The genome of Paenibacillus sabinae T27 provides insight into evolution, organization and functional elucidation of nif and nif-like genes
- Xinxin Li†1, 2,
- Zhiping Deng†1,
- Zhanzhi Liu†1,
- Yongliang Yan†3,
- Tianshu Wang1,
- Jianbo Xie1,
- Min Lin3,
- Qi Cheng3 and
- Sanfeng Chen1, 2Email author
© Li et al.; licensee BioMed Central Ltd. 2014
Received: 7 November 2013
Accepted: 31 July 2014
Published: 27 August 2014
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 T27. A total of 4,793 open reading frames were predicted from its 5.27 Mb genome. The genome of P. sabinae T27 contains fifteen nitrogen fixation (nif) genes, including three nifH, one nifD, one nifK, four nifB, two nifE, two nifN, one nifX and one nifV. Of the 15 nif genes, eight nif genes (nifB, nifH, nifD, nifK, nifE, nifN, nifX and nifV) and two non-nif genes (orf1 and hesA) 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 8 nif gene (nifB, nifH, nifD, nifK, nifE, nifN, nifX and nifV) products suggest that P. sabinae T27 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 JM109 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.
Biological nitrogen fixation, the conversion of atmospheric N2 to NH3, 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 [Mo-7Fe-9S-C-homocitrate] cluster which serves as the active site of substrate binding and reduction and the P-cluster, a [8Fe-7S] cluster which shuttles electrons to FeMo-co [2, 3]. 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 [2, 3].
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 W5 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 (NifHDK) . The last common ancestor (LCA) 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 1993 . 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 T27 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 (nifB, nifH, nifD, nifK, nifE, nifN, nifX, orf1, hesA and nifV) of P. sabinae T27 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 and discussion
General features of Paenibacillus sabinaeT27 genome
General features of the genome of P. sabinae T27
Complete genome size, bp
5, 270, 569
G + C%
Genes with assigned function
Genes with unknown function
Average CDs size
Percent of coding region%
No. of rRNAs
No. of tRNAs
Insertion sequence (IS) elements
Comparative genomics of P. sabinaeT27
P. sabinae T27 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 T27 contains the complete set of genes for the pentose phosphate pathway (PPP) (Additional file 1: Figure S1). 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 (TCA cycle), which is encoded by the P. sabinae T27 genome (Additional file 1: Figure S1). Although the gene coding for the classical malate dehydrogenase (MDH1, EC:18.104.22.168) in TCA cycle is absent, another malate dehydrogenase (MQO, EC:22.214.171.124) gene which might be involved in pyruvate metabolism pathway metabolizing oxaloacetate to malate, is found in the genome of P. sabinae T27.
Sucrose is the common carbon source used for isolation of P. sabinae T27 . 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 T27 contains an extensive set of 247 transport related genes. Of the 247 transport related genes, 64 are involved in carbohydrate transport, 66 encode components of amino acid transporters and 107 encode components of inorganic ion transporters. Importantly, Fe (iron), molybdenum, sulfate and NH4+ are related to nitrogen fixation and nitrogen metabolism.
Nitrogen fixation and nitrogenase-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 orf1, 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[19, 20]. 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 nifI1 and nifI2 located between nifH and nifDK in C. acetobutylicum and C. beijerinckii. Previous studies demonstrated that nifI1 and nifI2 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 nifI1 and nifI2 also exist between nifH and nifDK in these archaea.
IS may play important roles in the evolution of the nif and nif-like genes
Evolution of nif and nif-like genes of P. sabinaeT27
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 T27. Here we further constructed NifB, NifH and the concatenated NifEN phylogenetic trees (Additional files 4, 5, 6: Figures S2-S4) 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
Expressions of nifHDK and nifHDK-like genes in N2-fixing and non-N2-fixing conditions
Functional analysis of nifH/nifH-like and nifD/nifD-like genes in nitrogen fixation
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 (Additional file 7: Figure S7). 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 T27 does not contain the negative regulatory genes nifI1 and nifI2 (homologues of glnB), which are involved in post-translational regulation of nitrogenase activity in response to fixed nitrogen .
