nifH Fragments Research Articles

Identification of Cultured and Diazotrophic Bacterial Endophytes in Warm-Season Grasses

Endophytes can have positive effects on plant health and growth, but endophytes of warm-season grasses are largely understudied, and inocula are rarely applied to cultivated grasses. To identify endophytes in warm-season grasses, 35 endophytic bacterial isolates were cultured from the roots, rhizomes, and shoots of bermudagrass ( Cynodon dactylon), energy cane ( Saccharum spp.), Johnsongrass ( Sorghum halepense), napiergrass ( Cenchrus purpureus), perennial sorghum ( Sorghum bicolor × S. halepense), sorghum ( Sorghum bicolor), sorghum × sudangrass ( Sorghum × drummondii), and peanut (outgroup). Sequencing of the 16S rRNA fragment from the endophytes revealed that the bacterial sequences were similar to Bacillus spp. (19 isolates), Burkholderia spp. (4), Pantoea spp. (4), Pseudomonas spp. (3), Enterobacter spp. (2), Kosakonia spp . (2), and Sphingomonas sp. (1). To identify diazotrophic endophytes, DNA isolated from surface-disinfected tissue from warm-season grasses was used to amplify nifH. Bacteria containing nifH were similar to 13 genera, and sequences similar to Pseudolabrys sp. were present in the greatest number of warm-season grasses. Bacteria similar to Bradyrhizobium frederickii strain CNPSo 3447 were identified frequently from the leaves and roots of sorghum × sudangrass (and peanut roots). Using similarity to known nifH fragments, six genera were identified that had not been previously identified in grasses. Thus, a large number of endophytes were found in warm-season grasses and could enhance plant growth or grass nitrogen levels by using nitrogen fixation. [Formula: see text] The author(s) have dedicated the work to the public domain under the Creative Commons CC0 “No Rights Reserved” license by waiving all of his or her rights to the work worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law, 2023.

A Nodulation-Proficient Nonrhizobial Inhabitant of Pueraria phaseoloides.

Pueraria phaseoloides is a legume cover crop, found chiefly in the wet zone of Sri Lanka. Nitrogen fixation is performed by nodular inhabitants of this cover crop, comparable to the nodule-dwelling bacteria of most other legume plants. We isolated a bacterium (Sub1) from Pueraria phaseoloides, of coccobacillus cell shape, that showed nodulation, when assessed by hydroponics, showing nodules as early as 3 weeks after reinfection. When a nifH fragment from the genome of this bacterium was amplified using a pair of nifH primers, it yielded an amplicon of 360 bp that, when sequenced, helped us identify the bacterium, as belonging to a species of Pseudacidovorax intermedius, at 99% sequence identity. When we constructed a phylogenetic tree with neighboring sequences, we encountered nifH sequences of Pseudacidovorax, forming a monophyletic cluster, which too contained a single Azospirillum species. The genus Pseudacidovorax is a bacterium that, so far, has not been associated with legume nodules. Sub1 secreted a pair of enzymes to the extracellular medium to degrade cellulose and milk proteins. The Sub1 bacterium showed biofilm formation and secreted into the extracellular medium, indole acetic acid. Sub1 also showed a “bulls eye” swarming pattern for the chemoattractant proline, while showing no significant chemotaxis movement, for naringenin, quercetin, and glutamate. Sub1 too possesses the basic genetic foundation (nifH and nifD) to produce a molybdenum-dependent nitrogenase enzyme. We finally show that this rare nonrhizobial bacterium is able to impact, positively, nodulation and shoot length of Pueraria plants, demonstrating that this beta-proteobacterium can abet the biological vigor of this legume cover crop.

Distinct ecological niches of marine symbiotic N2 -fixing cyanobacterium Candidatus Atelocyanobacterium thalassa sublineages.

