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The International Center for Biotechnology, Osaka University, 2-1 Yamada-oka, Suita-shi, Osaka 565-0871, Japan
Correspondence
Hiroko Kawasaki
ICBKawasakiNakagawa{at}icb.osaka-u.ac.jp
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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The GenBank/EMBL/DDBJ accession number for the complete coding sequence of the nif gene cluster of Hbt. chlorum DSM 3682T is AB196525.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). This strictly anaerobic organism contained a novel photosynthetic pigment, bacteriochlorophyll g, which became a main characteristic of the family ‘Heliobacteriaceae’ (Brockmann & Lipinski, 1983; Madigan, 2001). Phylogenetically, heliobacteria belong to the group of low-G+C-content Gram-positive bacteria that includes Clostridium and Bacillus (Madigan, 1992, 2001), and they all have the capacity for endosporulation (Kimble-Long & Madigan, 2001). Hbt. chlorum was isolated from garden soil (Gest & Favinger, 1983), and later a large number of heliobacteria was isolated from rice fields (Beer-Romero & Gest, 1987; Ormerod et al., 1996; Stevenson et al., 1997), hot springs (Kimble et al., 1995; Stevenson et al., 1997) and the banks of soda lakes (Bryantseva et al., 1999, 2000); the main source of heliobacteria seems to be rice fields (Stevenson et al., 1997; Madigan, 2001). The link between the heliobacterial habitat (rice field) and the ability of heliobacteria to fix nitrogen both photosynthetically and in darkness suggested that they might be significant contributors of fixed nitrogen in the rice fields (Kimble & Madigan, 1992).
Nitrogen fixation is widely but sporadically distributed among both eubacteria and methanogenic archaea (Young, 1992; Raymond et al., 2004). The current understanding of nitrogenase diversity has been based largely on phylogenetic analyses of nifH and nifD, the nitrogenase structural genes (Zehr et al., 2003; Henson et al., 2004). Recently, Raymond et al. (2004) performed genomic analyses of nif genes encoding the core components of nitrogenase, including the NifH, NifD, NifK, NifE and NifN proteins, and proposed five groups: (1) typical Mo–Fe nitrogenases, predominantly composed of members of the proteobacterial and cyanobacterial phyla; (2) anaerobic Mo–Fe nitrogenases from predominantly anaerobic bacteria and several methanogens; (3) alternative nitrogenases, including the Mo-independent Anf and Vnf proteins (except VnfH, which is more similar to NifH rather than AnfH); (4) uncharacterized nif homologues detected only in methanogens and some anoxygenic photosynthetic bacteria; and (5) bacteriochlorophyll and chlorophyll biosynthesis genes common to all phototrophs. This grouping was largely consistent with the previous classification, in which the nitrogenase genes were divided into clusters I–IV (Zehr et al., 2003).
Phylogenetic analyses of NifH and NifD sequences of heliobacteria showed that heliobacteria form a distinct lineage in the nitrogenase phylogeny. Although heliobacteria are strictly anaerobic bacteria, they did not belong to group II of strictly anaerobic diazotrophs such as Clostridium, and were instead placed in group I (Enkh-Amgalan et al., 2005). Indeed, the approximately 50-residue conserved insertion in nifD shared by all members of group II was not found in heliobacteria. Moreover, the nifH and nifD genes of heliobacteria were contiguous, unlike nifH and nifD of members of group II, which are separated by two glnB-like genes (Chien & Zinder, 1996; Kessler et al., 1998; Kessler & Leigh, 1999; Sibold et al., 1991; Arcondeguy et al., 2001; Chen et al., 2001; unpublished genome survey) recently designated nifI1 and nifI2 (Arcondeguy et al., 2001). Another interesting finding was the specific relationship of heliobacteria with Geobacter species, which belong to the Deltaproteobacteria; the clade of heliobacteria was grouped with the Geobacter species clade in the NifH phylogeny, whereas no such grouping was formed in the NifD phylogeny, and both heliobacteria and Geobacter species formed independent clades (Enkh-Amgalan et al., 2005).
Consequently, we aimed to isolate and analyse other genes involved in nitrogen fixation of strictly anaerobic heliobacteria in order to understand fully their unique position in group I and to search for genes common to group I and/or group II diazotrophs. We selected Hbt. chlorum, the type species of Heliobacterium, for this study. Interestingly, sequencing results revealed similar features between the Hbt. chlorum nif cluster and group II of strictly anaerobic diazotrophs, i.e. small size and the presence of nifI genes (found upstream of nifH). The gene organization and phylogenetic analyses of the nifI and other nif genes of Hbt. chlorum in comparison with those of other diazotrophs are discussed in order to further understanding of nitrogenase evolution.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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.
DNA manipulations and cloning procedures.
