Natural association of Gluconacetobacter diazotrophicus and diazotrophic Acetobacter peroxydans with wetland rice

The family Acetobacteraceae currently includes three known nitrogen-fixing species, Gluconacetobacter diazotrophicus, G. johannae and G. azotocaptans. In the present study, acetic acid-producing nitrogen-fixing bacteria were isolated from four different wetland rice varieties cultivated in the state of Tamilnadu, India. Most of these isolates were identified as G. diazotrophicus on the basis of their phenotypic characteristics and PCR assays using specific primers for that species. Based on 16S rDNA partial sequence analysis and DNA: DNA reassociation experiments the remaining isolates were identified as Acetobacter peroxydans, another species of the Acetobacteraceae family, thus far never reported as diazotrophic. The presence of nifH genes in A. peroxydans was confirmed by PCR amplification with nifH specific primers. Scope for the findings: This is the first report of the occurrence and association of N2-fixing Gluconacetobacter diazotrophicus and Acetobacter peroxydans with wetland rice varieties. This is the first report of diazotrophic nature of A. peroxydans.     

A Comprehensive Evaluation of PCR Primers to Amplify the nifH Gene of Nitrogenase

The nifH gene is the most widely sequenced marker gene used to identify nitrogen-fixing Bacteria and Archaea. Numerous PCR primers have been designed to amplify nifH, but a comprehensive evaluation of nifH PCR primers has not been performed. We performed an in silico analysis of the specificity and coverage of 51 universal and 35 group-specific nifH primers by using an aligned database of 23,847 nifH sequences. We found that there are 15 universal nifH primers that target 90% or more of nitrogen fixers, but that there are also 23 nifH primers that target less than 50% of nifH sequences. The nifH primers we evaluated vary in their phylogenetic bias and their ability to recover sequences from commonly sampled environments. In addition, many of these primers will amplify genes that do not mediate nitrogen fixation, and thus it would be advisable for researchers to screen their sequencing results for the presence of non-target genes before analysis. Universal primers that performed well in silico were tested empirically with soil samples and with genomic DNA from a phylogenetically diverse set of nitrogen-fixing strains. This analysis will be of great utility to those engaged in molecular analysis of nifH genes from isolates and environmental samples.     

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.     

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.     

Nucleotide sequence of the R.meliloti nitrogenase reductase (nifH) gene.

The nucleotide sequence of the structural gene (nifH) of nitrogenase reductase (Fe protein) from R.meliloti 41 with its flanking ends is reported. The amino acid sequence of nitrogenase reductase was deduced from the DNA sequence. The predicted R.meliloti nitrogenase reductase protein consists of 297 amino acid residues, has a molecular weight of 32,740 daltons and contains 5 cysteine residues. The codon usage in the nifH gene is presented. In the 5' flanking region, sequences resembling to consensus sequences of bacterial control regions were found. Comparison of the R.meliloti nifH nucleotide and amino acid sequences with those from different nitrogen-fixing organisms showed that the amino acid sequences are more conserved than the nucleotide sequences. This structural conservation of nitrogenase reductase may be related to its function and may explain the conservation of the nifH gene during evolution.     

Isolation of nifH and part of nifD by modified capture polymerase chain reaction from a natural population of the marine cyanobacterium Trichodesmium sp.

Abstract A modified capture polymerase chain reaction (CPCR) technique was used to isolate the entire sequence of the nifH gene and its flanking regions from a natural population of Trichodesmium sp. A set of specific CPCR primers derived from a known 72-bp DNA segment of the nifH sequence permitted isolation of both the upstream and the downstream region of Trichodesmium sp. nifH . The 882-bp nifH gene presented here is the first full-length gene isolated from Trichodesmium sp. A sequence similar to a nif -like promoter was found in front of nifH . The nifH open reading frame of Trichodesmium sp. encoded 294 amino acids. Comparative analysis of the Trichodesmium sp. NifH sequence revealed strong similarity with 23 known NifH proteins. Amino acids postulated to be involved in binding of the 4Fe:4S cluster and those subjected to ADP-ribosylation were present. An open reading frame for the nifD gene was identified 189 bp downstream of nifH . A sequence similar to the consensus of the nif -like promoter was also found in front of nifD .     