A recently described symbiosis between the metabolically streamlined nitrogen-fixing cyanobacterium UCYN-A and a single-celled eukaryote prymnesiophyte alga is widely distributed throughout tropical and subtropical marine waters, and is thought to contribute significantly to nitrogen fixation in these regions. Several UCYN-A sublineages have been defined based on UCYN-A nitrogenase (nifH) sequences. Due to the low abundances of UCYN-A in the global oceans, currently existing molecular techniques are limited for detecting and quantifying these organisms. A targeted approach is needed to adequately characterize the diversity of this important marine cyanobacterium, and to advance understanding of its ecological importance. We present findings on the distribution of UCYN-A sublineages based on high throughput sequencing of UCYN-A nifH PCR amplicons from 78 samples distributed throughout many major oceanic provinces. These UCYN-A nifH fragments were used to define oligotypes, alternative taxonomic units defined by nucleotide positions with high variability. The data set was dominated by a single oligotype associated with the UCYN-A1 sublineage, consistent with previous observations of relatively high abundances in tropical and subtropical regions. However, this analysis also revealed for the first time the widespread distribution of the UCYN-A3 sublineage in oligotrophic waters. Furthermore, distinct assemblages of UCYN-A oligotypes were found in oligotrophic and coastally influenced waters. This unique data set provides a framework for determining the environmental controls on UCYN-A distributions and the ecological importance of the different sublineages.

Active nitrogen-fixing heterotrophic bacteria at and below the chemocline of the central Baltic Sea

The Baltic Sea receives large nitrogen inputs by diazotrophic (N2-fixing) heterocystous cyanobacteria but the significance of heterotrophic N2 fixation has not been studied. Here, the diversity, abundance and transcription of the nifH fragment of the nitrogenase enzyme in two basins of the Baltic Sea proper was examined. N2 fixation was measured at the surface (5 m) and in anoxic water (200 m). Vertical sampling profiles of >10 and <10 μm size fractions were collected in 2007, 2008 and 2011 at the Gotland Deep and in 2011 in the Bornholm Basin. Both of these stations are characterized by permanently anoxic bottom water. The 454-pyrosequencing nifH analysis revealed a diverse assemblage of nifH genes related to alpha-, beta- and gammaproteobacteria (nifH cluster I) and anaerobic bacteria (nifH cluster III) at and below the chemocline. Abundances of genes and transcripts of seven diazotrophic phylotypes were investigated using quantitative polymerase chain reaction revealing abundances of heterotrophic nifH phylotypes of up to 2.1 × 10(7) nifH copies l(-1). Abundant nifH transcripts (up to 3.2 × 10(4) transcripts l(-1)) within nifH cluster III and co-occurring N2 fixation (0.44±0.26 nmol l(-1) day(-1)) in deep water suggests that heterotrophic diazotrophs are fixing N2 in anoxic ammonium-rich waters. Our results reveal that N2 fixation in the Baltic Sea is not limited to illuminated N-deplete surface waters and suggest that N2 fixation could also be of importance in other suboxic regions of the world's oceans.

Effect of plant growth promoting rhizobia on seed germination, growth promotion and suppression of Fusarium wilt of fenugreek ( Trigonella foenum-graecum L.)

