Amplification, cloning, and sequencing of a nifH segment from aquatic microorganisms and natural communities.

By use of the polymerase chain reaction and degenerate oligonucleotide primers for highly conserved regions of nifH, a segment of nifH DNA was amplified from several aquatic microorganisms, including an N2-fixing bacterium closely associated with the marine filamentous cyanobacterium Trichodesmium sp., a heterotrophic isolate from the root/rhizome of the seagrass Ruppia maritima, and the heterocystous freshwater cyanobacterium Anabaena oscillarioides. nifH segments were amplified directly from DNA extracted from the rhizosphere of roots of the seagrass Halodule wrightii. The nifH fragments were then cloned and sequenced. The DNA and deduced amino acid sequences were compared with known sequences, revealing distinct differences between taxonomic groups. This technique was shown to be useful for (i) the detection of N2-fixing microorganisms and (ii) rapidly obtaining the DNA sequence of the nifH gene, which provides information about general taxonomic groups of N2-fixing microorganisms.     

Characterization of diatom-cyanobacteria symbioses on the basis of nifH, hetR and 16S rRNA sequences

Richelia intracellularis is a symbiotic heterocystous cyanobacterium that is capable of forming associations with several genera of diatoms. nifH, 16S rRNA and hetR sequences were amplified and cloned from field populations of Richelia associated with Hemiaulus hauckii (N. Atlantic), with Rhizosolenia clevei (N. Pacific), and from a cultivated isolate of Calothrix associated with Chaetoceros from station ALOHA (N. Pacific). Sequence identity was highest (98.2%) among the 16S rRNA sequences, and more divergent for the hetR (83.8%) and nifH (91.1%) sequences. The hetR and nifH DNA and amino acid sequences obtained from the symbionts associated with the three different diatom genera diverged into three separate lineages supported by high bootstrap values. The data indicate that symbionts in the different hosts are distinct species or strains. Furthermore, three previously unidentified heterocystous-like nifH sequence groups recently reported from station ALOHA in the subtropical Pacific, het-1, het-2 and het-3, were linked to Richelia associated with R. clevei, H. hauckii and the Calothrix symbiont of Chaetoceros sp. respectively.     

Genetic characterization of Cylindrospermopsis raciborskii (cyanobacteria) isolates from diverse geographic origins based on nifH and cpcBA-IGS nucleotide sequence analysis

Isolates of the toxic, N(2)-fixing species Cylindrospermopsis raciborskii from various geographic locations were analyzed with respect to their genetic diversity based on the nifH and cpcBA-IGS genes. Gene sequences clustered according to their geographic origin, with the nifH sequences separating into European, Australian, and American groups and the cpcBA-IGS sequences separating into American and European or Australian groups. PCR primers for both genes were designed to exclusively amplify DNA from Cylindrospermopsis species, and an additional primer set for cpcBA-IGS was designed to specifically amplify the American C. raciborskii strains.     

Genetic diversity of nodulating and non-nodulating rhizobia associated with wild soybean (Glycine soja Sieb. & Zucc.) in different ecoregions of China

Microorganisms are important in soil development, inputs and biogeochemical cycling of nutrients and organic matter during early stages of ecosystem development, but little is known about their diversity, distribution, and function in relation to the chemical and physical changes associated with the progress of succession. In this study, we characterized the community structure and activity of nitrogen-fixing microbes during primary succession on a copper tailings. Terminal fragment length polymorphism (T-RFLP) and clone sequencing of nifH genes indicated that different N(2) -fixing communities developed under primary succession. Phylogenetic analysis revealed a diversity of nifH sequences that were mostly novel, and many of these could be assigned to the taxonomic divisions Proteobacteria, Cyanobacteria, and Firmicutes. Members of the Cyanobacteria, mostly affiliated with Nostocales or not closely related to any known organisms, were detected exclusively in the biological soil crusts and represented a substantial fraction of the respective diazotrophic communities. Quantitative PCR (and statistical analyses) revealed that, overall, copy number of nifH sequences increased with progressing succession and correlated with changes in physiochemical properties (including elementary elements such as carbon and nitrogen) and the recorded nitrogenase activities of the tailings. Our study provides an initial insight into the biodiversity and community structure evolution of N(2) -fixing microorganisms in ecological succession of mine tailings.     

