BACKGROUND
biological nitrogen fixation, the conversion of atmospheric n <dig> to nh <dig>  plays an important role in the global nitrogen cycle and in world agriculture  <cit> .
most biological nitrogen fixation is catalyzed by the molybdenum nitrogenase.
this enzyme is a complex which contains the mofe protein encoded by nifdk and the fe protein encoded by nifh.
the mofe protein contains two metalloclusters: femo-co, a  cluster which serves as the active site of substrate binding and reduction and the p-cluster, a  cluster which shuttles electrons to femo-co  <cit> .
previous biochemical and genetic studies on klebsiella pneumoniae carrying twenty nif genes on 24-kb region genes and azotobacter vinelandii revealed that nifh, nifd, nifk, nife, nifn, nifx nifb, nifq, nifv, nify, nifu nifs, nifz and nifm contribute to the synthesis and maturation of nitrogenase  <cit> .
contents and organization of nif genes varied significantly among n2-fixing organisms.
for example, in k. pneumoniae, twenty nif genes are co-located within a ~24 kb cluster  <cit> , whereas in a. vinelandii the nif genes are more dispersed and distributed as two clusters in genome  <cit> .
there is usually only one nifh gene and the nifh, nifd and nifk genes are transcribed as a single unit in many diazotrophs, such as k. pneumoniae and a. vinelandii.
however, multiple nifh genes were found in a few diazotrophs.
for examples, rhizobium leguminosarum bv.
phaseoli possesses three nifh genes  <cit>  and clostridium pasteurianum w <dig> has six nifh homologs  <cit> .
nitrogen fixation is sporadically distributed among prokaryote families: proteobacteria, firmicutes, archaea, cyanobacteria and actinobacteria  <cit> .
the incomplete distribution pattern and the difference in contents and organization of nif genes raise the question of origins and evolution of mo-nitrogenase.
two conflicting hypotheses for the origin of mo-nitrogenase have been proposed on the basis of phylogenetic examination of mo-nitrogenase protein sequences   <cit> .
the last common ancestor  hypothesis implies that the mo-nitrogenase had its origin in a common ancestor of the bacterial and archaeal domains.
according to the lca model gene loss has been extensive and accounts for the fact that nitrogenase is found neither in eukaryotes nor in many entire phyla of prokaryotes.
the methanogen origin hypothesis implies that nitrogen fixation originated from methanogenic archaea and subsequently was transferred into a primitive bacterium via lateral gene transfer.
recent studies based on phylogenetic analysis of nifhdk sequences supported the methanogen origin hypothesis and implied that mo-nitrogenase evolved in the anaerobic and hydrogenotrophic methanogens with acquisition in the bacterial domain via lateral gene transfer involving an anaerobic member of the firmicutes  <cit> .
firmicutes have been thought to play an important role in evolution of nitrogen fixation.
studies on evolution of nitrogen fixation in firmicutes mainly focused on the anaerobic diazotrophic clostridia.
although paenibacillus is a genus of firmicute, its nitrogen fixation traits and evolution remains unclear.
it is well known that paenibacillus is a genus of gram-positive, facultative anaerobic, endospore-forming bacteria, originally included within the genus bacillus and then reclassified as a separate genus in  <dig>  <cit> .
bacteria belonging to this genus have been detected in a variety of environments such as soil, water, rhizosphere, vegetable matter, forage and insect larvae, as well as clinical samples  <cit> .
nitrogen-fixing paenibacillus species have great potential for use as a bacterial fertilizer in agriculture, but genomic information of these bacteria is lacking.
here we report the complete genome sequence of p. sabinae t <dig> which is a nitrogen-fixer isolated from the rhizosphere of plant sabina squamata by our laboratory  <cit> .
the whole genome analysis not only reveals the organization and distribution of nitrogen-fixing genes and nitrogenase-like genes, but also provides insight into the evolution of nif genes in paenibacillus.
furthermore, we demonstrate that the complete nif gene cluster consisting of ten genes  of p. sabinae t <dig> is a functional unit for nitrogen fixation.
here for the first time we demonstrated that nifhdk-like genes are not involved in nitrogen fixation.
RESULTS
general features of paenibacillus sabinaet <dig> genome
the complete genome of p. sabinae t <dig> is composed of a single circular molecule of  <dig> , <dig> base pairs  with an average g + c content of  <dig> %.
the circular chromosome has a total of  <dig>  putative protein-coding sequences ,  <dig> rrnas  and  <dig> trnas .
among the predicted genes,  <dig>  were assigned putative functions, covering  <dig> % of the genome, and  <dig>  encoded hypothetical proteins .
twenty eight insertion sequence  elements were identified in the p. sabinae t <dig> genome.table  <dig>
general features of the genome of
p. sabinae
t27
comparative genomics of p. sabinaet27
previous phylogeny based on nifhdk showed that firmicutes, cyanobacteria and actinobacteria are closely related  <cit> .
here we compared the genomes of p. sabinae t <dig>  clostridium acetobutylicum atcc  <dig> , frankia sp.
cci <dig>  and nostoc punctiforme pcc  <dig> .
the four species had the core genome of  <dig> putative protein-coding genes .
there are  <dig> genes which are shared by p. sabinae t27and c. acetobutylicum atcc  <dig>  there are  <dig> genes which are shared by p. sabinae t27and frankia sp.
cci <dig>  and there are  <dig> genes which are shared by p. sabinae t <dig> and n. punctiforme pcc  <dig>  the shared genes by p. sabinae t27and c. acetobutylicum are more than those shared by p. sabinae t27with frankia sp.
cci <dig> or n. punctiforme.
the results are consistent with the fact that paenibacillus is more closely related to clostridium than to frankia and cyanobacteria, since paenibacillus and clostridium belong to the same firmicutes.
furthermore, the genome of p. sabinae t <dig> was compared with those of the closely related paenibacillus azotofixans atcc <dig>   <cit>  and paenibacillu polymyxa sc <dig>   <cit>  .
genome sizes of p. sabinae t <dig>  p. azotofixans atcc <dig> and p. polymyxa sc <dig> are  <dig>  mb,  <dig>  mb and  <dig>  mb, respectively.
chromosome alignments showed higher level of conservation of genome architecture between p. sabinae t <dig> and p. polymyxa sc <dig> than that between p. sabinae t <dig> and p. azotofixans atcc <dig> figure  <dig>
comparative analysis of the complete genome sequence of
p. sabinae
t <dig>   alignment of the chromosomes from p. sabinae t <dig>  p. azotofixans atcc <dig> and p. polymyxa sc <dig>  generated using the artemis comparison tool.
the gray bands located at the top, middle and bottom represent the forward and reverse dna strands for the chromosome sequences.
the red lines correspond to regions of similarity between two chromosomes.
the blue lines correspond to regions of rearrangement and recombination between two chromosomes.
white regions are those that are unique to one strain.
venn diagram depicting the shared genes which were present in all the p. sabinae t <dig>  c. acetobutylicum atcc  <dig>  frankia sp.
cci <dig> and n. punctiforme pcc  <dig> and the specific genes which were present and absent from all the four species, and vice-versa.
central metabolism
p. sabinae t <dig> is a nitrogen-fixing bacterium isolated from the rhizosphere of the plant sabina squamata <cit> .
