Characterization and analysis of nifH genes from Paenibacillus sabinae T27

Paenibacillus sabinae T27 (CCBAU 10202=DSM 17841) is a gram-positive, spore-forming diazotroph with high nitrogenase activities. Three nifH clusters were cloned from P. sabinae T27. Phylogenetic analysis revealed that NifH1, NifH2 and NifH3 cluster with Cyanobacterium. Each of the coding regions of nifH1, nifH2 and nifH3 from P. sabinae T27 under the control of the nifH promoter of Klebsiella pneumoniae could partially restore nitrogenase activity of K. pneumoniae nifH^? mutant strain 1795, which has no nitrogenase activity. This suggests that the three nifH genes from P. sabinae T27 have some function in nitrogen fixation. RT-PCR showed that all three nifH genes were expressed under nitrogen-fixing growth conditions. Using promoter vectors which have promoterless lacZ gene, three putative promoter regions of nifH genes were identified.     

Phylogenetic Diversity of Nitrogen Fixation Genes in the Intestinal Tract of Reticulitermes chinensis Snyder

Wood-feeding termites live on cellulolytic materials that typically lack of nitrogen sources. It was reported that symbiotic microbes play important roles in the maintenance of a normal nitrogen contents in termite by different metabolisms including nitrogen fixation. In this study, the diversity of nitrogen-fixing organisms in the symbiotic intestinal microflora of Reticulitermes chinensis Snyder was investigated with culture independent method. Fragments of the nifH genes, which encode dinitrogenase reductase, were directly amplified from the DNA of the mixed microbial population in the termite gut with four sets of primers corresponding to the conserved regions of the genes. Clones were randomly selected and analyzed by RFLP. Sequence analysis revealed that a large number of nifH sequences retrieved from the termite gut were most closely related to strict anaerobic bacteria such as clostridia and spirochetes, some of the others were affiliated with proteobacteria, bacteroides, or methanogenic archaea. The results showed that there was a remarkable diversity of nitrogenase genes in the gut of Reticulitermes chinensis Snyder.     

Differences in the Faecal Microbiome in Schistosoma haematobium Infected Children vs. Uninfected Children

BackgroundSeveral infectious diseases and therapeutic interventions cause gut microbe dysbiosis and associated pathology. We characterised the gut microbiome of children exposed to the helminth Schistosoma haematobium pre- and post-treatment with the drug praziquantel (PZQ), with the aim to compare the gut microbiome structure (abundance and diversity) in schistosome infected vs. uninfected children.MethodsStool DNA from 139 children aged six months to 13 years old; with S. haematobium infection prevalence of 27.34% was extracted at baseline. 12 weeks following antihelminthic treatment with praziqunatel, stool DNA was collected from 62 of the 139 children. The 16S rRNA genes were sequenced from the baseline and post-treatment samples and the sequence data, clustered into operational taxonomic units (OTUs). The OTU data were analysed using multivariate analyses and paired T- test.ResultsPre-treatment, the most abundant phyla were Bacteroidetes, followed by Firmicutes and Proteobacteria respectively. The relative abundance of taxa among bacterial classes showed limited variation by age group or sex and the bacterial communities had similar overall compositions. Although there were no overall differences in the microbiome structure across the whole age range, the abundance of 21 OTUs varied significantly with age (FDR<0.05). Some OTUs including Veillonella, Streptococcus, Bacteroides and Helicobacter were more abundant in children ≤ 1 year old compared to older children. Furthermore, the gut microbiome differed in schistosome infected vs. uninfected children with 27 OTU occurring in infected but not uninfected children, for 5 of these all Prevotella, the difference was statistically significant (p <0.05) with FDR <0.05. PZQ treatment did not alter the microbiome structure in infected or uninfected children from that observed at baseline.ConclusionsThere are significant differences in the gut microbiome structure of infected vs. uninfected children and the differences were refractory to PZQ treatment.     