All DNA manipulations were performed using standard techniques (Sambrook et al., 1989) and according to instructions provided by the suppliers of the reagents. Chromosomal DNA was digested with appropriate restriction enzymes (TaKaRa Shuzo), fractionated by electrophoresis on 0·7 % agarose gel and transferred to Hybond-N+ nylon membrane (Amersham Biosciences) by capillary transfer. Hybridization probes from the PCR product were labelled using the DIG labelling kit (Roche Diagnostics), and hybridization signals were detected using the DIG luminescence detection kit (Roche Diagnostics) according to the manufacturer's instructions. We used four probes and the positions of the probes are shown in Fig. 1. DNA fragments that hybridized to the probes were recovered from agarose gel using the QIAEX II gel extraction kit (Qiagen) and cloned into pUC18 using the DNA ligation kit version 1 (TaKaRa Shuzo). Recombinant colonies were transferred onto Hybond-N+ nylon membrane (Amersham Biosciences), and hybridization and detection were performed as in Southern hybridization. Plasmid DNAs were purified using the QIAprep Spin Miniprep kit (Qiagen) for sequencing analysis.
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) with a BigDye Terminator cycle sequencing kit (PE Applied Biosystems) using an ABI PRISM 310 Genetic Analyzer (Perkin Elmer). Sequence data were analysed by the ABI PRISM sequence analysis program and assembled using the ABI Auto Assembler (Perkin Elmer). Nucleotide sequences were analysed with GENETYX-WIN software (version 3.1). Sequence similarity searches were performed via BLAST (Altschul et al., 1997) at both the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/) and the DNA Database of Japan (DDBJ; http://www.ddbj.nig.ac.jp/). Inferred amino acid sequences of individual genes were aligned with those of other bacteria and archaea by the CLUSTAL X program (Thompson et al., 1997; Jeanmougin et al., 1998) and checked by hand for proper alignment. Phylogenetic analysis was performed using the CLUSTAL X program. Alignment positions in which any of the sequences had a gap were discarded by using the ‘exclude positions with gaps' option, and evolutionary distances were corrected for multiple substitutions by using the appropriate option in the settings. The protein weight matrix GONNET 250 was used for sequence comparison, and phylogenetic trees were constructed by the neighbour-joining method (NJ; Saitou & Nei, 1987) with 1000 bootstrap replicates using default parameters. The concatenated NifHDKEN and NifI trees were degenerated by both the NJ and maximum-likelihood (ML) methods. The programs ProML, SeqBoot and CONSENSE from PHYLIP (Felsenstein, 2004) were used to infer and assemble ML trees (100 replicates), using the JTT model of amino acid substitution. To display and analyse the tree, NJPlot (Perrière & Gouy, 1996) and TreeView (Page, 1996) were used. |
RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). In this study, Hbt. chlorum genomic DNA was digested with BamHI and approximately 2·4 kb fragments were isolated and ligated into the BamHI site of pUC18. Colony hybridization resulted in five colonies giving a strong positive signal to the nifH probe. Plasmid DNA from the positive colonies was purified and then sequenced by primer walking and shown to carry the complete nifH gene and part of the nifD gene (Fig. 1
). Based on the sequence information obtained, specific probes were designed and the flanking regions were cloned and sequenced by chromosome walking until regions of genes not involved in nitrogen fixation were obtained. The probes used and the size and coding region of the fragments obtained are shown in Fig. 1. In this way, we obtained overlapping fragments with a total nucleotide sequence of 11 234 bp. Analysis of the entire nucleotide sequence revealed the presence of 11 open reading frames (ORFs). The amino acid sequences deduced from the nucleotide sequence of the ORFs showed remarkable similarity to the products of nitrogen-fixation genes in other diazotrophs (Table 1). The highest level of identity was detected with predicted gene products derived from a genomic sequence of Desulfitobacterium hafniense. Indeed, Desulfitobacterium is the genus that is most closely related to heliobacteria based on the 16S rRNA phylogeny, and these bacteria share some other similarities such as the formation of endospores and the absence of an outer membrane, despite negative Gram staining (Niggemyer et al., 2001). However, it should be noted that the capacity for nitrogen fixation has not yet been detected in the genus Desulfitobacterium.