the bacterium contains a wide spectrum of genes for carbon utilization and carbohydrate, amino acid and inorganic ion transport.
the genome of p. sabinae t <dig> contains the complete set of genes for the pentose phosphate pathway  .
in addition to the metabolism of pentose, the non-oxidative ppp allows the production of intermediates necessary for nucleic acid synthesis.
it contains the complete set of genes for the glycolysis pathway and allows production of acetyl-coa.
in the presence of external electron acceptors, acetyl-coa may be completely oxidized via the citrate cycle , which is encoded by the p. sabinae t <dig> genome .
although the gene coding for the classical malate dehydrogenase  in tca cycle is absent, another malate dehydrogenase  gene which might be involved in pyruvate metabolism pathway metabolizing oxaloacetate to malate, is found in the genome of p. sabinae t <dig>
sucrose is the common carbon source used for isolation of p. sabinae t <dig>  <cit> .
the genome of the bacterium has the sucrose-6-phosphate hydrolase and alpha-glucosidase for metabolizing sucrose to glucose and fructose.
transporter systems are an important element for bacteria to communicate with their environment.
the genome of p. sabinae t <dig> contains an extensive set of  <dig> transport related genes.
of the  <dig> transport related genes,  <dig> are involved in carbohydrate transport,  <dig> encode components of amino acid transporters and  <dig> encode components of inorganic ion transporters.
importantly, fe , molybdenum, sulfate and nh4+ are related to nitrogen fixation and nitrogen metabolism.
nitrogen fixation and nitrogenase-like genes
one of the most distinct features of p. sabinae t <dig> is its ability to fix nitrogen.
the genome of p. sabinae t <dig> contains fifteen nif genes, including four nifb, three nifh, one nifd, one nifk, two nife, two nifn, one nifx and one nifv.
of the  <dig> nif genes, eight nif genes  and two non-nif genes  which are located between nifx and nifv form a complete nif gene cluster, the four nif genes  are clustered together and the other three nif genes  are scattered at different locations .
in addition to the nif genes, there are nitrogenase-like genes, including two nifh-like and five pairs of nifdk-like genes.
our results are consistent with the reports that nifhdk-like genes existed in archaea and firmicutes  <cit> .
interestingly, genome of p. sabinae t <dig> does not contain transcription regulatory gene nifa which is found in almost all of gram-negative diazotrophs, such as in k. pneumoniae, a. vinelandii and pseudomonas stutzeri a <dig>  <cit> .
the lack of nifa suggests that there may be a different regulation mechanism of nitrogen fixation in p. sabinae t <dig> figure  <dig>
chromosome map and distribution of
nif
genes in
p
.
sabinae
t <dig>  a. chromosome map.
circles  <dig> and  <dig> are genes encoded by leading and lagging strands, respectively.
coding sequences are color-coded by cog categories.
circle  <dig> and  <dig> are distribution of trna  genes and rrna  genes in the + strand and – strand, respectively.
circle  <dig> and  <dig> show g + c content and gc skew , respectively.
the positions of nif genes and nif clusters are indicated in the chromosome map.
b. the organization of nif genes.
c. the organization of nif-like genes.
the content and organization of the complete nifgene cluster
bioinformatics analysis revealed that the ten genes nifbhdkenxorf1hesanifv within the complete nif gene cluster are organized as an operon within an 11 kb region.
the gene designated as hesa is also found in frankia <cit>  and cyanobacteria  <cit> .
the orf <dig>  whose predicted product is a hypothetical protein, is also found in several n2-fixing paenibacillus species  <cit> .
the predicted product of hesa shares ~ 45% identity with the putative molybdenum cofactor biosynthesis protein hesa.
hesa is a member of the thif-moeb-hesa family and contains an n-terminal nucleotide binding domain and a c-terminal moez/moeb-like domain.
the gene content and organization of the complete nif gene cluster is unique to paenibacillus <cit> .
although paenibacillus and clostridium are the members of the firmicute, their nif gene content and organization varied greatly.
for example, nifn-b fusion gene was found in the nif gene clusters of the three species of clostridia: c. acetobutylicum, c. beijerinckii, and c. pasteurianum.
also, there are two genes nifi <dig> and nifi <dig> located between nifh and nifdk in c. acetobutylicum and c. beijerinckii <cit> .
previous studies demonstrated that nifi <dig> and nifi <dig> are not essential for nitrogen fixation, but serve a regulatory function  <cit> .
actually, the nif gene content and organization of clostridium spp.
are more similar to those of methanosarcina acetovorans and methanococcus maripaudis, since two genes nifi <dig> and nifi <dig> also exist between nifh and nifdk in these archaea.
is may play important roles in the evolution of the nifand nif-like genes
as described above, twenty eight insertion sequence  elements, belonging to six transposase families were identified in p. sabinae t <dig> chromosome.
is elements were found to be located on the flanking region of the complete nif gene cluster, other nif genes and nif-like genes .
it is generally accepted that is abundance correlates positively with the frequency of horizontal gene transfer   <cit> .
is elements can mediate the transfer of genetic information  between genomes or between replicons of the same genome and they can also induce duplications, deletions, and rearrangements of genetic information  <cit> .
the existence of transposase in the flanking region of the complete nif gene cluster suggests that the nif cluster might be acquired in p. sabinae t <dig> by hgt event from other diazotrophs and the additional nifbhen genes and nifhdk-like genes might be horizontally transferred or duplicated.
the nif genes acquired by hgt were also reported in several diazotrophs.
for example, a sequence reminiscent of a transposase gene located just upstream the nif cluster in herbaspirillum seropedicae is an indicative of hgt event  <cit> .
it was generally recognized that variations of g + c contents between nif cluster and genome are indicative of hgt.
for example, g + c content of the nif gene cluster was higher than the average of the entire genome  in p. stutzeri a <dig>  <cit> .
however, we found that the g + c contents of the complete nif gene cluster of p. sabinae t <dig> is as same as the average of the entire genome , suggesting that the complete nif gene cluster of p. sabinae t <dig> has undergone a longer time of evolution.figure  <dig>
nif
and
nif
-like genes linked to transposonal elements.
evolution of nifand nif-like genes of p. sabinaet27
to further evaluate the evolution of nitrogen fixation in p. sabinae t <dig>  we reconstructed the phylogenies based on the concatenation of the nifbhdkenxv sequences .
notably, the nif protein sequences of p. sabinae t <dig> used for the phylogenetic trees were from the complete nif cluster.
the phylogenetic tree showed that paenibacillus and frankia are sister groups, suggesting that p. sabinae t <dig> may originate from a common ancestor with frankia.
also, we constructed the phylogenetic trees based on the hesa and orf <dig> sequences which are contained within the complete nif gene cluster.
the hesa phylogenetic tree revealed supported that paenibacillus and frankia are sister groups .
the orf <dig> phylogenetic tree showed that p. sabinae t <dig> is closely related to clostridium .
is element on the flanking region of the complete nif cluster suggested that the complete nif cluster may have been acquired in p. sabinae t <dig> by hgt.
