Phylogenetic Analysis of Microbial Communities in Different Regions of the Gastrointestinal Tract in Panaque nigrolineatus,a Wood-Eating Fish

The Neotropical detritivorous catfish Panaque nigrolineatus imbibes large quantities of wood as part of its diet. Due to the interest in cellulose, hemi-cellulose and lignin degradation pathways, this organism provides an interesting model system for the detection of novel microbial catabolism. In this study, we characterize the microbial community present in different regions of the alimentary tract of P. nigrolineatus fed a mixed diet of date palm and palm wood in laboratory aquaria. Analysis was performed on 16S rRNA gene clone libraries derived from anterior and posterior regions of the alimentary tract and the auxiliary lobe (AL), an uncharacterized organ that is vascularly attached to the midgut. Sequence analysis and phylogenetic reconstruction revealed distinct microbial communities in each tissue region. The foregut community shared many phylotypes in common with aquarium tank water and included Legionella and Hyphomicrobium spp. As the analysis moved further into the gastrointestinal tract, phylotypes with high levels of 16S rRNA sequence similarity to nitrogen-fixing Rhizobium and Agrobacterium spp. and Clostridium xylanovorans and Clostridium saccharolyticum, dominated midgut and AL communities. However, the hindgut was dominated almost exclusively by phylotypes with the highest 16S rRNA sequence similarity to the Cytophaga-Flavobacterium-Bacteroides phylum. Species richness was highest in the foregut (Chao₁ = 26.72), decreased distally through the midgut (Chao₁ = 25.38) and hindgut (Chao₁ = 20.60), with the lowest diversity detected in the AL (Chao₁ = 18.04), indicating the presence of a specialized microbial community. Using 16S rRNA gene phylogeny, we report that the P. nigrolineatus gastrointestinal tract possesses a microbial community closely related to microorganisms capable of cellulose degradation and nitrogen fixation. Further studies are underway to determine the role of this resident microbial community in Panaque nigrolineatus.     

nifH Sequences and Nitrogen Fixation in Type I and Type II Methanotrophs

Some methane-oxidizing bacteria (methanotrophs) are known to be capable of expressing nitrogenase and utilizing N₂ as a nitrogen source. However, no sequences are available for nif genes in these strains, and the known nitrogen-fixing methanotrophs are confined mainly to a few genera. The purpose of this work was to assess the nitrogen-fixing capabilities of a variety of methanotroph strains. nifH gene fragments from four type I methanotrophs and seven type II methanotrophs were PCR amplified and sequenced. Nitrogenase activity was confirmed in selected type I and type II strains by acetylene reduction. Activities ranged from 0.4 to 3.3 nmol/min/mg of protein. Sequence analysis shows that the nifH sequences from the type I and type II strains cluster with nifH sequences from other gamma proteobacteria and alpha proteobacteria, respectively. The translated nifH sequences from three Methylomonas strains show high identity (95 to 99%) to several published translated environmental nifH sequences PCR amplified from rice roots and a freshwater lake. The translated nifH sequences from the type II strains show high identity (94 to 99%) to published translated nifH sequences from a variety of environments, including rice roots, a freshwater lake, an oligotrophic ocean, and forest soil. These results provide evidence for nitrogen fixation in a broad range of methanotrophs and suggest that nitrogen-fixing methanotrophs may be widespread and important in the nitrogen cycling of many environments.     

Diversity of <Emphasis Type="Italic">nifH</Emphasis> gene amplified from plankton-community DNA in a shallow eutrophic lake (Lake Donghu,Wuhan, China)