Five bacterial strains (TR1 to TR5) isolated from root nodules of fenugreek ( Trigonella foenum-graecum) were tested for their plant growth promotory traits and biocontrol potential against Fusarium oxysporum. On the basis of morphological, physiological, biochemical and molecular characteristics, strains TR1 and TR3 – TR5 were identified as Ensifer meliloti, and TR2 as Rhizobium leguminosarum. All bacterial isolates utilized phosphate in vitro. Except TR5, all isolates produced IAA and none of them showed volatile cyanogens production. Except TR3, all isolates produced in vitro siderophore. Isolate TR1 and TR4 showed chitinase production while only TR2 showed β-1,3-glucanase activity. Isolates TR1, TR2 and TR5 exhibited ACC deaminase activity. Isolates TR1, TR2 and TR4 inhibited the growth of F. oxysporum, causing loss of structural integrity of the mycelium, hyphal perforation, lysis, fragmentation and degradation. The potential for nodulation and nitrogen fixation of the strains were confirmed by amplification of 500 bp nodC and 781 bp nifH fragments. The application of the TR1 + TR2 combination resulted in increased grain yield by 35% and 36% of fenugreek in two consecutive field trials, respectively as compared to control. Maximum increments in vigour index, nodule number and root and shoot biomass were recorded with seed inoculated with consortium (TR1 + TR2) followed by single inoculation as compared to control. The antibiotic resistant marker strain of E. meliloti TR1 strep+ and R. leguminosarum TR2 tet+ confirmed the efficient colonization of fenugreek roots. This study showed that these rhizobial isolates have properties of biocontrol agents and may be applied to promote the growth of fenugreek.

Occurrence and diversity of nitrogen-fixingSphingomonasbacteria associated with rice plants grown in Brazil

So far, the occurrence of nitrogen-fixing Sphingomonas bacteria has been restricted to three strains of Sphingomonas azotifigens. In this work, a group of 46 Sphingomonas-like isolates, which originated from two rice varieties grown in two soils in Brazil, were characterized based on morphological, physiological and genetic analyses. The PCR genus specifically applied indicated that all 46 isolates belonged to the Sphingomonas genus and confirmed the results based on the yellow pigment of the colonies grown on potato agar medium and the BIOLOG data. It was also observed that 22 isolates are nitrogen-fixing bacteria as determined by the acetylene reduction method and confirmed by nifH gene detection. The genetic diversity based on the 16S rRNA analysis (amplified rDNA restriction analysis) showed that the isolates formed two distinct groups at a similarity value of 60%. Furthermore, five clusters at 60% similarity were observed with the 16S-23S intergenic space (ribosomal intergenic space analysis) analysis. Sequencing of the 16S rRNA gene and nifH fragments showed that most of the 22 nitrogen-fixing isolates formed clusters apart from that of the S. azotifigens. This is the first report on the occurrence of nitrogen-fixing Sphingomonas bacteria associated with rice grown in Brazil.

Prevalence of Betaproteobacterial Sequences in nifH Gene Pools Associated with Roots of Modern Rice Cultivars

The diversity and function of nitrogen-fixing bacteria colonizing rice roots are not well understood. A field experiment was conducted to determine the diversity of diazotrophic communities associated with roots of modern rice cultivars using culture-independent molecular analyses of nitrogenase gene (nifH) fragments. Experimental treatments included four modern rice cultivars (Oryza sativa, one Indica, one Japonica and two hybrid rice varieties) and three levels (0, 50, and 100 kg N ha(-1)) of N (urea) fertilizer application. Cloning and sequencing of 103 partial nifH genes showed that a diverse community of diazotrophs was associated with rice roots. However, the nifH gene fragments belonging to betaproteobacteria were dominant, accounting for nearly half of nifH sequences analyzed across the clone libraries. Most of them were similar to nifH fragments retrieved from wild rice and Kallar grass, with Azoarcus spp. being the closest cultured relatives. Alphaproteobacteria were also detected, but their relative abundance in the nifH gene pools was dramatically decreased with N fertilizer application. In addition, a high fraction of nifH gene pools was affiliated with methylotrophs and methane oxidizers. The sequence analysis was consistent with the terminal restriction fragment-length polymorphism (T-RFLP) fingerprinting of the nifH gene fragments, which showed three of four dominant terminal restriction fragments were mainly related to betaproteobacteria based on in silico digestion of nifH sequences. T-RFLP analyses also revealed that the effects of N fertilizer on the nifH gene diversity retrieved from roots varied according to rice cultivars. In summary, the present study revealed the prevalence of betaproteobacterial sequences among the proteobacteria associated with roots of modern rice cultivars. This group of diazotrophs appeared less sensitive to N fertilizer application than diazotrophic alphaproteobacteria. Furthermore, methylotrophs may also play a role in nitrogen fixation on rice roots. However, it must be noted that due to the potential bias of polymerase chain reaction protocol, the significance of non-proteobacterial diazotrophs such as Firmicutes and anaerobic bacteria is possibly underestimated.