interestingly, these data revealed that although paenibacillus and clostridium are the members of the firmicutes, their nif genes are not very closely related.figure  <dig>
maximum-likelihood tree based on nifbhdkenxv protein sequences of
p. sabinae
t <dig> and the representative microorganisms.
the numbers at the nodes indicate levels of bootstrap support  based on a neighbor-joining analysis of  <dig> resampled datasets; only values at or above 50% are given, bar  <dig>  substitutions per amino acid position.
the complete genome sequence revealed that there are three nifh, one nifd, two nifh-like, five pairs of nifdk-like genes in p. sabinae t <dig>  here we constructed phylogenetic trees with real nifh/nifd/nifk and nifh/nifd/nifk-like sequences  and the phylogenetic tree revealed that nifh/nifd/nifk-like sequences are clearly divergent from conventional nitrogenase.
all nifh-like, nifd-like and nifk-like sequences are clustered together by themselves, suggesting that they may have been resulted from duplication.figure  <dig>
maximum-likelihood phylogenetic tree of nifhdk/nifhdk-like sequences.
nifhdk/nifhdk-like sequences were derived from p. sabinae t <dig> and the representative microorganisms.
the numbers at the nodes indicate levels of bootstrap support  based on a neighbor-joining analysis of  <dig> resampled datasets; only values at or above 50% are given, bar  <dig>  substitutions per amino acid position.
as described above, in addition to the ten genes nifbhdkenxorf1hesanifv within the complete nif gene cluster, three nifb, two nifh, one nife and one nifn genes exist in the genome of p. sabinae t <dig>  here we further constructed nifb, nifh and the concatenated nifen phylogenetic trees  and phylogenetic analysis revealed that these multiple nifb, nifh and nifen are clustered with their own corresponding genes within the complete nif gene cluster, suggesting that they may result from duplication of nifb, nifh, nife and nifn, respectively, of the complete nif gene cluster.
characterization of multiple nitrogenase-like genes
the nifhdk are structural genes of mo-nitrogenase, with the nifd and nifk genes encoding the α and β subunits, respectively, of the molybdenum iron protein  and the nifh the γ subunit of the iron protein .
the genome of p. sabinae t <dig> contains two nifh-like, five nifd-like and five nifk-like genes.
conserved residues in alignments of nifh-like sequences  with nifh sequences show that 4fe-4s iron sulfur cluster-ligating cysteines and the p-loop/mgatp binding motif are invariant, suggesting that these proteins may function analogously to dinitrogenase reductase.
conversely, nifd/nifk-like sequences are highly diverged from both the nitrogenase subunits.
femoco-ligating residues at αcys <dig> and αhis <dig> of nifd  are not conserved in nifd/nifk-like sequences, although several—but not all—conserved cysteines involved with p-cluster coordination are found in nifd/nifk-like sequences.figure  <dig>
alignment surrounding the mgatp binding motif  and 4fe-4s coordinating cysteines  for nifh and nifh-like protein sequences from
p. sabinae
t <dig>  numbering is based on
a. vinelandii
nifh.
ab = a. brasilense, av = a. vinelandii, ct tls = c. tepidum tls, cb = clostridium beijerinckii, cc 743b = c. cellulovorans 743b, c ucyn-a = cyanobacterium ucyn-a, c 51142 = c. sp.
atcc  <dig>  c 8802 = c. sp.
pcc  <dig>  fal = f. alni acn14a, fsp = frankia sp., f cci3 = f. sp.
cci <dig>  f ean1pec = f. sp.
ean1pec, f euik1 = f. sp.
euik <dig>  hc = h.chlorum, kp = k. pneumoniae  <dig>  mm = m. maripaludis, mm s2 = m. maripaludis s <dig>  na 0708 = n. azollae  <dig>  n 7120 = n. sp.
pcc  <dig>  pf = p. fujiensis, pt = p. terrae hpl- <dig>  ssp = s.
sp., s ja-3-3ab = s. sp.
ja-3-3ab, te = t. erythraeum ims <dig>
conservation in and around crucial residues  in nifd and nifd-like protein sequences from
p. sabinae
the p-cluster and femo-co ligands, based on
a vinelandii
numbering, are indicated with vertical arrows.
ab = a. brasilense, av = a. vinelandii, ct tls = c. tepidum tls, cb = clostridium beijerinckii, cc 743b = c. cellulovorans 743b, c ucyn-a = cyanobacterium ucyn-a, c 51142 = c. sp.
atcc  <dig>  c 8802 = c. sp.
pcc  <dig>  fal = f. alni acn14a, fsp = frankia sp., f cci3 = f. sp.
cci <dig>  f ean1pec = f. sp.
ean1pec, f euik1 = f. sp.
euik <dig>  hc = h.chlorum, kp = k. pneumoniae  <dig>  mm = m. maripaludis, mm s2 = m. maripaludis s <dig>  na 0708 = n. azollae  <dig>  n 7120 = n. sp.
pcc  <dig>  pf = p. fujiensis, pt = p. terrae hpl- <dig>  ssp = s.
sp., s ja-3-3ab = s. sp.
ja-3-3ab, te = t. erythraeum ims <dig>
conservation in and around crucial residues  in nifk and nifk-like protein sequences from
p. sabinae
t <dig>  the p-cluster ligands based on a. vinelandii numbering, are indicated with vertical arrows.
ab = a. brasilense, av = a. vinelandii, ct tls = c. tepidum tls, cb = clostridium beijerinckii, cc 743b = c. cellulovorans 743b, c ucyn-a = cyanobacterium ucyn-a, c 51142 = c. sp.
atcc  <dig>  c 8802 = c. sp.
pcc  <dig>  fal = f. alni acn14a, fsp = frankia sp., f cci3 = f. sp.
cci <dig>  f ean1pec = f. sp.
ean1pec, f euik1 = f. sp.
euik <dig>  hc = h.chlorum, kp = k. pneumoniae  <dig>  mm = m. maripaludis, mm s2 = m. maripaludis s <dig>  na 0708 = n. azollae  <dig>  n 7120 = n. sp.
pcc  <dig>  pf = p. fujiensis, pt = p. terrae hpl- <dig>  ssp = s.
sp., s ja-3-3ab = s. sp.
ja-3-3ab, te = t. erythraeum ims <dig>
expressions of nifhdkand nifhdk-like genes in n2-fixing and non-n2-fixing conditions
it is generally recognized that nif genes are expressed in n2-fixing conditions .
in order to examine whether the transcription of nifhdk and nifhdk-like genes is regulated by ammonium and oxygen in paenibacillus, expression levels of the p. sabinae t <dig> nifh, nifd, nifk, nifh-like, nifd-like and nifk-like genes were detected by the real-time quantitative rt-pcr method using rna isolated from cells grown under n2-fxing and non-n2-fxing conditions.
as shown in figure  <dig>  a large  increase in the transcript levels of the nifh, nifd and nifk genes was observed in n2-fixing conditions compared to those in the non- n <dig> fixing conditions.
especially, nifh <dig>  nifd <dig> and nifk <dig> within the complete nif cluster were significantly expressed in n2-fixing conditions compared to those in the non- n <dig> fixing conditions.
the data are consistent with the previous reports that the transcription of nifhdk genes is regulated by ammonium and oxygen in n2-fixing organisms, suggesting that the nifhdk genes of p. sabinae t <dig> are involved in nitrogen fixation.