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. A 1 bp overlap was observed between the 3' end of nifD and the 5' end of the next ORF, nifK, which indicates a possible translational coupling phenomenon (Oppenheim & Yanofsky, 1980). nifH, nifD and nifK started with the same start codon, ATG, and terminated with the stop codon TAA. The nifE gene, 109 bp downstream of nifK, had a less commonly used translational initiation codon, GTG, which is also found in some nif genes in various diazotrophs, for example, nifB and nifX of Frankia alni (Harriott et al., 1995) and nifN of Acetobacter diazotrophicus (Lee et al., 2000). Adjacent to the nifE gene (separated by 40 bp) was nifN, which started with the initiation codon ATG and terminated with the stop codon TAA. Analysis of the region immediately upstream of these five genes revealed the presence of putative ribosome-binding sites (AGGAGG, AAGAGG and AGGAGG) respectively located 8–10 bp from the start codons of nifH, nifD and nifE. In the region flanking the 3' end of nifK, an inverted-repeat sequence that might function as a transcription terminator was present 40 bp downstream from nifK. Such inverted repeats have been found in the nif operons of other diazotrophs and are suggested to have a transcriptional regulatory function (Brigle et al., 1985; Norel & Elmerich, 1987; Minerdi et al., 2001). However, the inverted-repeat structure was not found in the nifH–nifD intergenic region, unlike in the other diazotrophs. Downstream of the nifHDKEN genes were the following genes in this order: nifX, fdx, nifB and nifV. nifN and nifX were separated by 135 bp, 12 bp separated nifX and fdx, 170 bp separated fdx and nifB and nifV was 17 bp downstream of nifB. The inverted-repeat sequence was detected 105 bp downstream from the stop codon of nifV only.
Upstream of nifH, we detected two small ORFs encoding 105 and 127 amino acids. The products of these two ORFs exhibited significant similarity to those of the nifI1 and nifI2 genes present in the nif operon of methanogenic archaea (Chien & Zinder, 1996; Kessler et al., 1998; Kessler & Leigh, 1999; Sibold et al., 1991) and in some strictly anaerobic bacteria, Desulfovibrio gigas, Clostridium acetobutylicum, Clostridium cellobioparum (Arcondeguy et al., 2001), Clostridium beijerinckii (Chen et al., 2001), Desulfovibrio vulgaris and Chlorobium tepidum (unpublished genome survey), which belong to group II of nitrogenase. In all cases, nifI1 and nifI2 are located between nifH and nifD, suggesting their conserved function (Arcondeguy et al., 2001). However, in the previously demonstrated NifH- and NifD-based phylogeny, sequences of the strictly anaerobic heliobacteria were placed in nitrogenase group I, indicating that they bore less similarity to group II, which consists of strictly anaerobic diazotroph sequences (Enkh-Amgalan et al., 2005). Thus, the finding of nifI genes that exhibit striking similarity to the nifI genes of members of group II in both sequence identity and product size in heliobacteria was unexpected. Furthermore, in the Hbt. chlorum nif operon, nifI genes were found upstream of nifH; this is the first report of nifI genes located upstream of the nitrogenase structural genes. A BLAST search of the nifI1 and nifI2 amino acid sequences against the genome sequence of D. hafniense resulted in 74 and 71 % identical products, respectively, that were located upstream of nifH. The observation of nifI genes in the same location in the nif clusters of Hbt. chlorum and D. hafniense, together with the high identity of individual nif gene products and the similar overall organization of genes within the nif cluster in both organisms, suggested that these bacteria have remarkably similar nitrogenase systems.
Phylogenetic analysis
To study the evolutionary relationships between Hbt. chlorum and other nitrogen-fixing prokaryotes, products of nifH, nifD, nifK, nifE and nifN genes which encode the core components of nitrogenase were compared with corresponding sequences in the DDBJ/EMBL/GenBank databases and phylogenetic trees were generated (Fig. 2). Overall topologies of the five trees were significantly consistent with each other and, in particular, the phylogenetic position of Hbt. chlorum among other diazotrophs was consistently preserved in all trees. In the NifH, NifK, NifE and NifN trees, the sequences of Hbt. chlorum formed a cluster with sequences from D. hafniense and two metal-reducing bacteria in the Deltaproteobacteria, Geobacter sulfurreducens and Geobacter metallireducens, and the cluster was placed in group I, as expected. However, in the NifD tree, the two Geobacter species formed an independent cluster (with a low bootstrap value) which branched earlier than the cluster of Hbt. chlorum and D. hafniense. Nevertheless, these two clusters were placed as the deepest lineages in group I. Actually, D. hafniense was shown to bridge the gap between the group I and group II clades in the NifD, NifK, NifE and NifN trees, while NifH was found within group I but with poor bootstrap support (Raymond et al., 2004), which was probably because of the data available for the analysis.
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). Alternative nitrogenases or Anf (lack EN paralogues) and Vnf (VnfH clusters with NifH while VnfDK forms a separate clade) proteins and that of Rhodopseudomonas palustris, which is thought to have acquired the nifH gene by lateral transfer (Cantera et al., 2004), were excluded from this analysis. As shown in Fig. 3, the NJ and ML trees were consistent with each other and the phylogenetic relationships of diazotrophs were supported by the finding of higher bootstrap values than in the case of individual proteins. Sequences of Hbt. chlorum, D. hafniense, G. sulfurreducens and G. metallireducens formed a highly supported clade whose position was preserved as the basal lineage in group I. These data supported the phylogenetic position of Hbt. chlorum on the individual protein trees.