Diversity ofnifHgene pools in the rhizosphere of two cultivars of sorghum (Sorghum bicolor) treated with contrasting levels of nitrogen fertilizer

The diversity of nitrogen-fixing bacteria was assessed in the rhizospheres of two cultivars of sorghum (IS 5322-C and IPA 1011) sown in Cerrado soil amended with two levels of nitrogen fertilizer (12 and 120 kg ha(-1)). The nifH gene was amplified directly from DNA extracted from the rhizospheres, and the PCR products cloned and sequenced. Four clone libraries were generated from the nifH fragments and 245 sequences were obtained. Most of the clones (57%) were closely related to nifH genes of uncultured bacteria. NifH clones affiliated with Azohydromonas spp., Ideonella sp., Rhizobium etli and Bradyrhizobium sp. were found in all libraries. Sequences affiliated with Delftia tsuruhatensis were found in the rhizosphere of both cultivars sown with high levels of nitrogen, while clones affiliated with Methylocystis sp. were detected only in plants sown under low levels of nitrogen. Moreover, clones affiliated with Paenibacillus durus could be found in libraries from the cultivar IS 5322-C sown either in high or low amounts of fertilizer. This study showed that the amount of nitrogen used for fertilization is the overriding determinative factor that influenced the nitrogen-fixing community structures in sorghum rhizospheres cultivated in Cerrado soil.

Phylogeny and evolution of the family Ectothiorhodospiraceae based on comparison of 16S rRNA, cbbL and nifH gene sequences

The occurrence of genes encoding nitrogenase and ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) was investigated in the members of the family Ectothiorhodospiraceae. This family forms a separate phylogenetic lineage within the Gammaproteobacteria according to 16S rRNA gene sequence analysis and mostly includes photo- and chemoautotrophic halophilic and haloalkaliphilic bacteria. The cbbL gene encoding the large subunit of 'green-like' form I RubisCO was found in all strains, except the type strains of Alkalispirillum mobile and Arhodomonas aquaeolei. The nifH gene encoding nitrogenase reductase was present in all investigated species of the phototrophic genera Ectothiorhodospira, Halorhodospira and Thiorhodospira, but not of the genus Ectothiorhodosinus. Unexpectedly, nifH fragments were also obtained for the chemotrophic species Thioalkalispira microaerophila and Alkalilimnicola halodurans, for which diazotrophic potential has not previously been assumed. The cbbL-, nifH- and 16S rRNA gene-based trees were not highly congruent in their branching patterns since, in the 'RubisCO' and 'nitrogenase' trees, representatives of the Ectothiorhodospiraceae are divided in a number of broadly distributed clusters and branches. However, the data obtained may be regarded as evidence of the monophyletic origin of the cbbL and nifH genes in most species within the family Ectothiorhodospiraceae and mainly corresponded to the current taxonomic structure of this family. The cbbL phylogeny of the chemolithoautotrophic sulfur-oxidizers Thioalkalivibrio nitratireducens and Thioalkalivibrio paradoxus and the nitrifier Nitrococcus mobilis deviated significantly from the 16S-rRNA gene-based phylogeny. These species clustered with one of the duplicated cbbL genes of the purple sulfur bacterium Allochromatium vinosum, a member of the family Chromatiaceae.