in contrast to nifhdk, nifhdk-like genes of p. sabinae t <dig> were not significantly differently expressed in n2-fixing and non-n2-fixing conditions, suggesting that these nif-like genes did not function in nitrogen fixation.figure  <dig>
quantitative real-time rt-pcr analysis of transcripts of
nifhdk
and
nifhdk-
like genes of
p. sabinae
t <dig>
functional analysis of nifh/nifh-like and nifd/nifd-like genes in nitrogen fixation
to further comparatively study the functions of the nif and nif-like gene of p. sabinae t <dig>  k. pneumonia nifh− mutant strain  <dig> and nifd mutant strain iα423p  <cit> , both of which have no or very low nitrogenase activity, were complemented with the nifh/nifh-like and nifd/nifd-like genes of p. sabinae t <dig> under the control of k. pneumonia nifh promoter, respectively.
as shown in figure 10a, the complementary strains carrying nifh-like <dig> or nifh-like <dig> of p. sabinae t <dig> could not resumed the nitrogenase activity of k. pneumonia nifh− mutant strain  <dig>  while the nifh <dig> from the complete nif cluster of p. sabinae t <dig> could restore to nearly 50% of the wild-type strain m5al.
the data are consistent with our previous report that the three copies of nifh could restore nitrogenase activity of k. pneumonia nifh− mutant strain  <dig>  <cit> .
likewise, nifd of p. sabinae t <dig> could resumed the nitrogenase activity of k. pneumonia nifd− mutant strain iα423p, although k. pneumonia nifd enabled k. pneumonia nifd− mutant strain iα423p to have higher nitrogenase activity than p. sabinae t <dig> nifd did .
in contrast, none of nifd-like <dig>  nifd-like <dig>  nifd-like <dig>  nifd-like <dig> and nifd-like <dig> could restore the nitrogenase activity of k. pneumonia nifd− mutant.
these data suggest that nif-like genes may be not involved in nitrogen fixation.figure  <dig>
the nitrogenase activities of complementary strains and control strains.
kp m5a1: wild-type k. pneumonia; kp1795: k. pneumonia nifh mutant strain; iα423p: k. pneumonia nifd mutant strain.
complementation by nifh/nifh-like genes.
complementation by nifd/nifd-like genes.
the complete nifgene cluster is organized as an operon
bioinformatics analysis revealed that the ten genes nifbhdkenxorf1hesanifv within the complete nif gene cluster are organized as an operon.
here rt-pcr experiments using primers designed to span across intergenic regions indicated that the nine genes within the nif cluster are organized in a single operon .
single operon nif clusters have been reported in gram-positive prokaryotes and in the archaea, e.g.
heliobacterium chlorum <cit>  and methanococcus maripaludis <cit> .
however, in contrast to these nif clusters p. sabinae t <dig> does not contain the negative regulatory genes nifi <dig> and nifi <dig> , which are involved in post-translational regulation of nitrogenase activity in response to fixed nitrogen  <cit> .
the complete nifgene cluster of p. sabinaet <dig> has a σ70-dependent promoter
almost all of the nif genes in gram-negative nitrogen-fixing bacteria, such as k. pneumoniae and a. vinelandii, are transcribed from σ <dig> promoters  whose expression depends on activator nifa  <cit> .
however, the presumed promoter regions for the nif genes of p. sabinae t <dig> have sequences which are similar to the e. coli σ70-dependent − <dig> and − <dig> consensus promoter.
the following experiments demonstrated that the nif promoter of p. sabinae t <dig> is distinct from those of those of gram-negative nitrogen-fixing bacteria.
the transcriptional start site  of the nif gene cluster in p. sabinae t <dig> was determined by using the 5′-race  method.
the tss was located 222 bp upstream of the translational start site of nifb and a putative promoter was identified  <dig> nucleotides preceding the tss .
the − <dig>  and − <dig>  sequences in the nifb promoter were similar to the corresponding consensus sequences  of e. coli σ70-dependent promoters.
a σ54-dependent −24/- <dig> promoter sequence was not observed upstream of the nif cluster.
downstream of nifv, a potential transcriptional termination site was identified, containing two potential stem loops followed by a t-rich region .
these findings indicate that the nif genes in p. sabinae t <dig> are organized as a single operon containing  <dig> genes, which is transcribed from an rpod-dependent promoter.
to analyze the σ70-dependentcy of the nifb promoter, electrophoretic mobility shift assays  were carried out using either e. coli σ70-rnap  or σ <dig> from p. sabinae t <dig>  which was overexpressed and purified from e. coli .
emsa experiments revealed that both purified σ <dig> from p. sabinae t <dig> and e. coli σ70-rnap holoenzyme bind to the 45 bp nifb promoter fragment.
competition experiments with non-labelled nifb dna indicated that the e. coli rnap holoenzyme binds more tightly to this dna fragment, since higher concentrations of competitor were apparently required to dissociate the e. coli σ70-rnap .
these results are consistent with the ability of σa  of bacillus subtilis to bind to promoters independent of core rnap  <cit> .
the complete nifgene cluster of p. sabinaet <dig> enables e. colito fix nitrogen
we further cloned the 12-kb full-length nif gene cluster consisting of its own nif promoter and the contiguous nine genes nifbhdkenxorf1hesanifv into the wide-host plasmid pvk <dig> and then transformed this into e. coli jm <dig>  yielding the recombinant e. coli strain  <dig> .
to determine whether the paenibacillus nif gene cluster functions in e. coli, we employed two independent methods to assess nitrogenase activity: firstly, reduction of the alternative substrate acetylene to ethylene, which can be readily quantified by gas chromatography  <cit>  and secondly, a 15 n <dig> enrichment assay to directly measure the incorporation of this tracer into organic nitrogen  <cit> .
when grown anaerobically in nitrogen-deficient medium, p. sabinae t <dig> exhibits both acetylene reduction and 15 n <dig> incorporation .
the recombinant e. coli strain  <dig>  which expresses the nif genes from the native promoter showed approximately 10% of the specific activity for acetylene reduction when compared with paenibacillus and was competent to assimilate 15 n <dig>  the results demonstrated that the complete nif gene cluster is a functional unit.
CONCLUSIONS
in this study, we uncovered the contents and organization of nif and nif-like genes of p. sabinae t <dig> by completing its genome sequence.
the genome of p. sabinae t <dig> contains fifteen nitrogen fixation  genes, including three nifh, one nifd, one nifk, four nifb, two nife, two nifn, one nifx and one nifv.
of the  <dig> nif genes, eight nif genes  and two non-nif genes  form a complete nif gene cluster.
phylogenetic analysis suggests that the complete nif cluster of p. sabinae t <dig> was originated from a common ancestor with frankia.
multiple nifb, nifh, nife, nifn may result from duplication.
the complete nif gene cluster is organized in an operon as a functional unit for nitrogen fixation.
the complete nif gene cluster under the control of its σ70-dependent promoter enabled escherichia coli jm <dig> to fix nitrogen.
p. sabinae t <dig> contains two nifh-like genes and five pairs of nifdk-like genes.
unlike nif genes, the transcriptions of nifhdk-like genes were not regulated by ammonium and oxygen and nifhdk-like genes were not involved in nitrogen fixation.