Bacterial Life and Dinitrogen Fixation at a Gypsum Rock

The organisms of a bluish-green layer beneath the shards of a gypsum rock were characterized by molecular techniques. The cyanobacterial consortium consisted almost exclusively of Chroococcidiopsis spp. The organisms of the shards expressed nitrogenase activity (C₂H₂ reduction) aerobically and in light. After a prolonged period of drought at the rock, the cells were inactive, but they resumed nitrogenase activity 2 to 3 days after the addition of water. In a suspension culture of Chroococcidiopsis sp. strain PCC7203, C₂H₂ reduction required microaerobic conditions and was strictly dependent on low light intensities. Sequencing of a segment of the nitrogenase reductase gene (nifH) indicated that Chroococcidiopsis possesses the alternative molybdenum nitrogenase 2, expressed in Anabaena variabilis only under reduced O₂ tensions, rather than the widespread, common molybdenum nitrogenase. The shards apparently provide microsites with reduced light intensities and reduced O₂ tension that allow N₂ fixation to proceed in the unicellular Chroococcidiopsis at the gypsum rock, unless the activity is due to minute amounts of other, very active cyanobacteria. Phylogenetic analysis of nifH sequences tends to suggest that molybdenum nitrogenase 2 is characteristic of those unicellular or filamentous, nonheterocystous cyanobacteria fixing N₂ under microaerobic conditions only.     

Phylogenetic Diversity of Nitrogenase (nifH) Genes in Deep-Sea and Hydrothermal Vent Environments of the Juan de Fuca Ridge

The subseafloor microbial habitat associated with typical unsedimented mid-ocean-ridge hydrothermal vent ecosystems may be limited by the availability of fixed nitrogen, inferred by the low ammonium and nitrate concentrations measured in diffuse hydrothermal fluid. Dissolved N₂ gas, the largest reservoir of nitrogen in the ocean, is abundant in deep-sea and hydrothermal vent fluid. In order to test the hypothesis that biological nitrogen fixation plays an important role in nitrogen cycling in the subseafloor associated with unsedimented hydrothermal vents, degenerate PCR primers were designed to amplify the nitrogenase iron protein gene nifH from hydrothermal vent fluid. A total of 120 nifH sequences were obtained from four samples: a nitrogen-poor diffuse vent named marker 33 on Axial Volcano, sampled twice over a period of 1 year as its temperature decreased; a nitrogen-rich diffuse vent near Puffer on Endeavour Segment; and deep seawater with no detectable hydrothermal plume signal. Subseafloor nifH genes from marker 33 and Puffer are related to anaerobic clostridia and sulfate reducers. Other nifH genes unique to the vent samples include proteobacteria and divergent Archaea. All of the nifH genes from the deep-seawater sample are most closely related to the thermophilic, anaerobic archaeon Methanococcus thermolithotrophicus (77 to 83% amino acid similarity). These results provide the first genetic evidence of potential nitrogen fixers in hydrothermal vent environments and indicate that at least two sources contribute to the diverse assemblage of nifH genes detected in hydrothermal vent fluid: nifH genes from an anaerobic, hot subseafloor and nifH genes from cold, oxygenated deep seawater.     

The hindgut lumen prokaryotic microbiota of the termite Reticulitermes flavipes and its responses to dietary lignocellulose composition

Reticulitermes flavipes (Isoptera: Rhinotermitidae) is a highly eusocial insect that thrives on recalcitrant lignocellulosic diets through nutritional symbioses with gut‐dwelling prokaryotes and eukaryotes. In the R. flavipes hindgut, there are up to 12 eukaryotic protozoan symbionts; the number of prokaryotic symbionts has been estimated in the hundreds. Despite its biological relevance, this diverse community, to date, has been investigated only by culture‐ and cloning‐dependent methods. Moreover, it is unclear how termite gut microbiomes respond to diet changes and what roles they play in lignocellulose digestion. This study utilized high‐throughput 454 pyrosequencing of 16S V5‐V6 amplicons to sample the hindgut lumen prokaryotic microbiota of R. flavipes and to examine compositional changes in response to lignin‐rich and lignin‐poor cellulose diets after a 7‐day feeding period. Of the ~475 000 high‐quality reads that were obtained, 99.9% were annotated as bacteria and 0.11% as archaea. Major bacterial phyla included Spirochaetes (24.9%), Elusimicrobia (19.8%), Firmicutes (17.8%), Bacteroidetes (14.1%), Proteobacteria (11.4%), Fibrobacteres (5.8%), Verrucomicrobia (2.0%), Actinobacteria (1.4%) and Tenericutes (1.3%). The R. flavipes hindgut lumen prokaryotic microbiota was found to contain over 4761 species‐level phylotypes. However, diet‐dependent shifts were not statistically significant or uniform across colonies, suggesting significant environmental and/or host genetic impacts on colony‐level microbiome composition. These results provide insights into termite gut microbiome diversity and suggest that (i) the prokaryotic gut microbiota is much more complex than previously estimated, and (ii) environment, founding reproductive pair effects and/or host genetics influence microbiome composition.     