Diazotrophic community structure and function in two successional stages of biological soil crusts from the Colorado Plateau and Chihuahuan Desert

The objective of this study was to characterize the community structure and activity of N2-fixing microorganisms in mature and poorly developed biological soil crusts from both the Colorado Plateau and Chihuahuan Desert. Nitrogenase activity was approximately 10 and 2.5 times higher in mature crusts than in poorly developed crusts at the Colorado Plateau site and Chihuahuan Desert site, respectively. Analysis of nifH sequences by clone sequencing and the terminal restriction fragment length polymorphism technique indicated that the crust diazotrophic community was 80 to 90% heterocystous cyanobacteria most closely related to Nostoc spp. and that the composition of N2-fixing species did not vary significantly between the poorly developed and mature crusts at either site. In contrast, the abundance of nifH sequences was approximately 7.5 times greater (per microgram of total DNA) in mature crusts than in poorly developed crusts at a given site as measured by quantitative PCR. 16S rRNA gene clone sequencing and microscopic analysis of the cyanobacterial community within both crust types demonstrated a transition from a Microcoleus vaginatus-dominated, poorly developed crust to mature crusts harboring a greater percentage of Nostoc and Scytonema spp. We hypothesize that ecological factors, such as soil instability and water stress, may constrain the growth of N2-fixing microorganisms at our study sites and that the transition to a mature, nitrogen-producing crust initially requires bioengineering of the surface microenvironment by Microcoleus vaginatus.     

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.     

Biodiversity of denitrifying and dinitrogen-fixing bacteria in an acid forest soil

Isolated soil DNA from an oak-hornbeam forest close to Cologne, Germany, was suitable for PCR amplification of gene segments coding for the 16S rRNA and nitrogenase reductase (NifH), nitrous oxide reductase (NosZ), cytochrome cd(1)-containing nitrite reductase (NirS), and Cu-containing nitrite reductase (NirK) of denitrification. For each gene segment, diverse PCR products were characterized by cloning and sequencing. None of the 16S rRNA gene sequences was identical to any deposited in the data banks, and therefore each of them belonged to a noncharacterized bacterium. In contrast, the analyzed clones of nifH gave only a few different sequences, which occurred many times, indicating a low level of species richness in the N2-fixing bacterial population in this soil. Identical nifH sequences were also detected in PCR amplification products of DNA of a soil approximately 600 km distant from the Cologne area. Whereas biodiversity was high in the case of nosZ, only a few different sequences were obtained with nirK. With respect to nirS, cloning and sequencing of the PCR products revealed that many false gene segments had been amplified with DNA from soil but not from cultured bacteria. With the 16S rRNA gene data, many sequences of uncultured bacteria belonging to the Acidobacterium phylum and actinomycetes showed up in the PCR products when isolated DNA was used as the template, whereas sequences obtained for nifH and for the denitrification genes were closely related to those of the proteobacteria. Although in such an experimental approach one has to cope with the enormous biodiversity in soils and only a few PCR products can be selected at random, the data suggest that denitrification and N2 fixation are not genetic traits of most of the uncultured bacteria.     

Cloning of nifHD from Nostoc commune UTEX 584 and of a flanking region homologous to part of the Azotobacter vinelandii nifU gene.

The heterocystous cyanobacterium Nostoc commune UTEX 584 contains two nifH-like sequences (nifH1 and nifH2) in addition to nifHD. A region of DNA 1 kilobase upstream from the 5' end of nifH showed considerable sequence similarity to part of the published nifU sequences of Azotobacter vinelandii and Klebsiella pneumoniae.     

Phylogenetic diversity of nitrogen fixation genes in the symbiotic microbial community in the gut of diverse termites.