 biological nitrogen fixation, the conversion of atmospheric n to nh plays an important role in the global nitrogen cycle and in world agriculture. most biological nitrogen fixation is catalyzed by the molybdenum nitrogenase. this enzyme is a complex which contains the mofe protein encoded by nifdk and the fe protein encoded by nifh. the mofe protein contains two metalloclusters: femo-co, a cluster which serves as the active site of substrate binding and reduction and the p-cluster, a cluster which shuttles electrons to femo-co. previous biochemical and genetic studies on klebsiella pneumoniae carrying twenty nif genes on 24-kb region genes and azotobacter vinelandii revealed that nifh, nifd, nifk, nife, nifn, nifx nifb, nifq, nifv, nify, nifu nifs, nifz and nifm contribute to the synthesis and maturation of nitrogenase. contents and organization of nif genes varied significantly among n2-fixing organisms. for example, in k. pneumoniae, twenty nif genes are co-located within a ~24 kb cluster, whereas in a. vinelandii the nif genes are more dispersed and distributed as two clusters in genome. there is usually only one nifh gene and the nifh, nifd and nifk genes are transcribed as a single unit in many diazotrophs, such as k. pneumoniae and a. vinelandii. however, multiple nifh genes were found in a few diazotrophs. for examples, rhizobium leguminosarum bv. phaseoli possesses three nifh genes and clostridium pasteurianum w has six nifh homologs. nitrogen fixation is sporadically distributed among prokaryote families: proteobacteria, firmicutes, archaea, cyanobacteria and actinobacteria. the incomplete distribution pattern and the difference in contents and organization of nif genes raise the question of origins and evolution of mo-nitrogenase. two conflicting hypotheses for the origin of mo-nitrogenase have been proposed on the basis of phylogenetic examination of mo-nitrogenase protein sequences. the last common ancestor hypothesis implies that the mo-nitrogenase had its origin in a common ancestor of the bacterial and archaeal domains. according to the lca model gene loss has been extensive and accounts for the fact that nitrogenase is found neither in eukaryotes nor in many entire phyla of prokaryotes. the methanogen origin hypothesis implies that nitrogen fixation originated from methanogenic archaea and subsequently was transferred into a primitive bacterium via lateral gene transfer. recent studies based on phylogenetic analysis of nifhdk sequences supported the methanogen origin hypothesis and implied that mo-nitrogenase evolved in the anaerobic and hydrogenotrophic methanogens with acquisition in the bacterial domain via lateral gene transfer involving an anaerobic member of the firmicutes. firmicutes have been thought to play an important role in evolution of nitrogen fixation. studies on evolution of nitrogen fixation in firmicutes mainly focused on the anaerobic diazotrophic clostridia. although paenibacillus is a genus of firmicute, its nitrogen fixation traits and evolution remains unclear. it is well known that paenibacillus is a genus of gram-positive, facultative anaerobic, endospore-forming bacteria, originally included within the genus bacillus and then reclassified as a separate genus in. bacteria belonging to this genus have been detected in a variety of environments such as soil, water, rhizosphere, vegetable matter, forage and insect larvae, as well as clinical samples. nitrogen-fixing paenibacillus species have great potential for use as a bacterial fertilizer in agriculture, but genomic information of these bacteria is lacking. here we report the complete genome sequence of p. sabinae t which is a nitrogen-fixer isolated from the rhizosphere of plant sabina squamata by our laboratory. the whole genome analysis not only reveals the organization and distribution of nitrogen-fixing genes and nitrogenase-like genes, but also provides insight into the evolution of nif genes in paenibacillus. furthermore, we demonstrate that the complete nif gene cluster consisting of ten genes of p. sabinae t is a functional unit for nitrogen fixation. here for the first time we demonstrated that nifhdk-like genes are not involved in nitrogen fixation.  general features of paenibacillus sabinaet genome the complete genome of p. sabinae t is composed of a single circular molecule of, base pairs with an average g + c content of %. the circular chromosome has a total of putative protein-coding sequences, rrnas and trnas. among the predicted genes, were assigned putative functions, covering % of the genome, and encoded hypothetical proteins. twenty eight insertion sequence elements were identified in the p. sabinae t genome.table general features of the genome of p. sabinae t27 comparative genomics of p. sabinaet27 previous phylogeny based on nifhdk showed that firmicutes, cyanobacteria and actinobacteria are closely related. here we compared the genomes of p. sabinae t clostridium acetobutylicum atcc, frankia sp. cci and nostoc punctiforme pcc. the four species had the core genome of putative protein-coding genes. there are genes which are shared by p. sabinae t27and c. acetobutylicum atcc there are genes which are shared by p. sabinae t27and frankia sp. cci and there are genes which are shared by p. sabinae t and n. punctiforme pcc the shared genes by p. sabinae t27and c. acetobutylicum are more than those shared by p. sabinae t27with frankia sp. cci or n. punctiforme. the results are consistent with the fact that paenibacillus is more closely related to clostridium than to frankia and cyanobacteria, since paenibacillus and clostridium belong to the same firmicutes. furthermore, the genome of p. sabinae t was compared with those of the closely related paenibacillus azotofixans atcc and paenibacillu polymyxa sc. genome sizes of p. sabinae t p. azotofixans atcc and p. polymyxa sc are  mb,  mb and  mb, respectively. chromosome alignments showed higher level of conservation of genome architecture between p. sabinae t and p. polymyxa sc than that between p. sabinae t and p. azotofixans atcc figure comparative analysis of the complete genome sequence of p. sabinae t alignment of the chromosomes from p. sabinae t p. azotofixans atcc and p. polymyxa sc generated using the artemis comparison tool. the gray bands located at the top, middle and bottom represent the forward and reverse dna strands for the chromosome sequences. the red lines correspond to regions of similarity between two chromosomes. the blue lines correspond to regions of rearrangement and recombination between two chromosomes. white regions are those that are unique to one strain. venn diagram depicting the shared genes which were present in all the p. sabinae t c. acetobutylicum atcc frankia sp. cci and n. punctiforme pcc and the specific genes which were present and absent from all the four species, and vice-versa. central metabolism p. sabinae t is a nitrogen-fixing bacterium isolated from the rhizosphere of the plant sabina squamata. the bacterium contains a wide spectrum of genes for carbon utilization and carbohydrate, amino acid and inorganic ion transport. the genome of p. sabinae t contains the complete set of genes for the pentose phosphate pathway. in addition to the metabolism of pentose, the non-oxidative ppp allows the production of intermediates necessary for nucleic acid synthesis. it contains the complete set of genes for the glycolysis pathway and allows production of acetyl-coa. in the presence of external electron acceptors, acetyl-coa may be completely oxidized via the citrate cycle, which is encoded by the p. sabinae t genome. although the gene coding for the classical malate dehydrogenase in tca cycle is absent, another malate dehydrogenase gene which might be involved in pyruvate metabolism pathway metabolizing oxaloacetate to malate, is found in the genome