Diazotrophic bacterioplankton in a coral reef lagoon: phylogeny, diel nitrogenase expression and response to phosphate enrichment

We investigated diazotrophic bacterioplankton assemblage composition in the Heron Reef lagoon (Great Barrier Reef, Australia) using culture-independent techniques targeting the nifH fragment of the nitrogenase gene. Seawater was collected at 3 h intervals over a period of 72 h (i.e. over diel as well as tidal cycles). An incubation experiment was also conducted to assess the impact of phosphate (PO(4)3*) availability on nifH expression patterns. DNA-based nifH libraries contained primarily sequences that were most similar to nifH from sediment, microbial mat and surface-associated microorganisms, with a few sequences that clustered with typical open ocean phylotypes. In contrast to genomic DNA sequences, libraries prepared from gene transcripts (mRNA amplified by reverse transcription-polymerase chain reaction) were entirely cyanobacterial and contained phylotypes similar to those observed in open ocean plankton. The abundance of Trichodesmium and two uncultured cyanobacterial phylotypes from previous studies (group A and group B) were studied by quantitative-polymerase chain reaction in the lagoon samples. These were detected as transcripts, but were not detected in genomic DNA. The gene transcript abundance of these phylotypes demonstrated variability over several diel cycles. The PO(4)3* enrichment experiment had a clearer pattern of gene expression over diel cycles than the lagoon sampling, however PO(4)3* additions did not result in enhanced transcript abundance relative to control incubations. The results suggest that a number of diazotrophs in bacterioplankton of the reef lagoon may originate from sediment, coral or beachrock surfaces, sloughing into plankton with the flooding tide. The presence of typical open ocean phylotype transcripts in lagoon bacterioplankton may indicate that they are an important component of the N cycle of the coral reef.

A nifH-based Oligonucleotide Microarray for Functional Diagnostics of Nitrogen-fixing Microorganisms

Nitrogen fixation is an important process in biogeochemical cycles exclusively carried out by prokaryotes, mostly by an evolutionarily conserved nitrogenase protein complex, of which one of the structural genes (nifH) is highly valuable for phylogenetic and diversity analyses. We developed a nifH-based short oligonucleotide microarray (nifH diagnostic microarray) as a rapid tool to effectively monitor nitrogen-fixing diazotrophic populations in a wide range of environments. Taking account of the overwhelming predominance of environmental nifH fragments from uncultivated microorganisms in public databases, our nifH microarray is mainly based on nifH sequences from as yet unidentified prokaryotes. Standard conditions for microarray performance were determined, and criteria for the design of specific oligonucleotides were defined. A primary set of 56 oligonucleotides was validated with fluorescence-labeled single-stranded nifH targets from five reference strains, 26 environmental clones, and artificial mixtures of reference strains. The nifH microarray was applied to analyze the diversity (based on DNA) and activity (based on mRNA) of diazotrophs in roots of wild rice samples from Namibia. Results demonstrated that only a small subset of diazotrophs being present in the sample were actually fixing nitrogen actively. Our data suggest that the developed nifH microarray is a highly reproducible and semiquantitative method for mapping the variability of diazotrophic diversity, allowing rapid comparisons of the relative abundance and activity of diazotrophic prokaryotes in the environment. A further refined nifH microarray comprising of 194 oligonucleotide probes now covers more than 90% of sequences in our nifH database.

Cloning and sequencing of nifBHDKENX genes of Paenibacillus massiliensis T7 and its nif promoter analysis

A 324 bp of nifH fragment was PCR amplified from Paenibacillus massiliensis T7 using the universal degenerate primers. The PCR-amplified nifH fragment was labeled with DIG and then used as a probe in Southern blot analysis. Southern blot result showed that there were two positive signals, indicating that there might be two copies of nifH in P. massiliensis T7. A total of 10254 bp DNA sequence containing purD and nifBHDKENX was obtained by five rounds of inverse-PCR amplification. The predicted proteins of nifBHDKENX had high homology with those from other nitrogen-fixing bacteria. Only one putative sigma54-dependent promoter sequence was detected upstream of the nifB gene and nifBHDKENX were likely to be organized in one operon. Assays of 3-galactosidase activity of P. massiliensis T7PB carrying a nifB-lacZ fusion under different concentrations of NH4+ and O2 showed that the expression of nifB-lacZ was strongly inhibited by O2.

