6 of them produced IAA (values ranging from 1,7 to 2,5 //g/mL): Gluconacetobacter iazotrophicus PAl-5, Gluconacetobacter diazotrophicus 1-05, Gluconacetobacter diazotrophicus 4-02, 17, 30 and 305. It was demonstrated that culture medium nutrient sources and pH affectedthe nitrogenase activity of the strains representing the endophytic community.

Scavenging of nickel and chromium toxicity in Aulosira fertilissima by immobilization: effect on nitrogen assimilating enzymes

The ubiquity of heavy metals in the biosphere results in the introduction of high amounts of toxic metals into the food chain from various sources. In the present study, one of the strongest nitrogen fixing cyanobacterium of the rice fields, Aulosira fertilissima, was subjected to nickel and chromium stress and the ameliorating effect of immobilization was investigated. Cell immobilization could protect the organism's growth against the toxicity of both heavy metals at LC50 as compared to lethal concentrations. The nitrate reductase activity in free cells treated with the metals was substantially inhibited but immobilized cells treated with 0.1 ppm nickel was not affected by the metal treatment. Cell immobilization also resulted in a significant protection against sub-lethal concentration of chromium but to a lesser degree than it did with sub- lethal levels of nickel. Control immobilized cells also had higher Nitrogenase activity than control free cells. Nickel and chromium addition markedly decreased the enzyme activity in free cells but immobilized cells exposed to sublethal concentrations of both metals could overcome this decrease. Glutamine synthetase showed similar response under immobilized conditions compared to free cells with both metals. The addition of algal filtrate in 3:1 ratio further increased the nitrogenase activity compared with immobilized cells treated with sublethal doses of both metals. Immobilization facilitated higher uptake of nickel as compared to chromium. The observations of the present study clearly demonstrate the protective effect of immobilization on Aulosira fertilissima against Nickel and chromium toxicity. Rice field ecosystem thus possess a bidirectional natural metal ameliorating system where Aulosira mats act as a naturally immobilized system and the decay of Aulosira along with other cyanobacteria act as natural chelators protecting the rice plants from deleterious effects of the heavy metals. Most importantly is...

Can mushrooms fix atmospheric nitrogen?

It is generally reported that fungi like Pleurotus spp. can fix nitrogen (N2). The way they do it is still not clear. The present study hypothesized that only associations of fungi and diazotrophs can fix N2. This was tested in vitro. Pleurotus ostreatus was inoculated with a bradyrhizobial strain nodulating soybean and P. ostreatus with no inoculation was maintained as a control. At maximum mycelial colonization by the bradyrhizobial strain and biofilm formation, the cultures were subjected to acetylene reduction assay (ARA). Another set of the cultures was evaluated for growth and nitrogen accumulation. Nitrogenase activity was present in the biofilm, but not when the fungus or the bradyrhizobial strain was alone. A significant reduction in mycelial dry weight and a significant increase in nitrogen concentration were observed in the inoculated cultures compared to the controls. The mycelial weight reduction could be attributed to C transfer from the fungus to the bradyrhizobial strain, because of high C cost of biological N2 fixation. This needs further investigations using 14C isotopic tracers. It is clear from the present study that mushrooms alone cannot fix atmospheric N2. But when they are in association with diazotrophs, nitrogenase activity is detected because of the diazotrophic N2 fixation. It is not the fungus that fixes N2 as reported earlier. Effective N2 fixing systems, such as the present one, may be used to increase protein content of mushrooms. Our study has implications for future identification of as yet unidentified N2 systems occurring in the environment.

Dual Inoculation of Native Rhizobium spp. and Arbuscular Mycorrhizal Fungi: An Impact Study for Enhancement of Pulse Production

Fifteen Rhizobium spp. from nodules of 6 common pulses collected from 6 districts of Assam were studied for their infectivity, intrinsic antibiotic resistance, nitrogenase activity and effect of dual inoculation with two native Arbuscular Mycorrhizal Fungi viz. Glomus mosseae(GM) and Gigaspora gilmarie(GG). Out of the 15 isolates 9 were found nodulation positive and 6 of them(AR1, BR8, BR12, AR10, UR10 & GR21) were subjected to intrinsic antibiotic sensitivity test of which AR1 showed resistance against all the 9 test antibiotics. Isolates AR1 and GR21 showed the highest(4.25 mole, gm(-1)hour(-1)) and the lowest(1.05 mole, gm(-1)hour(-1)) nitrogenase activity respectively. In Most Probable Number count, the maximum Rhizobium population 5.8x10(5), was found in both Blackgram and Greengram variety of pulses. The maximum dry weight of nodules(3.14 g), dry weight of shoot(10.08 g), nitrogen content(7.68 mg, plant(-1)), chlorophyll content(1.89 mg, g(-1)), phosphorus content of shoot(6.17 mg, g(-1)) and yield(535.67 kg, Ha(-1)) were found when AR1 dually inoculated with GM in Blackgram.