of p. sabinae t sucrose is the common carbon source used for isolation of p. sabinae t. the genome of the bacterium has the sucrose-6-phosphate hydrolase and alpha-glucosidase for metabolizing sucrose to glucose and fructose. transporter systems are an important element for bacteria to communicate with their environment. the genome of p. sabinae t contains an extensive set of transport related genes. of the transport related genes, are involved in carbohydrate transport, encode components of amino acid transporters and encode components of inorganic ion transporters. importantly, fe, molybdenum, sulfate and nh4+ are related to nitrogen fixation and nitrogen metabolism. nitrogen fixation and nitrogenase-like genes one of the most distinct features of p. sabinae t is its ability to fix nitrogen. the genome of p. sabinae t contains fifteen nif genes, including four nifb, three nifh, one nifd, one nifk, two nife, two nifn, one nifx and one nifv. of the nif genes, eight nif genes and two non-nif genes which are located between nifx and nifv form a complete nif gene cluster, the four nif genes are clustered together and the other three nif genes are scattered at different locations. in addition to the nif genes, there are nitrogenase-like genes, including two nifh-like and five pairs of nifdk-like genes. our results are consistent with the reports that nifhdk-like genes existed in archaea and firmicutes. interestingly, genome of p. sabinae t does not contain transcription regulatory gene nifa which is found in almost all of gram-negative diazotrophs, such as in k. pneumoniae, a. vinelandii and pseudomonas stutzeri a. the lack of nifa suggests that there may be a different regulation mechanism of nitrogen fixation in p. sabinae t figure chromosome map and distribution of nif genes in p. sabinae t a. chromosome map. circles and are genes encoded by leading and lagging strands, respectively. coding sequences are color-coded by cog categories. circle and are distribution of trna genes and rrna genes in the + strand and – strand, respectively. circle and show g + c content and gc skew, respectively. the positions of nif genes and nif clusters are indicated in the chromosome map. b. the organization of nif genes. c. the organization of nif-like genes. the content and organization of the complete nifgene cluster bioinformatics analysis revealed that the ten genes nifbhdkenxorf1hesanifv within the complete nif gene cluster are organized as an operon within an 11 kb region. the gene designated as hesa is also found in frankia and cyanobacteria. the orf whose predicted product is a hypothetical protein, is also found in several n2-fixing paenibacillus species. the predicted product of hesa shares ~ 45% identity with the putative molybdenum cofactor biosynthesis protein hesa. hesa is a member of the thif-moeb-hesa family and contains an n-terminal nucleotide binding domain and a c-terminal moez/moeb-like domain. the gene content and organization of the complete nif gene cluster is unique to paenibacillus. although paenibacillus and clostridium are the members of the firmicute, their nif gene content and organization varied greatly. for example, nifn-b fusion gene was found in the nif gene clusters of the three species of clostridia: c. acetobutylicum, c. beijerinckii, and c. pasteurianum. also, there are two genes nifi and nifi located between nifh and nifdk in c. acetobutylicum and c. beijerinckii. previous studies demonstrated that nifi and nifi are not essential for nitrogen fixation, but serve a regulatory function. actually, the nif gene content and organization of clostridium spp. are more similar to those of methanosarcina acetovorans and methanococcus maripaudis, since two genes nifi and nifi also exist between nifh and nifdk in these archaea. is may play important roles in the evolution of the nifand nif-like genes as described above, twenty eight insertion sequence elements, belonging to six transposase families were identified in p. sabinae t chromosome. is elements were found to be located on the flanking region of the complete nif gene cluster, other nif genes and nif-like genes. it is generally accepted that is abundance correlates positively with the frequency of horizontal gene transfer. is elements can mediate the transfer of genetic information between genomes or between replicons of the same genome and they can also induce duplications, deletions, and rearrangements of genetic information. the existence of transposase in the flanking region of the complete nif gene cluster suggests that the nif cluster might be acquired in p. sabinae t by hgt event from other diazotrophs and the additional nifbhen genes and nifhdk-like genes might be horizontally transferred or duplicated. the nif genes acquired by hgt were also reported in several diazotrophs. for example, a sequence reminiscent of a transposase gene located just upstream the nif cluster in herbaspirillum seropedicae is an indicative of hgt event. it was generally recognized that variations of g + c contents between nif cluster and genome are indicative of hgt. for example, g + c content of the nif gene cluster was higher than the average of the entire genome in p. stutzeri a. however, we found that the g + c contents of the complete nif gene cluster of p. sabinae t is as same as the average of the entire genome, suggesting that the complete nif gene cluster of p. sabinae t has undergone a longer time of evolution.figure nif and nif -like genes linked to transposonal elements. evolution of nifand nif-like genes of p. sabinaet27 to further evaluate the evolution of nitrogen fixation in p. sabinae t we reconstructed the phylogenies based on the concatenation of the nifbhdkenxv sequences. notably, the nif protein sequences of p. sabinae t used for the phylogenetic trees were from the complete nif cluster. the phylogenetic tree showed that paenibacillus and frankia are sister groups, suggesting that p. sabinae t may originate from a common ancestor with frankia. also, we constructed the phylogenetic trees based on the hesa and orf sequences which are contained within the complete nif gene cluster. the hesa phylogenetic tree revealed supported that paenibacillus and frankia are sister groups. the orf phylogenetic tree showed that p. sabinae t is closely related to clostridium. is element on the flanking region of the complete nif cluster suggested that the complete nif cluster may have been acquired in p. sabinae t by hgt. interestingly, these data revealed that although paenibacillus and clostridium are the members of the firmicutes, their nif genes are not very closely related.figure maximum-likelihood tree based on nifbhdkenxv protein sequences of p. sabinae t and the representative microorganisms. the numbers at the nodes indicate levels of bootstrap support based on a neighbor-joining analysis of resampled datasets; only values at or above 50% are given, bar substitutions per amino acid position. the complete genome sequence revealed that there are three nifh, one nifd, two nifh-like, five pairs of nifdk-like genes in p. sabinae t here we constructed phylogenetic trees with real nifh/nifd/nifk and nifh/nifd/nifk-like sequences and the phylogenetic tree revealed that nifh/nifd/nifk-like sequences are clearly divergent from conventional nitrogenase. all nifh-like, nifd-like and nifk-like sequences are clustered together by themselves, suggesting that they may have been resulted from duplication.figure maximum-likelihood phylogenetic tree of nifhdk/nifhdk-like sequences. nifhdk/nifhdk-like sequences were derived from p. sabinae t and the representative microorganisms. the numbers at the nodes indicate levels of bootstrap support based on a neighbor-joining analysis of resampled datasets; only values at or above 50% are given, bar substitutions per amino acid position. as described