Occurrence and distribution of nitrogen fixing bacterial community associated with oat ( Avena sativa) assessed by molecular and microbiological techniques

Molecular and microbiological techniques were used to evaluate the nitrogen fixing bacterial community associated with oat ( Avena sativa) in different soil management systems of Rio Grande do Sul State. The conventional microbiological approach made use of semi-solid nitrogen-free and solid selective media to isolate some of these nitrogen fixing bacteria. For the molecular approach, a PCR-RFLP strategy was adopted to amplify a segment of the nifH gene, which encodes nitrogenase reductase, from DNA samples extracted from rhizosphere soils, roots and leaves of oat. The amplified nifH fragments were cleaved with TaqI and HaeIII endonucleases and the products analyzed in polyacrylamide gels. One sample was analyzed in more detail. From this site rhizosphere soil and leaf samples PCR fragments were cloned into pUC18, generating 55 nifH positive clones, from which 19 different RFLP patterns were obtained. These patterns were compared with RFLP patterns generated from DNA obtained from pure strains or by theoretical digestion data. To identify and confirm the RFLP patterns obtained, at least one clone of each was sequenced and the nucleotide sequences were compared with the GenBank nucleotide database. As the partial nifH sequences analyzed had shown homologies with several known and unknown diazotrophs, we attempted to identify the diazotrophs through the positioning of the clones in the NifH partial sequence-derived tree. Through the molecular biological analysis we were able to identify a reasonable occurrence of diazotrophs in the rhizosphere soil and leaf samples analyzed, 9 and 13 different genera, respectively. Of these, two genera were particularly abundant: Azospirillum and Herbaspirillum. These two genera were also the two most abundant genera isolated by conventional microbiological techniques. Both approaches indicated that the nitrogen-fixing communities were different in soils under cultivation and permanent pasture from these under cultivation without live stock. Our results showed that while the molecular approach gives us a general view of the variability of nitrogen fixers in the community, the conventional methodology allows us to isolate those bacteria and to select some of them that have showed the highest nitrogen fixation capacity and auxin production.

Influence of different Oryza cultivars on expression of nifH gene pools in roots of rice

Cultivation-independent studies suggest that roots of rice (Oryza sativa) are colonized by a diverse community of nitrogen-fixing bacteria. Here we report for the first time mRNA-based profiling of nitrogenase (nifH) genes, to study the impact of lowland-rice genotypes at the cultivar level on the functional diversity of root-associated diazotrophs. Root RNA extracts from all plants contained nifH mRNA at levels detectable by reverse transcription polymerase chain reaction (RT-PCR). Terminal restriction fragment length polymorphism (T-RFLP) analysis of RT-PCR products showed only small interplant variations. However, RNA- and DNA-based profiles obtained from the same root extractions differed from each other, suggesting that presence of diazotrophs did not necessarily coincide with active transcription of nif genes. Application of N-fertilizer at planting had a long-term effect on the profile of expressed nitrogenase genes. Phylogenetic analysis of a clone library constructed for nifH fragments expressed in wild species of rice roots indicated that active diazotrophs were not related to cultured strains. The composition of active diazotrophic communities was compared for six related cultivars of O. sativa, wild species Oryza brachyantha, and a genetic cross between it and cv. IR56, grown under identical conditions in rice field soil in the Philippines without N-fertilizer application. Remarkable varietal differences in root associated nifH-gene expressing communities were detected. This underlines the importance of mRNA-based approaches to study functional diversity and eventually identify key diazotrophs in a particular environment.

Endophytic colonization of rice by a diazotrophic strain of Serratia marcescens.