Nitrogen fixation by the microorganisms in the gut of termites is one of the crucial aspects of symbiosis, since termites usually thrive on a nitrogen-poor diet. The phylogenetic diversity of the nitrogen-fixing organisms within the symbiotic community in the guts of various termite species was investigated without culturing the resident microorganisms. A portion of the dinitrogenase reductase gene (nifH) was directly amplified from DNA extracted from the mixed population in the termite gut. Analysis of deduced amino acid sequences of the products of the clonally isolated nifH genes revealed the presence of diverse nifH sequences in most of the individual termite species, and their constituents were considerably different among termite species. A majority of the nifH sequences from six lower termites, which showed significant levels of nitrogen fixation activity, could be assigned to either the anaerobic nif group (consisting of clostridia and sulfur reducers) or the alternative nif methanogen group among the nifH phylogenetic groups. In the case of three higher termites, which showed only low levels of nitrogen fixation activity, a large number of the sequences were assigned to the most divergent nif group, probably functioning in some process other than nitrogen fixation and being derived from methanogenic archaea. The nifH groups detected were similar within each termite family but different among the termite families, suggesting an evolutionary trend reflecting the diazotrophic habitats in the symbiotic community. Within these phylogenetic groups, the sequences from the termites formed lineages distinct from those previously recognized in studies using classical microbiological techniques, and several sequence clusters unique to termites were found. The results indicate the presence of diverse potentially nitrogen-fixing microbial assemblages in the guts of termites, and the majority of them are as yet uncharacterized.     

Diversity of diazotrophic unicellular cyanobacteria in the tropical North Atlantic Ocean

We present data on the genetic diversity and phylogenetic affinities of N2-fixing unicellular cyanobacteria in the plankton of the tropical North Atlantic Ocean. Our dinitrogenase gene (nifH) sequences grouped together with a group of cyanobacteria from the subtropical North Pacific; another subtropical North Pacific group was only distantly related. Most of the 16S ribosomal DNA sequences from our tropical North Atlantic samples were closely allied with sequences from a symbiont of the diatom Climacodium frauenfeldianum. These findings suggest a complex pattern of evolutionary and ecological divergence among unicellular cyanobacteria within and between ocean basins.     

Isolation and gene quantification of heterotrophic N2-fixing bacterioplankton in the Baltic Sea

Cyanobacteria are regarded as the main N(2)-fixing organisms in marine waters. However, recent clone libraries from various oceans show a wide distribution of the dinitrogenase reductase gene (nifH) originating from heterotrophic bacterioplankton. We isolated heterotrophic N(2)-fixing bacteria from Baltic Sea bacterioplankton using low-nitrogen plates and semi-solid diazotroph medium (SSDM) tubes. Isolates were analysed for the nitrogenase (nifH) gene and active N(2) fixation by nested polymerase chain reaction (PCR) and acetylene reduction respectively. A primer-probe set targeting the nifH gene from a gamma-proteobacterial isolate, 97% 16S rDNA similarity to Pseudomonas stutzeri, was designed for measuring in situ dynamics using quantitative real-time PCR. This nifH gene sequence was detected at two of 11 stations in a Baltic Proper transect at abundances of 3 x 10(4) and 0.8 x 10(3) copies per litre seawater respectively. Oxygen requirements of isolates were examined by cultivation in SSDM tubes where oxygen gradients were determined with microelectrodes. Growth, and thereby N(2) fixation, was observed as horizontal bands formed at oxygen levels of 0-6% air saturation. The apparent microaerophilic or facultative anaerobic nature of the isolates explains why the SSDM approach is the most appropriate isolation method. Our study illustrates how combined isolation, functional analyses and in situ quantification yielded insights into the oxygen requirements of heterotrophic N(2)-fixing bacterioplankton isolates, which were confirmed to be present in situ.     

Detection and characterization of cyanobacterial nifH genes.