A comparative study of gut microbiota profiles of earthworms fed in three different substrates

The gut microbiome of earthworms has a complex interdependence with the host. When the soil minerals pass through earthworm’s gut, they may affect the gut microbiota. To gain insight into the response of gut microbiota to the passed minerals, we fed earthworm (Eisenia fetida) on nutrient-poor soil and ore powder, and used high throughput sequencing to characterize the earthworm intestinal microbial community to find evidence for a core bacterial community of the E. fetida. The results showed that earthworms’ gut maintained a core microbiome that appeared in all samples. These core microbiota may play a significant role in a species’ environmental interactions. The composition of intestinal microbiomes varied with substrates. The earthworm guts from two nutrient-poor substrates had similar microbial communities and they were different from nutrient-rich substrate. Proteobacteria and Bacteroidetes were more abundant in the gut of earthworms kept on a nutrient-poor substrate such as ore powder or mineral soil than in the gut of earthworms kept in organic-rich compost soil; some of these microorganisms may help earthworms survive in nutrient-poor substrates.     

Phylogeny of 16S rRNA and nifH genes and regulation of nitrogenase activity by oxygen and ammonium in the genus Paenibacillus

All Paenibacillus 16S rDNA sequences, except for that of Paenibacillus massiliensis T7, formed a coherent cluster, distinct from gram-positive nitrogen-fixing Clostridium pasteurianum and Heliobacterium chlorum. All Paenibacillus NifH sequences formed two main clusters. Cluster I encompassing the NifH sequences from most of members of Paenibacillus spp., such as Paenibacillus azotofixans NifH1 and NifH2, Paenibacillus polymyxa and Paenibacillus macerans. Cluster II including only P. azotofixans NifH3. Curiously, three copies of nifH genes of Paenibacillus sabine T27 clustered within P. azotofixans cluster I (NifH1 and NifH2). The effect of O₂ and ammonium on nitrogenase activity was studied with 14 different nitrogenfixing Paenibacillus strains. The optimal oxygen concentration level for all Paenibacillus strains is in the 0 to 0.05% range, similar to that for Klebsiella pneumoniae. In all Paenibacillus strains, the highest nitrogenase activity is obtained in the condition of 0?C0.1 mM NH₄Cl and the increase of NH₄Cl from 0.1 to 5 mM caused a rapid inhibition of nitrogenase activity. However, the inhibition was reversible in the presence of 200 mM NH₄Cl in some Paenibacillus strains. It is the first time to use almost all of the recognized nitrogen-fixing Paenibacilus spp. to investigate the phylogeny of 16S rRNA and nifH genes. The data that the inhibition of O₂ and ammonium on nitrogenase acitivity will provide a base for studying the molecular regulatory mechanism of nitrogen fixation in the genus Paenibacillus.     

Role of bacteria in maintaining the redox potential in the hindgut of termites and preventing entry of foreign bacteria

