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.

Temporal assessment of microbial communities in soils of two contrasting mangroves

ABSTRACT Variations in microbial communities promoted by alterations in environmental conditions are reflected in similarities/differences both at taxonomic and functional levels. Here we used a natural gradient within mangroves from seashore to upland, to contrast the natural variability in bacteria, cyanobacteria and diazotroph assemblages in a pristine area compared to an oil polluted area along a timespan of three years, based on ARISA (bacteria and cyanobacteria) and nifH T-RFLP (diazotrophs) fingerprinting. The data presented herein indicated that changes in all the communities evaluated were mainly driven by the temporal effect in the contaminated area, while local effects were dominant on the pristine mangrove. A positive correlation of community structure between diazotrophs and cyanobacteria was observed, suggesting the functional importance of this phylum as nitrogen fixers in mangroves soils. Different ecological patterns explained the microbial behavior in the pristine and polluted mangroves. Stochastic models in the pristine mangrove indicate that there is not a specific environmental factor that determines the bacterial distribution, while cyanobacteria and diazotrophs better fitted in deterministic model in the same area. For the contaminated mangrove site, deterministic models better represented the variations in the communities, suggesting that the presence of oil might change the microbial ecological structures over time. Mangroves represent a unique environment threatened by global change, and this study contributed to the knowledge of the microbial distribution in such areas and its response on persistent contamination historic events.

Effect of biofertilizer on the diversity of nitrogen - fixing bacteria and total bacterial community in lowland paddy fields in Sukabumi West Java, Indonesia

Aims: Some of methanotrophic bacteria and nitrous oxide (N2O) reducing bacteria have been proven able to support the plant growth and increase productivity of paddy. However, the effect of application of the methanotrophics and N2O reducing bacteria as a biofertilizer to indigenous nitrogen-fixing bacteria and total bacterial community are still not well known yet. The aim of the study was to analyze the diversity of nitrogen-fixing bacteria and total bacterial communty in lowland paddy soils. Methodology and results: Soil samples were taken from lowland paddy fields in Pelabuhan Ratu, Sukabumi, West Java, Indonesia. There were two treatments applied to the paddy field i.e biofertilizer-treated field (biofertilizer with 50 kg/ha NPK) and control (250 kg/ha NPK fertilizer). There were nine different nifH bands which were successfully sequenced and most of them were identified as unculturable bacteria and three of them were closely related to Sphingomonas sp., Magnetospirillum sp. and Ideonella dechloratans respectively. In addition, there were 20 different 16S rDNA bands which were successfully sequenced. Phylogenetic analysis of the sequence showed that there were 5 phyla of bacteria, i.e. Proteobacteria (Alphaproteobacteria and Gammaproteobacteria), Chlorofexi, Gemmatimonadetes, Clostridia, and Bacteroidetes respectively. Alphaproteobacteria was the most dominant group in lowland paddy field. Microbial diversities in the biofertilizer-treated field were lower than that of 100% fertilizer-treated field either based on nifH and 16S rDNA genes. Conclusion, significance and impact study: Biofertilizer treatment has lower microbial diversity than control, either based on nifH and 16S rDNA genes.

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.

Functional divergence outlines the evolution of novel protein function in NifH/BchL protein family.

Biological nitrogen fixation is accomplished by prokaryotes through the catalytic action of complex metalloenzyme, nitrogenase. Nitrogenase is a two-protein component system comprising MoFe protein (NifD&K) and Fe protein (NifH). NifH shares structural and mechanistic similarities as well as evolutionary relationships with light-independent protochlorophyllide reductase (BchL), a photosynthesis-related metalloenzyme belonging to the same protein family. We performed a comprehensive bioinformatics analysis of the NifH/BchL family in order to elucidate the intrinsic functional diversity and the underlying evolutionary mechanism among the members. To analyse functional divergence in the NifH/ BchL family, we have conducted pair-wise estimation in altered evolutionary rates between the member proteins. We identified a number of vital amino acid sites which contribute to predicted functional diversity.We have also made use of the maximum likelihood tests for detection of positive selection at the amino acid level followed by the structure-based phylogenetic approach to draw conclusion on the ancient lineage and novel characterization of the NifH/BchL protein family. Our investigation provides ample support to the fact that NifH protein and BchL share robust structural similarities and have probably deviated from a common ancestor followed by divergence in functional properties possibly due to gene duplication.

Diversification of nitrogen fixing bacterial community using nifH gene as a biomarker in different geographical soils of Western Indian Himalayas.

Six soil samples (Pantnagar, Chamoli, Almora, Ranichauri, Pithoragarh and Badrinath) belonging to different geographical locations of Western Himalayas in India, were analyzed to diversify the nitrogen fixing bacterial community using nifH gene biomarker. DNA from soil samples were isolated and amplified using nifH gene specific primers. Genomic DNA and PCR amplified products were then individually subjected to restriction digestion with tetra to octacutter enzymes (AluI, MspI, BglII, XbaI, HindIII, HaeIII, AluI, MspI and PasI. Further, restriction pattern was studied by preparing dendograms on the basis of similarity matrix and compared for the nifH community. It was observed that temperate region soils (Ranichauri and Pithoragarh) were negative for nifH marker while subalpine region (Badrinath) and tarai region soils (Pantnagar) documented similar nifH community. Moreover, the direct genomic DNA restriction analysis indicated that subalpine region soil (Badrinath) was most diversified.