above, in addition to the ten genes nifbhdkenxorf1hesanifv within the complete nif gene cluster, three nifb, two nifh, one nife and one nifn genes exist in the genome of p. sabinae t here we further constructed nifb, nifh and the concatenated nifen phylogenetic trees and phylogenetic analysis revealed that these multiple nifb, nifh and nifen are clustered with their own corresponding genes within the complete nif gene cluster, suggesting that they may result from duplication of nifb, nifh, nife and nifn, respectively, of the complete nif gene cluster. characterization of multiple nitrogenase-like genes the nifhdk are structural genes of mo-nitrogenase, with the nifd and nifk genes encoding the α and β subunits, respectively, of the molybdenum iron protein and the nifh the γ subunit of the iron protein. the genome of p. sabinae t contains two nifh-like, five nifd-like and five nifk-like genes. conserved residues in alignments of nifh-like sequences with nifh sequences show that 4fe-4s iron sulfur cluster-ligating cysteines and the p-loop/mgatp binding motif are invariant, suggesting that these proteins may function analogously to dinitrogenase reductase. conversely, nifd/nifk-like sequences are highly diverged from both the nitrogenase subunits. femoco-ligating residues at αcys and αhis of nifd are not conserved in nifd/nifk-like sequences, although several—but not all—conserved cysteines involved with p-cluster coordination are found in nifd/nifk-like sequences.figure alignment surrounding the mgatp binding motif and 4fe-4s coordinating cysteines for nifh and nifh-like protein sequences from p. sabinae t numbering is based on a. vinelandii nifh.  ab = a. brasilense, av = a. vinelandii, ct tls = c. tepidum tls, cb = clostridium beijerinckii, cc 743b = c. cellulovorans 743b, c ucyn-a = cyanobacterium ucyn-a, c 51142 = c. sp. atcc c 8802 = c. sp. pcc fal = f. alni acn14a, fsp = frankia sp., f cci3 = f. sp. cci f ean1pec = f. sp. ean1pec, f euik1 = f. sp. euik hc = h.chlorum, kp = k. pneumoniae mm = m. maripaludis, mm s2 = m. maripaludis s na 0708 = n. azollae n 7120 = n. sp. pcc pf = p. fujiensis, pt = p. terrae hpl- ssp = s. sp., s ja-3-3ab = s. sp. ja-3-3ab, te = t. erythraeum ims conservation in and around crucial residues in nifd and nifd-like protein sequences from p. sabinae the p-cluster and femo-co ligands, based on a vinelandii numbering, are indicated with vertical arrows. ab = a. brasilense, av = a. vinelandii, ct tls = c. tepidum tls, cb = clostridium beijerinckii, cc 743b = c. cellulovorans 743b, c ucyn-a = cyanobacterium ucyn-a, c 51142 = c. sp. atcc c 8802 = c. sp. pcc fal = f. alni acn14a, fsp = frankia sp., f cci3 = f. sp. cci f ean1pec = f. sp. ean1pec, f euik1 = f. sp. euik hc = h.chlorum, kp = k. pneumoniae mm = m. maripaludis, mm s2 = m. maripaludis s na 0708 = n. azollae n 7120 = n. sp. pcc pf = p. fujiensis, pt = p. terrae hpl- ssp = s. sp., s ja-3-3ab = s. sp. ja-3-3ab, te = t. erythraeum ims conservation in and around crucial residues in nifk and nifk-like protein sequences from p. sabinae t the p-cluster ligands based on a. vinelandii numbering, are indicated with vertical arrows. ab = a. brasilense, av = a. vinelandii, ct tls = c. tepidum tls, cb = clostridium beijerinckii, cc 743b = c. cellulovorans 743b, c ucyn-a = cyanobacterium ucyn-a, c 51142 = c. sp. atcc c 8802 = c. sp. pcc fal = f. alni acn14a, fsp = frankia sp., f cci3 = f. sp. cci f ean1pec = f. sp. ean1pec, f euik1 = f. sp. euik hc = h.chlorum, kp = k. pneumoniae mm = m. maripaludis, mm s2 = m. maripaludis s na 0708 = n. azollae n 7120 = n. sp. pcc pf = p. fujiensis, pt = p. terrae hpl- ssp = s. sp., s ja-3-3ab = s. sp. ja-3-3ab, te = t. erythraeum ims expressions of nifhdkand nifhdk-like genes in n2-fixing and non-n2-fixing conditions it is generally recognized that nif genes are expressed in n2-fixing conditions. in order to examine whether the transcription of nifhdk and nifhdk-like genes is regulated by ammonium and oxygen in paenibacillus, expression levels of the p. sabinae t nifh, nifd, nifk, nifh-like, nifd-like and nifk-like genes were detected by the real-time quantitative rt-pcr method using rna isolated from cells grown under n2-fxing and non-n2-fxing conditions. as shown in figure  a large increase in the transcript levels of the nifh, nifd and nifk genes was observed in n2-fixing conditions compared to those in the non- n fixing conditions. especially, nifh nifd and nifk within the complete nif cluster were significantly expressed in n2-fixing conditions compared to those in the non- n fixing conditions. the data are consistent with the previous reports that the transcription of nifhdk genes is regulated by ammonium and oxygen in n2-fixing organisms, suggesting that the nifhdk genes of p. sabinae t are involved in nitrogen fixation. in contrast to nifhdk, nifhdk-like genes of p. sabinae t were not significantly differently expressed in n2-fixing and non-n2-fixing conditions, suggesting that these nif-like genes did not function in nitrogen fixation.figure quantitative real-time rt-pcr analysis of transcripts of nifhdk and nifhdk- like genes of p. sabinae t functional analysis of nifh/nifh-like and nifd/nifd-like genes in nitrogen fixation to further comparatively study the functions of the nif and nif-like gene of p. sabinae t k. pneumonia nifh− mutant strain and nifd mutant strain iα423p, both of which have no or very low nitrogenase activity, were complemented with the nifh/nifh-like and nifd/nifd-like genes of p. sabinae t under the control of k. pneumonia nifh promoter, respectively. as shown in figure 10a, the complementary strains carrying nifh-like or nifh-like of p. sabinae t could not resumed the nitrogenase activity of k. pneumonia nifh− mutant strain while the nifh from the complete nif cluster of p. sabinae t could restore to nearly 50% of the wild-type strain m5al. the data are consistent with our previous report that the three copies of nifh could restore nitrogenase activity of k. pneumonia nifh− mutant strain. likewise, nifd of p. sabinae t could resumed the nitrogenase activity of k. pneumonia nifd− mutant strain iα423p, although k. pneumonia nifd enabled k. pneumonia nifd− mutant strain iα423p to have higher nitrogenase activity than p. sabinae t nifd did. in contrast, none of nifd-like nifd-like nifd-like nifd-like and nifd-like could restore the nitrogenase activity of k. pneumonia nifd− mutant. these data suggest that nif-like genes may be not involved in nitrogen fixation.figure the nitrogenase activities of complementary strains and control strains. kp m5a1: wild-type k. pneumonia; kp1795: k. pneumonia nifh mutant strain; iα423p: k. pneumonia nifd mutant strain. complementation by nifh/nifh-like genes. complementation by nifd/nifd-like genes. the complete nifgene cluster is organized as an operon bioinformatics analysis revealed that the ten genes nifbhdkenxorf1hesanifv within the complete nif gene cluster are organized as an operon. here rt-pcr experiments using primers designed to span across intergenic regions indicated that the nine genes within the nif cluster are organized in a single operon. single operon nif clusters have been reported in gram-positive prokaryotes and in the archaea, e.g. heliobacterium chlorum and methanococcus maripaludis. however, in contrast to these nif clusters p. sabinae t does not contain the negative regulatory genes nifi and nifi, which are involved in post-translational regulation of nitrogenase activity in response to fixed nitrogen. the complete nifgene cluster of p. sabinaet has a σ70-dependent promoter almost all of the nif genes in gram-negative nitrogen-fixing bacteria, such as k. pneumoniae and a. vinelandii, are transcribed from σ promoters whose expression depends on activator nifa. however, the presumed promoter regions for the nif genes of p. sabinae t have sequences which