Analysis of a diverse assemblage of diazotrophic bacteria from Spartina alterniflora using DGGE and clone library screening

Methods to assess the diversity of the diazotroph assemblage in the rhizosphere of the salt marsh cordgrass, Spartina alterniflora were examined. The effectiveness of nifH PCR-denaturing gradient gel electrophoresis (DGGE) was compared to that of nifH clone library analysis. Seventeen DGGE gel bands were sequenced and yielded 58 nonidentical nifH sequences from a total of 67 sequences determined. A clone library constructed using the GC-clamp nifH primers that were employed in the PCR-DGGE (designated the GC-Library) yielded 83 nonidentical sequences from a total of 257 nifH sequences. A second library constructed using an alternate set of nifH primers (N-Library) yielded 83 nonidentical sequences from a total of 138 nifH sequences. Rarefaction curves for the libraries did not reach saturation, although the GC-Library curve was substantially dampened and appeared to be closer to saturation than the N-Library curve. Phylogenetic analyses showed that DGGE gel band sequencing recovered nifH sequences that were frequently sampled in the GC-Library, as well as sequences that were infrequently sampled, and provided a species composition assessment that was robust, efficient, and relatively inexpensive to obtain. Further, the DGGE method permits a large number of samples to be examined for differences in banding patterns, after which bands of interest can be sampled for sequence determination.     

Nucleotide sequence of the gene encoding the nitrogenase iron protein of Thiobacillus ferrooxidans.

The DNA sequence was determined for the cloned Thiobacillus ferrooxidans nifH and part of the nifD genes. A putative T. ferrooxidans nifH promoter was identified whose sequences showed perfect consensus with those of the Klebsiella pneumoniae nif promoter. Two putative consensus upstream activator sequences were also identified. The amino acid sequence was deduced from the DNA sequence. In a comparison of nifH DNA sequences from T. ferrooxidans and eight other nitrogen-fixing microbes, a Rhizobium sp. isolated from Parasponia andersonii showed the greatest homology (74%) and Clostridium pasteurianum (nifH 1) showed the least homology (54%). In a comparison of the amino acid sequences of the Fe proteins, the Rhizobium sp. and Rhizobium japonicum showed the greatest homology (both 86%) and C. pasteurianum (nifH 1 gene product) demonstrated the least homology (56%) to the T. ferrooxidans Fe protein.     

Characterization of amplified polymerase chain reaction glnB and nifH gene fragments of nitrogen-fixing Burkholderia species

AIMS: To clone and sequence polymerase chain reaction (PCR)-amplified glnB and nifH genes of the nitrogen-fixing bacteria Burkholderia brasilensis strain M130, B. tropicalis strain PPe8 and B. kururiensis strain KP23. METHODS AND RESULTS: The glnB and nifH gene fragments were amplified by PCR using universal degenerated primers. A very high percentage of similarity for the nifH (100%) and glnB (96%) genes was observed between strains M130 and KP23. A similarity of 100% for the nifH gene was also observed between strains M130 and PPe8. However, the identity for the glnB gene was 98% and the similarity 88%. The phylogenetic tree of the nifH gene showed a very high degree of similarity to the 16S rDNA gene. CONCLUSIONS: The nitrogen-fixing bacteria of the Burkholderia genus formed a cluster separated from the other species of the genus mainly when the nifH rather than the glnB gene was used to construct the phylogenetic tree. Significance and Impact of the Study: Knowledge of the nifH and glnB gene sequences of B. brasilensis, B. tropicalis and B. kururiensis will support new studies on the diversity of these diazotrophs in natural environments.     

A system of oligonucleotide primers for amplifying nifH genes from various 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.     