Biological nitrogen fixation (BNF) is one of the major nitrogen inputs into the biosphere, and the nitrogenase iron protein (nifH) gene plays important roles in regulating the molecular nitrogen (N₂) fixation process. The nifH gene has also been extensively used to study the diversity and function of nitrogen-fixing microorganisms. In this study, we investigated the diversity of the nifH gene by culture-independent methods to analysis the planktonic nitrogen-fixing organisms in Lake Donghu, Wuhan, the largest urban lake in China. Results indicate that nifH gene sequences cloned from planktonic-community DNA showed high similarity to the uncultured cyanobacterial sequences deposited in the GenBank database. Phylogenetic analysis on the basis of the translated amino acid sequences further showed that most nifH clones were closely related to the reported cyanobacterial nifH gene sequences. Results also indicate that there are similar planktonic nitrogen-fixing organisms in the relatively independent areas of Lake Donghu, even though different regions showed a wide gradient in trophic status. These and other observations led us to believe that studies on nifH gene diversity and expression will increase our ability to understand the ecological function of target nitrogen-fixing groups in aquatic ecosystems.     

A System of Oligonucleotide Primers for the Amplification of nifH Genes of Different Taxonomic Groups of Prokaryotes

Based on the analysis of the nifH gene nucleotide sequences from GenBank, a system of primers was developed that makes it possible to obtain 370- and 470-bp PCR fragments of the nifH gene of nitrogen-fixing bacteria and archaea. The effectiveness of the proposed system for revealing the presence of nifH genes was demonstrated by PCR on the DNA isolated from nitrogen-fixing prokaryotes for which the primary structure of these genes is known and which belong to different taxonomic groups. nifH sequences of nitrogen-fixing prokaryotes of the genera Xanthobacter, Beijerinckia, and Methanosarcina, for which the capacity for nitrogen fixation was demonstrated earlier, but no data existed on the nucleotide composition of these genes, were determined and deposited in GenBank.     

Inside the guts of wood-eating catfishes: can they digest wood?

To better understand the structure and function of the gastrointestinal (GI) tracts of wood-eating catfishes, the gross morphology, length, and microvilli surface area (MVSA) of the intestines of wild-caught Panaque nocturnus, P. cf. nigrolineatus “Marañon”, and Hypostomus pyrineusi were measured, and contrasted against these same metrics of a closely related detritivore, Pterygoplichthys disjunctivus. All four species had anatomically unspecialized intestines with no kinks, valves, or ceca of any kind. The wood-eating catfishes had body size-corrected intestinal lengths that were 35% shorter than the detritivore. The MVSA of all four species decreased distally in the intestine, indicating that nutrient absorption preferentially takes place in the proximal and mid-intestine, consistent with digestive enzyme activity and luminal carbohydrate profiles for these same species. Wild-caught Pt. disjunctivus, and P. nigrolineatus obtained via the aquarium trade, poorly digested wood cellulose (<33% digestibility) in laboratory feeding trials, lost weight when consuming wood, and passed stained wood through their digestive tracts in less than 4 h. Furthermore, no selective retention of small particles was observed in either species in any region of the gut. Collectively, these results corroborate digestive enzyme activity profiles and gastrointestinal fermentation levels in the fishes’ GI tracts, suggesting that the wood-eating catfishes are not true xylivores such as beavers and termites, but rather, are detritivores like so many other fishes from the family Loricariidae.     

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.     

New Nitrogen-Fixing Microorganisms Detected in Oligotrophic Oceans by Amplification of Nitrogenase (nifH) Genes