A novel strategy of oxygen restriction by some diazotrophic enteric bacteria.

Protection of nitrogenase against oxygen inactivation in diazotrophs involves numerous strategies. Glutathione is known to play an important role in scavenging oxyradicals in many living systems. The involvement of glutathione (reduced) (GSH), glutathione peroxidase (GPX) and glutathione reductase (GR) in the protection of nitrogenase in free living diazotrophs is reported here for the first time. Reduced glutathione content and the activity of glutathione peroxidase and glutathione reductase increased with increase in oxygen concentration under nitrogen fixing conditions but decreased under anaerobic and nitrogenase repressed conditions. This correlation is used to postulate a protecting role for GSH-GPX-GR system against oxygen inactivation of nitrogenase.

Nitrogenase activity of Beijerinckia derxii is preserved under adverse conditions for its growth

In order to evaluate the response of Beijerinckia derxii ICB-10 to different environmental factors, growth curves and specific nitrogenase activity were studied. Tested conditions were as follows: a) media with different pH values (2.5, 2.8, 4.2 and 5.7); b) medium supplemented with 230 æM aluminium sulphate; c) media with two different potassium phosphate concentrations (50 mM and 100 mM); d) shaken or still cultures; e) medium supplemented with 40 mM sodium thiosulphate. Growth curves and specific nitrogenase activity at pH 4.2 were closely similar to those for cultures at pH 5.7 (standard condition), whereas no growth occurred at pH 2.5. Changes in growth curves and/or specific nitrogenase activity were observed under the following conditions: I) pH 2.8 (decrease in initial CFU number, reduction of maximum specific growth rate, reduced number of generations and stimulation of nitrogenase activity), II) presence of aluminium (early death phase), III) 50 mM PO4(3-) (reduction of maximum specific growth rate), IV) 100 mM PO4(3-) (reduction of both number of generations and maximum specific growth rate as well as early death phase), V) low O2 availability (increasing nitrogenase activity), and VI) presence of thiosulphate (reduction of maximum specific growth rate; early death phase and high stimulation of nitrogenase activity). The data obtained showed the high variability of the cell growth response to environmental factors. Nitrogenase activity was always preserved even when population growth was affected

Reversibility of oxygen induced inactivation of nitrogenase in some enterobacteria.

Aerobic and microaerobic diazotrophs possess numerous oxygen restriction strategies to protect nitrogenase from inactivation by oxygen without interfering with energy generation through oxidative phosphorylation. Protection by conformational change in nitrogenase was first detected and described in Azotobacter. This strategy once considerd unique for Azotobacter has been shown in this study to occur in Citrobacterfreundii (Braak) Werkman and Gillen and Klebsiella pneumoniae subspecies rhinoscleromatis (Trevisan) Migula also. However, in these enteric bacteria the entire enzyme is not protected probably due to the absence of any respiratory protection similar to that found in the aerobe, Azotobacter.

Metalaxyl effect on nitrogenase activity (acetylene reduction) and yield of mungbean (Vigna radiata (L.) wilzek).

In an experiment, application of different levels of metalaxyl to a sandy loam soil significantly affected the nodulation and nitrogenase activity of mungbean. In both the compost amended and unamended soils, 0.5 mg kg(-1) of metalaxyl enhanced acetylene reduction activity and yield of mungbean, where as higher concentrations (1 mg and 2.5 mg kg(-1) of fungicide) inhibited the nodulation traits as well as economic traits of mungbean.

"Beijerinckia derxii" stimulates the viability of non-N(2)-fixing bacteria in nitrogen-free media

The interactions between the nitrogen-fixing microorganism "Beijerinckia derxii" with two non-diazotrophic bacteria, either "Escherichia coli" or a faculty sulphur-oxidizing chemolitroph, were studied in mixed cultures. Direct and indirect contact between "B. derxii" and "E. coli" were tested. "B. derxii" increased CFU numbers and/or maintained the viability of the non-diazotrophic bacteria, but neither growth nor nitrogenase activity of the nitrogen-fixing bacterium were affected by either partner

Analogue-resistant mutants of Azotobacter chroococcum derepressed for nitrogenase activity and early ammonia excretion having potential as inoculants for cereal crops.

Spontaneous mutants resistant to methionine sulfoximine (Msx), methyl alanine (Mal) and methyl ammonium chloride (Mac) were derived from A. chroococcum strain A103. Msx and Mal-resistant mutants expressed 1.73 to 10.98% of the fully derepressed nitrogenase activity when grown in Burk's medium containing ammonium acetate. Mac-resistant mutants did not express nitrogenase activity in ammonium acetate supplemented medium. The mutants excreted ammonia even after 2 days of growth and some mutants excreted more ammonia as compared to the parent. Selected mutants were inoculated on wheat (Triticum aestivum) and barley (Hordeum vulgare) under field conditions. Majority of the derepressed mutants increased grain yield of wheat and barley varying from 1.2 to 33.3%. However, host-dependent effects on grain yield were observed with different mutants. Two mutants, Mal 27 and Mac 19 showed significant increase in grain yields of both the crops. The results suggest that metabolic analogue-resistant mutants of Azotobacter have potential for use as a biofertilizer for cereal crops.