Diazotrophic diversity and distribution in the tropical and subtropical Atlantic Ocean

To understand the structure of marine diazotrophic communities in the tropical and subtropical Atlantic Ocean, the molecular diversity of the nifH gene was studied by nested PCR amplification using degenerate primers, followed by cloning and sequencing. Sequences of nifH genes were amplified from environmental DNA samples collected during three cruises (November-December 2000, March 2002, and October-November 2002) covering an area between 0 to 28.3 degrees N and 56.6 to 18.5 degrees W. A total of 170 unique sequences were recovered from 18 stations and 23 depths. Samples from the November-December 2000 cruise contained both unicellular and filamentous cyanobacterial nifH phylotypes, as well as gamma-proteobacterial and cluster III sequences, so far only reported in the Pacific Ocean. In contrast, samples from the March 2002 cruise contained only phylotypes related to the uncultured group A unicellular cyanobacteria. The October-November 2002 cruise contained both filamentous and unicellular cyanobacterial and gamma-proteobacterial sequences. Several sequences were identical at the nucleotide level to previously described environmental sequences from the Pacific Ocean, including group A sequences. The data suggest a community shift from filamentous cyanobacteria in surface waters to unicellular cyanobacteria and/or heterotrophic bacteria in deeper waters. With one exception, filamentous cyanobacterial nifH sequences were present within temperatures ranging between 26.5 and 30 degrees C and where nitrate was undetectable. In contrast, nonfilamentous nifH sequences were found throughout a broader temperature range, 15 to 30 degrees C, more often in waters with temperature of <26 degrees C, and were sometimes recovered from waters with detectable nitrate concentrations.     

A putative new endophytic nitrogen-fixing bacterium Pantoea sp. from sugarcane

AIMS: To isolate and identify endophytic nitrogen-fixing bacteria in sugarcane growing in Cuba without chemical fertilizers. METHODS AND RESULTS: Two N2-fixing isolates, 9C and T2, were obtained from surface-sterilized stems and roots, respectively, of sugarcane variety ML3-18. Both isolates showed acetylene reduction and H2 production in nitrogen-free media. Nitrogenase activity measured by H2 production was about 15 times higher for isolate 9C than for T2 or for Gluconoacetobacter diazotrophicus (PAL-5 standard strain, ATCC 49037). The nifH gene segment was amplified from both isolates using specific primers. Classification of both T2 and 9C was made on the basis of morphological, biochemical, PCR tests and 16S rDNA sequence analysis. CONCLUSIONS: Isolate 9C was identified as a Pantoea species from its 16S rDNA, but showed considerable differences in physiological properties from previously reported species of this genus. For example, 9C can be cultured over a wide range of temperature, pH and salt concentration, and showed high H2 production (up to 67.7 nmol H2 h(-1) 10(10) cell(-1)). Isolate T2 was a strain of Gluconacetobacter diazotrophicus. SIGNIFICANCE AND IMPACT OF THE STUDY: A new N2-fixing endophyte, i.e. Pantoea, able to produce H2 and to grow in a wide range of conditions, was isolated from sugarcane stem tissue and characterized. The strain with these attributes may well be valuable for agriculture.     

Phylogenetic diversity of nitrogen-fixing bacteria and the nifH gene from mangrove rhizosphere soil

Nine types of nitrogen-fixing bacterial strains were isolated from 3 rhizosphere soil samples taken from mangrove plants in the Dongzhaigang National Mangrove Nature Reserve of China. Most isolates belonged to Gammaproteobacteria Pseudomonas, showing that these environments constituted favorable niches for such abundant nitrogen-fixing bacteria. New members of the diazotrophs were also found. Using a soil DNA extraction and PCR-cloning-sequencing approach, 135 clones were analyzed by restriction fragment length polymorphism (RFLP) analysis, and 27 unique nifH sequence phylotypes were identified, most of which were closely related to sequences from uncultured bacteria. The diversity of nitrogen-fixing bacteria was assessed by constructing nifH phylogenetic trees from sequences of all isolates and clones in this work, together with related nifH sequences from other mangrove ecosystems in GenBank. The nifH diversity varied among soil samples, with distinct biogeochemical properties within a mangrove ecosystem. When comparing different mangrove ecosystems, the nifH gene sequences from a specific site tended to cluster as individual groups. The results provided interesting data and novel information on our understanding of diazotroph community diversity in the mangrove ecosystems.     