Oligotrophic oceanic waters of the central ocean gyres typically have extremely low dissolved fixed inorganic nitrogen concentrations, but few nitrogen-fixing microorganisms from the oceanic environment have been cultivated. Nitrogenase gene (nifH) sequences amplified directly from oceanic waters showed that the open ocean contains more diverse diazotrophic microbial populations and more diverse habitats for nitrogen fixers than previously observed by classical microbiological techniques. Nitrogenase genes derived from unicellular and filamentous cyanobacteria, as well as from the α and γ subdivisions of the class Proteobacteria, were found in both the Atlantic and Pacific oceans. nifH sequences that cluster phylogenetically with sequences from sulfate reducers or clostridia were found associated with planktonic crustaceans. Nitrogenase sequence types obtained from invertebrates represented phylotypes distinct from the phylotypes detected in the picoplankton size fraction. The results indicate that there are in the oceanic environment several distinct potentially nitrogen-fixing microbial assemblages that include representatives of diverse phylotypes.The productivity of the oceans controls the fluxes of many biogeochemically important compounds, including the rate of exchange of carbon dioxide between the open ocean and the atmosphere. In turn, oceanic carbon fixation is limited by the bioavailability of nutrients, including nitrogen, phosphorus, and iron (9, 10, 20). In contrast to the biogeochemical cycles of phosphorus and iron, nitrogen is present in relatively high concentrations in seawater as gaseous N₂. Gaseous nitrogen is available only to microorganisms with the capability of biological nitrogen fixation, the reduction of atmospheric N₂ to ammonium. Although large areas of the world’s oceans are virtually devoid of fixed dissolved inorganic nitrogen and primary production may be nitrogen limited, very few species of nitrogen-fixing organisms have been identified or isolated from the plankton. Trichodesmium, a filamentous aggregate-forming cyanobacterium, is an abundant diazotroph in tropical and subtropical waters (3, 5), but few other examples of diazotrophs from the open ocean are known (21, 35). The seeming low diversity of known nitrogen-fixing organisms in the open ocean stands in stark contrast to the presumptive nitrogen limitation in the world’s oceans and presents an evolutionary paradox.Recently, biological nitrogen fixation has gained recognition as an important source of nitrogen for supporting oceanic primary production (3, 11, 18, 22). The nitrogen budget for the Atlantic Ocean does not balance because a source of nitrogen cannot be accounted for by current knowledge of fluxes and pools of nitrogen, even after including nitrogen fixation by Trichodesmium (22). It is speculated that rates of nitrogen fixation by known diazotrophic organisms have been underestimated (17), or as yet unidentified diazotrophic organisms are active in the ocean (18). Conventional nitrogenase, the enzyme that catalyzes biological dinitrogen reduction to ammonium, is composed of two highly conserved proteins: the iron (Fe) protein (encoded by the nifH gene) and the molybdenum iron (MoFe) protein (encoded by the nifDK genes). The nitrogenase enzyme is present in diverse lineages of prokaryotes and is generally believed to be ancient (38). Evolutionarily conserved amino acid sequences within the nifH (which encodes the Fe protein component of nitrogenase) gene have been exploited to design PCR primers to detect the genetic potential for nitrogen fixation in the marine environment (39). With this approach, the diversity of nitrogen-fixing microorganisms in oceanic water and marine plankton was determined. This report shows that there are far more diverse nitrogen-fixing populations and diverse habitats which can support nitrogen fixation in the open ocean than previously documented.     

Environment-Dependent Distribution of the Sediment nifH-Harboring Microbiota in the Northern South China Sea

The South China Sea (SCS), the largest marginal sea in the Western Pacific Ocean, is a huge oligotrophic water body with very limited influx of nitrogenous nutrients. This suggests that sediment microbial N₂ fixation plays an important role in the production of bioavailable nitrogen. To test the molecular underpinning of this hypothesis, the diversity, abundance, biogeographical distribution, and community structure of the sediment diazotrophic microbiota were investigated at 12 sampling sites, including estuarine, coastal, offshore, deep-sea, and methane hydrate reservoirs or their prospective areas by targeting nifH and some other functional biomarker genes. Diverse and novel nifH sequences were obtained, significantly extending the evolutionary complexity of extant nifH genes. Statistical analyses indicate that sediment in situ temperature is the most significant environmental factor influencing the abundance, community structure, and spatial distribution of the sediment nifH-harboring microbial assemblages in the northern SCS (nSCS). The significantly positive correlation of the sediment pore water NH₄⁺ concentration with the nifH gene abundance suggests that the nSCS sediment nifH-harboring microbiota is active in N₂ fixation and NH₄⁺ production. Several other environmental factors, including sediment pore water PO₄³⁻ concentration, sediment organic carbon, nitrogen and phosphorus levels, etc., are also important in influencing the community structure, spatial distribution, or abundance of the nifH-harboring microbial assemblages. We also confirmed that the nifH genes encoded by archaeal diazotrophs in the ANME-2c subgroup occur exclusively in the deep-sea methane seep areas, providing for the possibility to develop ANME-2c nifH genes as a diagnostic tool for deep-sea methane hydrate reservoir discovery.     