Evaluation of N2-fixation efficiency of azotobacter in Alginate-encapsulated and free cell systems

The nitrogenase activity of alginate encapsulated and non- encapsulated cells of six Azotobacter strains was compared in liquid medium. The nitrogenase activity [expressed as n moles C2H4/hr/ml] of the non-encapsulated cells of the best strain [strain No. 6] was 85 n moles C2H4/hr/ml. However, alginate encapsulated cells tremendously increased the nitrogenase activity to 5050 n moles C2H4/hr/ml. In pots experiment, the results showed that inoculant cells introduced to wheat plants in alginate are protected from adverse soil effects over 60 days. Cell number in the rhizosphere soil of plants inoculated with encapsulated cells reached 210 x 106/g, whereas the number at the same time in plants inoculated with free cells was 79 x 106/g. Sufficient cells were able to reach and colonize wheat roots 20 days following introduction of beads. Plants inoculated with encapsulated cells had significantly higher dry weight and higher nitrogen content as compared to those inoculated with free cells

Effect of hydration and dehydration on initiation and dynamics of some physiological reactions in desiccation tolerant cyanobacterium Scytonema geitleri.

The effect of hydration and dehydration has been studied on extent and recovery of some metabolic reactions in desiccation tolerant terrestrial cyanobacterium Scytonema geitleri. The results show that the energy transducing reactions like photochemical reactions of photosynthesis recover first, followed by increase in ATP pool size. During later phase of hydration, appearance of energy consuming processes such as CO2 fixation and nitrogen fixation have been observed. Sensitivity of reactions during dehydration followed the pattern reverse to recovery processes.

Nitrogen fixation efficiency of rhizobia nodulating Apios americana.

In this study, the efficiency of Apios rhizobial isolates in N2 fixation was compared with a typical Bradyrhizobium strain [USDA 3451] in a greenhouse pot experiment. The study exhibited marked differences among Apios isolates in a nodule number and nodule mass top dry weight and total uptake of nitrogen by-plant. There were approximately two-fold differences in nitrogen accumulated within the plant between the most and the least effective isolate. The study also revealed that all isolates of Apios rhizobia [B. Sp.] gave a significant growth response despite the presence of indigenous B. Sp. [Vigna]. The differentiation between Apios isolates in their efficiency to fix atmospheric N was assessed using acetylene reduction technique for measuring N-ase activity. Data also showed that there were significant differences in the amount of N fixed between the different isolates of Apios

Expression of Rhizobium meliloti symbiotic promoters in Azorhizobium caulinodans.

Plasmids containing Rhizobium meliloti symbiotic promoters P1 (promoter of nifHDK) and P2 (promoter of fixABCX) when mobilized into the cells of Azorhizobium caulinodans strain IRBG 46 showed strong expression of these promoters under free-living microaerobic as well as symbiotic conditions. Under free-living conditions microaerobiosis (3% or less O2) was found to be sufficient to activate these promoters; expression being higher at 1% than at 3% O2 concentration. Under symbiotic conditions the expression was much more stronger-with bacteroids in stem nodules showing higher expression than those in root nodules. Under both the conditions expression of the promoters in the native R. meliloti strain Rm102F34 was lower than that in the A. caulinodans strain IRBG 46. The results suggest a functional homology of these promoters in the heterologous background of A. caulinodans.

Cloning and sequencing of the nitrogenase structural genes nifHDK of Herbaspirillum seropedicae

The nitrogenase structural genes (nifHDK) of the endophytic diazotroph Herbaspirillum seropedicae were isolated from a genomic bank by plate hybridization. Sequence analysis of the DNA showed a consensus promoter region upstream from the nifH gene containing a -24/-12 type promoter together with NifA- and integration host factor (IHF)- binding sites. The derived protein sequences of NifH, NifD and NifK contained conserved cysteine residues for binding iron-sulfur clusters and the iron-molybdenum cofactor. These protein sequences showed the strongest similarities to the nifHDK gene products of the symbiotic diazotroph Bradyrhizobium japonicum (93.5 per cent, 91.3 per cent and 83.3 per cent, respectively), the plant-associated diazotrophAzospirillum brasilense (90.0 per cent, 83.7 per cent and 75.1 per cent, respectively) and to Thiobacillus ferrooxidans (91.0 per cent, 83.4 per cent and 81.1 per cent, respectively) of the same phylogenetic group of the protobacteria.