are similar to the e. coli σ70-dependent − and − consensus promoter. the following experiments demonstrated that the nif promoter of p. sabinae t is distinct from those of those of gram-negative nitrogen-fixing bacteria. the transcriptional start site of the nif gene cluster in p. sabinae t was determined by using the 5′-race method. the tss was located 222 bp upstream of the translational start site of nifb and a putative promoter was identified nucleotides preceding the tss. the − and − sequences in the nifb promoter were similar to the corresponding consensus sequences of e. coli σ70-dependent promoters. a σ54-dependent −24/- promoter sequence was not observed upstream of the nif cluster. downstream of nifv, a potential transcriptional termination site was identified, containing two potential stem loops followed by a t-rich region. these findings indicate that the nif genes in p. sabinae t are organized as a single operon containing genes, which is transcribed from an rpod-dependent promoter. to analyze the σ70-dependentcy of the nifb promoter, electrophoretic mobility shift assays were carried out using either e. coli σ70-rnap or σ from p. sabinae t which was overexpressed and purified from e. coli. emsa experiments revealed that both purified σ from p. sabinae t and e. coli σ70-rnap holoenzyme bind to the 45 bp nifb promoter fragment. competition experiments with non-labelled nifb dna indicated that the e. coli rnap holoenzyme binds more tightly to this dna fragment, since higher concentrations of competitor were apparently required to dissociate the e. coli σ70-rnap. these results are consistent with the ability of σa of bacillus subtilis to bind to promoters independent of core rnap. the complete nifgene cluster of p. sabinaet enables e. colito fix nitrogen we further cloned the 12-kb full-length nif gene cluster consisting of its own nif promoter and the contiguous nine genes nifbhdkenxorf1hesanifv into the wide-host plasmid pvk and then transformed this into e. coli jm yielding the recombinant e. coli strain. to determine whether the paenibacillus nif gene cluster functions in e. coli, we employed two independent methods to assess nitrogenase activity: firstly, reduction of the alternative substrate acetylene to ethylene, which can be readily quantified by gas chromatography and secondly, a 15 n enrichment assay to directly measure the incorporation of this tracer into organic nitrogen. when grown anaerobically in nitrogen-deficient medium, p. sabinae t exhibits both acetylene reduction and 15 n incorporation. the recombinant e. coli strain which expresses the nif genes from the native promoter showed approximately 10% of the specific activity for acetylene reduction when compared with paenibacillus and was competent to assimilate 15 n the results demonstrated that the complete nif gene cluster is a functional unit.  in this study, we uncovered the contents and organization of nif and nif-like genes of p. sabinae t by completing its genome sequence. the genome of p. sabinae t contains fifteen nitrogen fixation genes, including three nifh, one nifd, one nifk, four nifb, two nife, two nifn, one nifx and one nifv. of the nif genes, eight nif genes and two non-nif genes form a complete nif gene cluster. phylogenetic analysis suggests that the complete nif cluster of p. sabinae t was originated from a common ancestor with frankia. multiple nifb, nifh, nife, nifn may result from duplication. the complete nif gene cluster is organized in an operon as a functional unit for nitrogen fixation. the complete nif gene cluster under the control of its σ70-dependent promoter enabled escherichia coli jm to fix nitrogen. p. sabinae t contains two nifh-like genes and five pairs of nifdk-like genes. unlike nif genes, the transcriptions of nifhdk-like genes were not regulated by ammonium and oxygen and nifhdk-like genes were not involved in nitrogen fixation. 
is elements on the flanking regions of nif and nif-like genes imply that these genes might have been obtained by horizontal gene transfer. most biological nitrogen fixation is catalyzed by the molybdenum nitrogenase. of the nif genes, eight nif genes and two non-nif genes form a complete nif gene cluster. in addition to the nif genes, there are nitrogenase-like genes, including two nifh-like genes and five pairs of nifdk-like genes. also, here for the first time we demonstrated that unlike nif genes, the transcriptions of nifhdk-like genes were not regulated by ammonium and oxygen, and nifh-like or nifd-like gene could not restore the nitrogenase activity of klebsiella pneumonia nifh− and nifd− mutant strains, respectively, suggesting that nifhdk-like genes were not involved in nitrogen fixation. our data and analysis reveal the contents and distribution of nif and nif-like genes and contribute to the study of evolutionary history of nitrogen fixation in paenibacillus. in addition to nifhdk, nifhdk-like genes were found in some archaea and firmicutes, but their function is unclear. for the first time we demonstrated that the transcriptions of nifhdk-like genes were not regulated by ammonium and oxygen and nifhdk-like genes were not involved in nitrogen fixation. rt-pcr analysis showed that the complete nif gene cluster is organized as an operon. we demonstrated that the complete nif gene cluster under the control of σ70-dependent promoter enabled escherichia coli jm to fix nitrogen. the genome of p. sabinae t contains fifteen nitrogen fixation genes, including three nifh, one nifd, one nifk, four nifb, two nife, two nifn, one nifx and one nifv. this enzyme is a complex which contains the mofe protein encoded by nifdk and the fe protein encoded by nifh.
 most biological nitrogen fixation is catalyzed by the molybdenum nitrogenase. this enzyme is a complex which contains the mofe protein encoded by nifdk and the fe protein encoded by nifh. in addition to nifhdk, nifhdk-like genes were found in some archaea and firmicutes, but their function is unclear.  we sequenced the genome of paenibacillus sabinae t a total of open reading frames were predicted from its mb genome. the genome of p. sabinae t contains fifteen nitrogen fixation genes, including three nifh, one nifd, one nifk, four nifb, two nife, two nifn, one nifx and one nifv. of the nif genes, eight nif genes and two non-nif genes form a complete nif gene cluster. in addition to the nif genes, there are nitrogenase-like genes, including two nifh-like genes and five pairs of nifdk-like genes. is elements on the flanking regions of nif and nif-like genes imply that these genes might have been obtained by horizontal gene transfer. phylogenies of the concatenated nif gene products suggest that p. sabinae t is closely related to frankia. rt-pcr analysis showed that the complete nif gene cluster is organized as an operon. we demonstrated that the complete nif gene cluster under the control of σ70-dependent promoter enabled escherichia coli jm to fix nitrogen. also, here for the first time we demonstrated that unlike nif genes, the transcriptions of nifhdk-like genes were not regulated by ammonium and oxygen, and nifh-like or nifd-like gene could not restore the nitrogenase activity of klebsiella pneumonia nifh− and nifd− mutant strains, respectively, suggesting that nifhdk-like genes were not involved in nitrogen fixation.  our data and analysis reveal the contents and distribution of nif and nif-like genes and contribute to the study of evolutionary history of nitrogen fixation in paenibacillus. for the first time we demonstrated that the transcriptions of nifhdk-like genes were not regulated by ammonium and oxygen and nifhdk-like genes were not involved in nitrogen fixation. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords paenibacillus sabinae t27nif gene clusternif-like genegenome sequenceissue-copyright-statement© the author 2014 