The hindgut of the lower termites, Mastotermes darwiniensis and Coptotermes lacteus and the higher termite Nasutitermes exitiosus were made aerobic by exposure of the termites to pure oxygen, a procedure which killed their spirochaetes and their protozoa (lower termites only). The time taken for the hindgut to become anaerobic after the termites were restored to normal atmospheric conditions ranged from 2 to 4.5 hr. After oxygen treatment the number of gut bacteria increased some six- to ten-fold in all termite species, indicating that the bacteria are poised to use oxygen entering the gut. Removal of all the hindgut microbiota by feeding tetracycline caused the hindgut to become aerobic in M. darwiniensis and N. exitiosus. The transferring of M. darwiniensis to fresh wood, free of antibiotic, resulted in the return of the normal flora and the eventual establishment of anaerobic conditions in the hindgut. Thus the bacteria appear to be important in maintaining anaerobic conditions in the gut. Attempts to determine whether the protozoa (in the lower termites) played any part in maintaining the Eh of the hindgut were unsuccessful. Serratia marcescens failed to colonise the gut of normal C. lacteus and transiently colonized (for 5 days) the gut of normal N. exitiosus. Transient colonization by S. marcescens (from 6 to 10 days) occurred in N. exitiosus when its hindgut spirochaetes were killed and in C. lacteus when its spirochaetes and protozoa were killed, indicating a possible role for the spirochaetes and/or protozoa in influencing the bacteria allowed to reside in the hindgut. Exposure of normal termites to Serratia provoked an increase in the numbers of the normal gut bacteria.     

Coliform Bacteria and Nitrogen Fixation in Pulp and Paper Mill Effluent Treatment Systems

The majority of pulp and paper mills now biotreat their combined effluents using activated sludge. On the assumption that their wood-based effluents have negligible fixed N, and that activated-sludge microorganisms will not fix significant N, these mills routinely spend large amounts adding ammonia or urea to their aeration tanks (bioreactors) to permit normal biomass growth. N₂ fixation in seven Eastern Canadian pulp and paper mill effluent treatment systems was analyzed using acetylene reduction assays, quantitative nitrogenase (nifH) gene probing, and bacterial isolations. In situ N₂ fixation was undetectable in all seven bioreactors but was present in six associated primary clarifiers. One primary clarifier was studied in greater detail. Approximately 50% of all culturable cells in the clarifier contained nifH, of which >90% were Klebsiella strains. All primary-clarifier coliform bacteria growing on MacConkey agar were identified as klebsiellas, and all those probed contained nifH. In contrast, analysis of 48 random coliform isolates from other mill water system locations showed that only 24 (50%) possessed the nifH gene, and only 13 (27%) showed inducible N₂-fixing activity. Thus, all the pulp and paper mill primary clarifiers tested appeared to be sites of active N₂ fixation (0.87 to 4.90 mg of N liter⁻¹ day⁻¹) and a microbial community strongly biased toward this activity. This may also explain why coliform bacteria, especially klebsiellas, are indigenous in pulp and paper mill water systems.     

Metagenomic Insights into Metabolic Capacities of the Gut Microbiota in a Fungus-Cultivating Termite (Odontotermes yunnanensis)

Macrotermitinae (fungus-cultivating termites) are major decomposers in tropical and subtropical areas of Asia and Africa. They have specifically evolved mutualistic associations with both a Termitomyces fungi on the nest and a gut microbiota, providing a model system for probing host-microbe interactions. Yet the symbiotic roles of gut microbes residing in its major feeding caste remain largely undefined. Here, by pyrosequencing the whole gut metagenome of adult workers of a fungus-cultivating termite (Odontotermes yunnanensis), we showed that it did harbor a broad set of genes or gene modules encoding carbohydrate-active enzymes (CAZymes) relevant to plant fiber degradation, particularly debranching enzymes and oligosaccharide-processing enzymes. Besides, it also contained a considerable number of genes encoding chitinases and glycoprotein oligosaccharide-processing enzymes for fungal cell wall degradation. To investigate the metabolic divergence of higher termites of different feeding guilds, a SEED subsystem-based gene-centric comparative analysis of the data with that of a previously sequenced wood-feeding Nasutitermes hindgut microbiome was also attempted, revealing that SEED classifications of nitrogen metabolism, and motility and chemotaxis were significantly overrepresented in the wood-feeder hindgut metagenome, while Bacteroidales conjugative transposons and subsystems related to central aromatic compounds metabolism were apparently overrepresented here. This work fills up our gaps in understanding the functional capacities of fungus-cultivating termite gut microbiota, especially their roles in the symbiotic digestion of lignocelluloses and utilization of fungal biomass, both of which greatly add to existing understandings of this peculiar symbiosis.     