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). Interestingly, the G. sulfurreducens and G. metallireducens nif operons lacked the nifI genes, but the draT and draG genes, which are involved in the bacterial switch-off, were found.
NifI proteins belong to the PII signal transduction protein family, which consists of a large number of GlnB, GlnK and NifI proteins (Ninfa & Atkinson, 2000; Arcondeguy et al., 2001). We explored the phylogeny based on NifI together with GlnB and GlnK sequences using the NJ and ML methods (Fig. 4). Both phylogenetic trees were divided into three subfamilies, NifI1, NifI2 and GlnB with GlnK, and this division was in agreement with phylogenetic trees drawn by other authors (Chien & Zinder, 1996; Noda et al., 1999). The Hbt. chlorum NifI1 and NifI2 sequences fell into the NifI1 and NifI2 subfamilies, respectively. Within each subfamily, the Hbt. chlorum NifI sequences formed a clade with that of D. hafniense, and this clade was distinct from clades formed by members of nitrogenase group I and group II. The presence of nifI genes in both the Archaea and Bacteria suggests the early origin of these genes, preceding the divergence of the two groups of prokaryotes, and the last common ancestor (LCA) most likely had nifI genes in its nitrogenase family. During the divergence between groups I and II, which resulted from the development of oxygenic photosynthesis and the subsequent aerobic/anaerobic segregation of environments, nifI genes were lost, although the reason for this remains unclear. Genome analyses showed that nifI genes have not been found in aerobic diazotrophs as yet. That the nifI genes have an early origin was also assumed to be true in the hypothesis holding that nitrogen fixation first arose in methanogenic archaea (Raymond et al., 2004), since all types of nitrogenases of methanogens in the cluster have nifI genes. Surprisingly, the alternative nitrogenases found in aerobic diazotrophs and presumed to have transferred from methanosarcina in both the nitrogen-fixing LCA and methanogen-origin hypothesis (Raymond et al., 2004) did not carry nifI genes. This finding again suggests the influence of oxygen on nifI evolution.
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) and Chlorobium tepidum (Wahlund & Madigan, 1993), although the mechanism or proteins involved are still unknown. This regulation was also observed in Clostridium beijerinckii and it was speculated that products of nifI genes play a role in it, since Clostridium pasteurianum, which lacks nifI genes in the genome, did not show similar regulation of nitrogenase to ammonia (Chen, 2004). Although NifI proteins are highly similar to GlnB and GlnK, they differ significantly in the T-loop region where the interaction with other proteins occurs. The conserved site (within the T-loop) for uridylylation or phosphorylation is absent in NifI (Kessler et al., 2001), and this is also true for the Hbt. chlorum and D. hafniense NifI. This suggests that Hbt. chlorum NifI might have functional similarity to the archaeal NifI, although the detailed mechanism is still unknown.
Conclusions
The Hbt. chlorum nif gene cluster was concise, and consisted of 11 genes arranged within a 10 kb region (Fig. 1). Actually, among the diazotrophs, the smallest number of genes required for nitrogen fixation has been found in strictly anaerobic prokaryotes; for example, the Methanococcus maripaludis nif cluster contains eight genes (Kessler et al., 1998) and the nif cluster of Clostridium acetobutylicum consists of nine genes (Chen, 2004) (Fig. 5). The universal presence of nifI genes in strictly anaerobic prokaryotes suggested the essential role of these genes in nitrogen fixation, probably in the regulation of nitrogenase protein as in Methanococcus maripaludis. The relatively ‘simple’ nitrogenase system of the strict anaerobes is regulated by NifI, although the mechanism is unknown. On the contrary, the extensive nif clusters of aerobic diazotrophs, for example the nif cluster from Azotobacter vinelandii, lack nifI genes but have acquired other regulatory genes (Fig. 5). The highly conserved nitrogen-fixation cluster in Hbt. chlorum was found to be acquired through vertical transfer from the LCA or the methanogen, and its phylogenetic position as an intermediate between group I, consisting of aerobic diazotrophs, and group II, consisting of strictly anaerobic prokaryotes, may reflect an evolutionary stage of a divergence of the two nitrogenase groups.
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ACKNOWLEDGEMENTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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 J. A. Dodsworth and J. A. Leigh Regulation of nitrogenase by 2-oxoglutarate-reversible, direct binding of a PII-like nitrogen sensor protein to dinitrogenase PNAS, June 27, 2006; 103(26): 9779 - 9784. [Abstract] [Full Text] [PDF]
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indicates more than five ORFs.
54. Functions of regulatory elements are shown in boxes and predicted functions are shown in dotted boxes. For Azotobacter vinelandii, the gene and ORF designations are as presented by Jacobson et al. (1989)