Six closely related N2-fixing bacterial strains were isolated from surface-sterilized roots and stems of four different rice varieties. The strains were identified as Serratia marcescens by 16S rRNA gene analysis. One strain, IRBG500, chosen for further analysis showed acetylene reduction activity (ARA) only when inoculated into media containing low levels of fixed nitrogen (yeast extract). Diazotrophy of IRBG500 was confirmed by measurement of 15N2 incorporation and by sequence analysis of the PCR-amplified fragment of nifH. To examine its interaction with rice, strain IRBG500 was marked with gusA fused to a constitutive promoter, and the marked strain was inoculated onto rice seedlings under axenic conditions. At 3 days after inoculation, the roots showed blue staining, which was most intense at the points of lateral root emergence and at the root tip. At 6 days, the blue precipitate also appeared in the leaves and stems. More detailed studies using light and transmission electron microscopy combined with immunogold labeling confirmed that IRBG500 was endophytically established within roots, stems, and leaves. Large numbers of bacteria were observed within intercellular spaces, senescing root cortical cells, aerenchyma, and xylem vessels. They were not observed within intact host cells. Inoculation of IRBG500 resulted in a significant increase in root length and root dry weight but not in total N content of rice variety IR72. The inoculated plants showed ARA, but only when external carbon (e.g., malate, succinate, or sucrose) was added to the rooting medium.

Classification of rhizobia based on nodC and nifH gene analysis reveals a close phylogenetic relationship among Phaseolus vulgaris symbionts.

The nodC and nifH genes were characterized in a collection of 83 rhizobial strains which represented 23 recognized species distributed in the genera Rhizobium, Sinorhizobium, Mesorhizobium and Bradyrhizobium, as well as unclassified rhizobia from various host legumes. Conserved primers were designed from available nucleotide sequences and were able to amplify nodC and nifH fragments of about 930 bp and 780 bp, respectively, from most of the strains investigated. RFLP analysis of the PCR products resulted in a classification of these rhizobia which was in general well-correlated with their known host range and independent of their taxonomic status. The nodC and nifH fragments were sequenced for representative strains belonging to different genera and species, most of which originated from Phaselous vulgaris nodules. Phylogenetic trees were constructed and revealed close relationships among symbiotic genes of the Phaseolus symbionts, irrespective of their 16S-rDNA-based classification. The nodC and nifH phylogenies were generally similar, but cases of incongruence were detected, suggesting that genetic rearrangements have occurred in the course of evolution. The results support the view that lateral genetic transfer across rhizobial species and, in some instances, across Rhizobium and Sinorhizobium genera plays a role in diversification and in structuring the natural populations of rhizobia.

Molecular evidence for zooplankton-associated nitrogen-fixing anaerobes based on amplification of the nifH gene

A fragment of the nifH gene was amplified from anaerobic microbial enrichments initiated with marine planktonic invertebrates, including copepods and euphausids. A number of the nifH sequences from these enrichments were phylogenetically related to Cluster III nifH sequences of Clostridium pasteurianum and Desulfovibrio gigas. nifH fragments were cloned and sequenced from strains of Desulfobacter curvatus, Desulfonema limicola, and Chromatium purpuratum in order to provide the basis for identifying nifH sequences amplified from uncultivated microorganisms in the enrichments. Nitrogenase genes from Desulfonema limicola and Desulfobacter curvatus clustered with the sequence from Desulfovibrio gigas. Some of the nifH gene sequences from the zooplankton-derived microbial enrichments clustered with these sequences as well as a number of previously reported sequences from marine microbial mats, termite guts and intact invertebrate zooplankton. Some of the nitrogen-fixing phylotypes in the enrichments appeared to be active, since nitrogenase activity was detected by the acetylene reduction technique in the enrichments. Multiple copies of nifH were amplified from the Desulfobacter curvatus culture, with one of the sequences clustering with non-vanadium second alternative anfH sequences, but no anfH sequences were recovered from the enrichments. The results demonstrate that there are nitrogen-fixing anaerobes associated with planktonic crustacea, and the identified nifH phylotypes could be important in oceanic nitrogen fixation.