Use of degenerate oligonucleotides for amplification of the nifH gene from the marine cyanobacterium Trichodesmium thiebautii.

Trichodesmium spp. are marine filamentous, nonheterocystous, nitrogen-fixing cyanobacteria which are an important component of marine ecosystems. This organism has never been maintained in axenic culture, and there has remained some doubt as to the identity of the organism responsible for nitrogen fixation in Trichodesmium aggregates. By using degenerate oligonucleotide primers, it has been possible to amplify, clone, and sequence a segment of the nifH gene from a natural assemblage of Trichodesmium thiebautii. Examination of the DNA and presumed amino acid sequence shows that the gene is most closely related to that of Anabaena spp. and therefore is most likely a cyanobacterial nifH gene. The use of degenerate oligonucleotides, in concert with the polymerase chain reaction, can be a powerful tool for the cloning and sequencing of a variety of genes from microorganisms in the environment.     

Use of degenerate oligonucleotides for amplification of the nifH gene from the marine cyanobacterium Trichodesmium thiebautii

Trichodesmium spp. are marine filamentous, nonheterocystous, nitrogen-fixing cyanobacteria which are an important component of marine ecosystems. This organism has never been maintained in axenic culture, and there has remained some doubt as to the identity of the organism responsible for nitrogen fixation in Trichodesmium aggregates. By using degenerate oligonucleotide primers, it has been possible to amplify, clone, and sequence a segment of the nifH gene from a natural assemblage of Trichodesmium thiebautii. Examination of the DNA and presumed amino acid sequence shows that the gene is most closely related to that of Anabaena spp. and therefore is most likely a cyanobacterial nifH gene. The use of degenerate oligonucleotides, in concert with the polymerase chain reaction, can be a powerful tool for the cloning and sequencing of a variety of genes from microorganisms in the environment.     

New evidence for nitrogen fixation within the Italian white truffle Tuber magnatum

Diversity of nitrogen-fixing bacteria and the nitrogen-fixation activity was investigated in Tuber magnatum, the most well-known prized species of Italian white truffle. Degenerate PCR primers were applied to amplify the nitrogenase gene nifH from T. magnatum ascomata at different stages of maturation. Putative amino acid sequences revealed mainly the presence of Alphaproteobacteria belonging to Bradyrhizobium spp. and expression of nifH genes from Bradyrhizobia was detected. The nitrogenase activity evaluated by acetylene reduction assay was 0.5-7.5μmolC(2)H(4)h(-1)g(-1), comparable with early nodules of legumes associated with specific nitrogen-fixing bacteria. This is the first demonstration of nitrogenase expression gene and activity within truffle.     

Isolation and identification of nitrogen-fixing bacilli from plant rhizospheres in Beijing region

AIMS: To isolate and identify nitrogen-fixing bacilli from the plant rhizospheres in Beijing region of China. METHODS AND RESULTS: A total of 29 isolates were selectively obtained from the rhizospheres of wheat, maize, ryegrass and willow based on their growth on nitrogen-free medium and their resistance to 100 degrees C for 10 min. Of the 29 isolates, seven had nifH gene determined by PCR amplification. The seven isolates were found to belong to the genera Bacillus and Paenibacillus based on phenotypic characterization, 16S rDNA sequence, G+C content and DNA-DNA hybridization. Isolates T1 and W5 were identified as Bacillus cereus and Bacillus marisflavi respectively. Isolates G1, C4 and C5 were identified as Bacillus megaterium. Isolate G2 was identified as Paenibacillus polymyxa and isolate T7 as Paenibacillus massiliensis. CONCLUSIONS: This study suggests that nifH gene could be detected in the both genera Bacillus and Paenibacillus. These degenerate primers for nifH gene fragment used in this study were shown to be useful for identifying nitrogen-fixing bacilli. SIGNIFICANCE AND IMPACT OF THE STUDY: It is the first demonstration that nitrogen fixation exists in B. marisflavi and P. massiliensis and the first report of the sequences of the nifH gene from B. megaterium and B. cereus. The nitrogen-fixing bacilli obtained in this study will be used in our future research for investigating the mechanisms of nitrogen fixation in bacilli.