The DNA sequence of a 359-bp fragment of nifH was determined for the heterocystous strains Anabaena sp. strain CA (ATCC 33047), Nostoc muscorum UTEX 1933, a Nostoc sp., Gloeothece sp. strain ATCC 27152, Lyngbya lagerheimii UTEX 1930, and Plectonema boryanum IU 594. Results confirmed that the DNA sequence of the 359-bp segment is sufficiently variable to distinguish cyanobacterial nifH genes from other eubacterial and arachaeobacterial nifH genes, as well as to distinguish heterocystous from nonheterocystous nifH genes. Nonheterocystous cyanobacterial nifH sequences were greater than 70 and 82% identical on the DNA and amino acid levels, respectively, whereas corresponding values for heterocystous cyanobacterial nifH sequences were 84 and 91%. The amplified nifH fragments can be used as DNA probes to differentiate between species, although there was substantial cross-reactivity between the nifH amplification products of some strains. However, an oligonucleotide designed from a sequence conserved within the heterocystous cyanobacteria hybridized primarily with the amplification product from heterocystous strains. The use of oligonucleotides designed from amplified nifH sequences shows great promise for characterizing assemblages of diazotrophs.     

Proof that Burkholderia strains form effective symbioses with legumes: a study of novel Mimosa-nodulating strains from South America

Twenty Mimosa-nodulating bacterial strains from Brazil and Venezuela, together with eight reference Mimosa-nodulating rhizobial strains and two other beta-rhizobial strains, were examined by amplified rRNA gene restriction analysis. They fell into 16 patterns and formed a single cluster together with the known beta-rhizobia, Burkholderia caribensis, Burkholderia phymatum, and Burkholderia tuberum. The 16S rRNA gene sequences of 15 of the 20 strains were determined, and all were shown to belong to the genus Burkholderia; four distinct clusters could be discerned, with strains isolated from the same host species usually clustering very closely. Five of the strains (MAP3-5, Br3407, Br3454, Br3461, and Br3469) were selected for further studies of the symbiosis-related genes nodA, the NodD-dependent regulatory consensus sequences (nod box), and nifH. The nodA and nifH sequences were very close to each other and to those of B. phymatum STM815, B. caribensis TJ182, and Cupriavidus taiwanensis LMG19424 but were relatively distant from those of B. tuberum STM678. In addition to nodulating their original hosts, all five strains could also nodulate other Mimosa spp., and all produced nodules on Mimosa pudica that had nitrogenase (acetylene reduction) activities and structures typical of effective N2-fixing symbioses. Finally, both wild-type and green fluorescent protein-expressing transconjugant strains of Br3461 and MAP3-5 produced N2-fixing nodules on their original hosts, Mimosa bimucronata (Br3461) and Mimosa pigra (MAP3-5), and hence this confirms strongly that Burkholderia strains can form effective symbioses with legumes.     

Relative abundance of denitrifying and dinitrogen-fixing bacteria in layers of a forest soil

The populations of N(2)-fixing and denitrifying bacteria in an acid forest soil near Cologne were characterized by gene probing. The DNA isolated from the soil for this purpose was suitable for DNA-DNA hybridization using 0.4-0.7-kb probes targeting denitrification enzymes, dinitrogenase reductase (nifH) and eubacterial 16S rRNA. The densitometrical comparison of band intensities obtained in these Southern hybridizations indicated that the highest number of total bacteria, of denitrifying and N(2)-fixing microorganisms always occurred in the upper ( approximately 5 cm) soil layer. The concentration of all these organisms decreased in parallel with the soil depth. The soil investigated was rich in nitrate in all layers, and the availability of nitrate apparently did not govern the distribution of denitrifying and N(2)-fixing bacteria in this soil. Soil cores investigated in the laboratory formed N(2)O on addition of nitrate irrespective of the presence of C(2)H(2). Hybridization intensities, with a gene probe for the 16S rRNA, and MPN numbers were generally higher in soil samples taken from the roots of plants than in the bulk soil. There was no selective enrichment of denitrifying or N(2)-fixing bacteria at the roots. The data obtained by hybridizing isolated soil DNA generally matched previous results obtained with culturable bacteria.