Molecular evidence for zooplankton-associated nitrogen-fixing anaerobes based on amplification of the nifH gene

A fragment of the nifH gene was amplified from anaerobic microbial enrichments initiated with marine planktonic invertebrates, including copepods and euphausids. A number of the nifH sequences from these enrichments were phylogenetically related to Cluster III nifH sequences of Clostridium pasteurianum and Desulfovibrio gigas. nifH fragments were cloned and sequenced from strains of Desulfobacter curvatus, Desulfonema limicola, and Chromatium purpuratum in order to provide the basis for identifying nifH sequences amplified from uncultivated microorganisms in the enrichments. Nitrogenase genes from Desulfonema limicola and Desulfobacter curvatus clustered with the sequence from Desulfovibrio gigas. Some of the nifH gene sequences from the zooplankton-derived microbial enrichments clustered with these sequences as well as a number of previously reported sequences from marine microbial mats, termite guts and intact invertebrate zooplankton. Some of the nitrogen-fixing phylotypes in the enrichments appeared to be active, since nitrogenase activity was detected by the acetylene reduction technique in the enrichments. Multiple copies of nifH were amplified from the Desulfobacter curvatus culture, with one of the sequences clustering with non-vanadium second alternative anfH sequences, but no anfH sequences were recovered from the enrichments. The results demonstrate that there are nitrogen-fixing anaerobes associated with planktonic crustacea, and the identified nifH phylotypes could be important in oceanic nitrogen fixation.

N2-Fixing Microbial Consortia Associated with the Ice Cover of Lake Bonney, Antarctica.

Abstract Nitrogen (N) availability is a key nutritional factor controlling microbial production in Antarctic freshwater and soil habitats. Since there are no significant sources of biologically available N entering these ecosystems, nitrogen fixation may be a major source of "new" N supporting primary and secondary production. The role of N2 fixation was examined in cyanobacteria-dominated microbial aggregates embedded in the permanent ice cover of Lake Bonney, McMurdo Dry Valley (Victoria Land) lakes area, and in cyanobacterial mats found in soils adjacent to the ice edge. Nitrogenase activity was extremely low compared to temperate and tropical systems, but N2 fixation was found at all study sites. N2 fixation occurred under both dark and light conditions, indicating the potential involvement of both phototrophic and heterotrophic diazotrophs. Nitrogenase activity measurements (acetylene reduction assay) and molecular characterization (PCR amplification of nifH fragments) demonstrated a diverse and periodically active (when liquid water is present) diazotrophic community in this arid, nutrient-limited environment. As a result of the close proximity to other microorganisms and the nutritional constraints of this environment, these diazotrophs may be involved in mutually beneficial consortial relationships that enhance their growth when water is available.

Diversity of heterotrophic nitrogen fixation genes in a marine cyanobacterial mat

The diversity of nitrogenase genes in a marine cyanobacterial mat was investigated through amplification of a fragment of nifH, which encodes the Fe protein of the nitrogenase complex. The amplified nifH products were characterized by DNA sequencing and were compared with the sequences of nitrogenase genes from cultivated organisms. Phylogenetic analysis showed that similar organisms clustered together, with the exception that anaerobic bacteria clustered together, even though they represented firmicutes, (delta)-proteobacteria, and (gamma)-proteobacteria. Mat nifH sequences were most closely related to those of the anaerobes, with a few being most closely related to the cluster of (gamma)-proteobacteria containing Klebsiella and Azotobacter species. No cyanobacterial nifH sequences were found from the mat collected in November when Microcoleus chthonoplastes was the dominant cyanobacterium, but sequences closely related to the cyanobacterium Lyngbya lagerheimeii were found during summer, when a Lyngbya strain was dominant. The results indicate that there is a high diversity of heterotrophic nitrogen-fixing organisms in marine cyanobacterial mats.