Homogenisation of water and sediment bacterial communities in a shallow lake (lake Balihe,China)

1. Planktonic and benthic bacterial communities hold central roles in the functioning of freshwater ecosystems and mediate key ecosystem services such as primary production and nutrient remineralisation. Although it is clear that such communities vary in composition both within and between lakes, the environmental factors and processes shaping the diversity and composition of freshwater bacteria are still not fully understood.
2. In order to assess seasonal and spatial variability in lake bacterial communities and identify environmental factors underpinning biogeographical patterns, we performed a large-scale sampling campaign with paired water and sediment sample collection at 18 locations during four seasons in Lake Balihe, a subtropical shallow fish-farming lake in mid-eastern China.
3. Pelagic and benthic bacterial communities were distinctly different in terms of diversity, taxonomic composition and community structure, with Actinobacteria, Bacteroidetes, Cyanobacteria and Alphaproteobacteria dominating lake water, and Acidobacteria, Bacteroidetes, Chloroflexi, Gammaproteobacteria and Deltaproteobacteria dominating sediment. Nevertheless, these two communities had stronger spatial concordance and overlap in taxa during spring and autumn seasons. Together, the main drivers of both the spatial and temporal variations in Lake Balihe bacterial communities were identified as water temperature, turbidity, nitrogen and phosphorus availability, and thermal stratification controlled by wind-mixing and activity of the dense farmed fish populations. Notably, populations affiliated with Firmicutes, known to be abundant in fish gut microbiome, were especially abundant in the summer season and locations where high fish biomass was found, suggesting a potential link between fish gut microbiome and the pelagic bacterial communities.
4. Our findings demonstrated seasonal homogenisation of pelagic and benthic bacterial communities linked to marked shifts in a set of seasonally-driven environmental variables including water temperature and nutrient availability.

     

The Microbial Community in the Feces of the White Rhinoceros (Ceratotherium simum) as Determined by Barcoded Pyrosequencing Analysis

As a non-ruminant herbivore, the white rhinoceros has the ability to utilize fibrous plant matter through microbial fermentation in the hindgut. So far, there has been no report using molecular techniques to study the gut microbiota of the white rhinoceros. We used barcoded pyrosequencing to characterize 105,651 sequences of 16S rRNA genes obtained from fecal samples from five white rhinoceroses. Results showed that Firmicutes and Bacteroidetes were the predominant phyla in the samples, which were comprised largely of unclassified bacteria. The microbiota of one animal treated with drug therapy differed from those in other healthy animals, and was dominated by Aerococcus -related bacteria. The core microbiota in the healthy rhinoceros were dominated by phyla Firmicutes and Bacteroidetes, represented by the Ruminococcaceae, Lachnospiraceae, Rikenellaceae and Prevotellaceae families. The present work provides a phylogenetic framework for understanding the complex microbial community of the rhinoceros; however, further studies are required to link the distinctive microbiota with their digestive role in the hindgut of the white rhinoceros.     

Diversity of Nitrogen-Fixing Bacteria Associated with Switchgrass in the Native Tallgrass Prairie of Northern Oklahoma

Switchgrass (Panicum virgatum L.) is a perennial C₄ grass native to North America that is being developed as a feedstock for cellulosic ethanol production. Industrial nitrogen fertilizers enhance switchgrass biomass production but add to production and environmental costs. A potential sustainable alternative source of nitrogen is biological nitrogen fixation. As a step in this direction, we studied the diversity of nitrogen-fixing bacteria (NFB) associated with native switchgrass plants from the tallgrass prairie of northern Oklahoma (United States), using a culture-independent approach. DNA sequences from the nitrogenase structural gene, nifH, revealed over 20 putative diazotrophs from the alpha-, beta-, delta-, and gammaproteobacteria and the firmicutes associated with roots and shoots of switchgrass. Alphaproteobacteria, especially rhizobia, predominated. Sequences derived from nifH RNA indicated expression of this gene in several bacteria of the alpha-, beta-, delta-, and gammaproteobacterial groups associated with roots. Prominent among these were Rhizobium and Methylobacterium species of the alphaproteobacteria, Burkholderia and Azoarcus species of the betaproteobacteria, and Desulfuromonas and Geobacter species of the deltaproteobacteria.