Diazotrophic diversity in the Caribbean coral, Montastraea cavernosa

Previous research on the Caribbean coral Montastraea cavernosa reported the presence of cyanobacterial endosymbionts and nitrogen fixation in orange, but not brown, colonies. We compared the diversity of nifH gene sequences between these two color morphs at three locations in the Caribbean and found that the nifH sequences recovered from M. cavernosa were consistent with previous studies on corals where members of both the α-proteobacteria and cyanobacteria were recovered. A number of nifH operational taxonomic units (OTUs) were significantly more abundant in the orange compared to the brown morphs, and one specific OTU (OTU 17), a cyanobacterial nifH sequence similar to others from corals and sponges and related to the cyanobacterial genus Cyanothece, was found in all orange morphs of M. cavernosa at all locations. The nifH diversity reported here, from a community perspective, was not significantly different between orange and brown morphs of M. cavernosa.     

Diversity of nifH Genotypes in Floating Periphyton Mats Along a Nutrient Gradient in the Florida Everglades

Periphyton mats are an important component of many wetland ecosystems, performing a range of vital ecosystem functions, including nitrogen fixation. The composition and integrity of these mats are affected by nutrient additions, which might result in changes in their function. The overall objective of this study was to investigate the distribution of nifH sequences in floating periphyton mats collected along a nutrient gradient in the Florida Everglades. Distribution of nifH clone libraries indicated nutrient enrichment selected primarily for sequences branching deeply within the heterocystous cyanobacteria and within a novel group of cyanobacteria; sequences from low-nutrient sites were broadly distributed, with no clear dominance of sequences associated with heterocystous and nonheterocystous cyanobacteria and alpha-, gamma-, and delta-proteobacteria. The dominance of heterocystous cyanobacteria in nutrient-enriched sites and the lack of clear dominance by heterocystous cyanobacteria is consistent with previously reported diurnal cycles of nitrogen fixation rates in these systems. Sequences clustering with those harbored by methanotrophs were also identified; sequences from nutrient-impacted and transition regions clustered with those characteristic of type II methanotrophs, and sequences from oligotrophic regions clustered with type I methanotrophs.     

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.     

Diversity of diazotrophic gut inhabitants of pikas (Ochotonidae) revealed by PCR-DGGE analysis

Diazotrophic gut symbionts are considered to act as nitrogen providers for their hosts, as was shown for various termite species. Although the diet of lagomorphs, like pikas or rabbits, is very poor in nitrogen and energy, their fecal matter contains 30–40% of protein. Since our hypothesis was that pikas maintained a diazotrophic consortium in their gastrointestinal tract, we conducted the first investigation of microbial diversity in pika guts. We obtained gut samples from animals of several Ochotona species, O. hyperborea (Northern pika), O. mantchurica (Manchurian pika), and O. dauurica (Daurian pika), in order to retrieve and compare the nitrogen-fixing communities of different pika species. The age and gender of the animals were taken into consideration. We amplified 320-bp long fragments of the nifH gene using the DNA extracted directly from the colon and cecum samples of pika’s gut, resolved them by DGGE, and performed phylogenetic reconstruction of 51 sequences obtained from excised bands. No significant difference was detected between the nitrogen-fixing gut inhabitants of different pika species. NifH sequences fell into two clusters. The first cluster contained the sequences affiliated with NifH Cluster I (Zehr et al., 2003) with similarity to Sphingomonas sp., Bradyrhizobium sp., and various uncultured bacteria from soil and rhizosphere. Sequences from the second group were related to Treponema sp., Fibrobacter succinogenes, and uncultured clones from the guts of various termites and belonged to NifH Cluster III. We suggest that diazotrophic organisms from the second cluster are genuine endosymbionts of pikas and provide nitrogen for further synthesis processes thus allowing these animals not to be short of protein.     

Diazotrophic diversity, nifH gene expression and nitrogenase activity in a rice paddy field in Fujian, China

The diazotrophic communities in a rice paddy field were characterized by a molecular polyphasic approach including DNA/RNA-DGGE fingerprinting, real time RT-PCR analysis of nifH gene and the measurement of nitrogen fixation activities. The investigation was performed on a diurnal cycle and comparisons were made between bulk and rhizosphere / root soil as well as between fertilized / unfertilized soils. Real time RT-PCR showed no significant difference in the total quantity of nifH expression under the conditions investigated. The functional diversity and dynamics of the nifH gene expressing diazotroph community investigated using RT-PCR-DGGE revealed high diurnal variations, as well as variation between different soil types. Most of the sequence types recovered from the DGGE gels and clone libraries clustered within nifH Cluster I and III (65 different nifH sequences in total). Sequence types most similar to Azoarcus spp., Metylococcus spp., Rhizobium spp., Methylocystis spp., Desulfovibrio spp., Geobacter spp., Chlorobium spp., were abundant and indicate that these species may be responsible for the observed diurnal variation in the diazotrophic community structure in these rice field samples. Previously described diazotrophic cyanobacterial genera in rice fields, such as Nostoc and Cyanothece, were present in the samples but not detectable in RT-PCR assays.     

Microbial diversity and diazotrophy associated with the freshwater non-heterocyst forming cyanobacterium Lyngbya robusta

Lake Atitlan, Guatemala, a freshwater lake in South America, experiences annually recurring blooms comprised of the planktic filamentous cyanobacterium Lyngbya robusta. Previous physiochemical characterisation of the bloom identified diurnal nitrogenase activity typical of non-heterocystous cyanobacteria, in addition to the low-level detection of the cyanotoxins cylindrospermopsin and saxitoxin. A molecular approach, combining deep sequencing of the 16S rRNA and nifH genes, was applied to a cyanobacteria-dominated sample collected during the extensive 2009 bloom. Lyngbya accounted for over 60 % of the total 16S rRNA sequences with the only other cyanobacterial species detected being the picophytoplankton Synechococcus. The remaining bacterial population was comprised of organisms typical of other eutrophic freshwater bodies, although the proportionate abundances were atypical. An obligate anaerobe Opitutus, not typically found in freshwater systems, was identified within the community which suggests it may have a role in enhancing nitrogen fixation. Primary nitrogen fixation was attributed to Lyngbya, with other putative nitrogen fixers, Desulfovibrio, Clostridium and Methylomonas, present at very low abundance.     

Evaluation of the diversity of nitrogen-fixing bacteria in soybean rhizosphere by <Emphasis Type="Italic">nifH</Emphasis> gene analysis

Biological nitrogen fixation plays an important role in the nitrogen balance of agricultural ecosystems and provides an essential part of nitrogen nutrition for plants, even in conditions of intensive fertilization. The main agrobiotechnological method for soybean cultivation (Glycine max (L.) Merril) is an application of microbial preparations based on Bradyrhizobium japonicum. Successful inoculation strongly depends on the interactions between the introduced microorganism and the aboriginal rhizosphere microorganisms. To evaluate the composition of diazotrophic communities, a study of the diversity of the molecular marker for nitrogen fixation, the nifH gene, in the samples of soybean rhizosphere soil was carried out. Experiments were performed in the variants when soybean was cultivated without inoculation and after adding bacterial preparations, as well as in enrichment cultures of diazotrophs. The revealed diazotrophic microorganisms demonstrated low level of similarity to the known microorganisms (74–95% identity by nucleotides), and were identified as species of the phyla Firmicutes and Proteobacteria. In the composition of nitrogen-fixing communities in the rhizosphere soil, the microorganisms of the genera Clostridium, Paenibacillus, and Spirochaeta were shown to prevail.