The complete nif gene cluster of P. sabinae T27 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 σ54 promoters (−24/-12) whose expression depends on activator NifA . However, the presumed promoter regions for the nif genes of P. sabinae T27 have sequences which are similar to the E. coli σ70-dependent −35 and −10 consensus promoter. The following experiments demonstrated that the nif promoter of P. sabinae T27 is distinct from those of those of Gram-negative nitrogen-fixing bacteria.
The transcriptional start site (TSS) of the nif gene cluster in P. sabinae T27 was determined by using the 5′-RACE (Rapid Amplification of cDNA Ends) method. The TSS was located 222 bp upstream of the translational start site of nifB and a putative promoter was identified 6 nucleotides preceding the TSS (Additional file 8: Figure S8A). The −35 (TTGACG) and −10 (TATGAT) sequences in the nifB promoter were similar to the corresponding consensus sequences (TTGACA and TATAAT respectively) of E. coli σ70-dependent promoters. A σ54-dependent −24/-12 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 (Additional file 8: Figure S8A). These findings indicate that the nif genes in P. sabinae T27 are organized as a single operon containing 9 genes, which is transcribed from an rpoD-dependent promoter.
To analyze the σ70-dependentcy of the nifB promoter, electrophoretic mobility shift assays (EMSA) were carried out using either E. coli σ70-RNAP (RNA polymerase) or σ70 from P. sabinae T27, which was overexpressed and purified from E. coli (Additional file 8: Figure S8B). EMSA experiments revealed that both purified σ70 from P. sabinae T27 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 (Additional file 8: Figure S8C and D). These results are consistent with the ability of σA (σ70) of Bacillus subtilis to bind to promoters independent of core RNAP [32, 33].
The complete nif gene cluster of P. sabinae T27 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 pVK100 and then transformed this into E. coli JM109, yielding the recombinant E. coli strain 27 (Additional file 9: Figure S9). 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 [34, 35] and secondly, a 15 N2 enrichment assay to directly measure the incorporation of this tracer into organic nitrogen . When grown anaerobically in nitrogen-deficient medium, P. sabinae T27 exhibits both acetylene reduction and 15 N2 incorporation (Additional file 9: Figure S9). The recombinant E. coli strain 27, 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 N2. 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 T27 by completing its genome sequence. The genome of P. sabinae T27 contains fifteen nitrogen fixation (nif) genes, including three nifH, one nifD, one nifK, four nifB, two nifE, two nifN, one nifX and one nifV. Of the 15 nif genes, eight nif genes (nifB, nifH, nifD, nifK, nifE, nifN, nifX and nifV) and two non-nif genes (orf1 and hesA) form a complete nif gene cluster. Phylogenetic analysis suggests that the complete nif cluster of P. sabinae T27 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 JM109 to fix nitrogen. P. sabinae T27 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.
Strains and media
Strains used in this study is listed in Additional file 10: Table S1. P. sabinae T27 and the recombinant E. coli strains were routinely grown in LD medium (per liter contains: 2.5 g NaCl, 5 g yeast and 10 g tryptone) at 30°C with shaking. When appropriate, antibiotics were added in the following concentrations: 100 μg∕ml ampicillin, and 20 μg∕ml tetracycline for maintenance of plasmids.
Nitrogen-free and nitrogen-deficient media were used in this study. Nitrogen-free medium contained (per liter) 10.4 g Na2HPO4, 3.4 g KH2PO4, 26 mg CaCl2• 2H2O, 30 mg MgSO4, 0.3 mg MnSO4, 36 mg Ferric citrate, 7.6 mg Na2MoO4 · 2H2O, 10 μg p-aminobenzoic acid, 5 μg biotin and 4 g glucose as carbon source. Nitrogen-deficient medium contained 2 mM glutamate as nitrogen source in nitrogen-free medium .
Genome sequencing, genome annotation and analysis
Genomic DNA of P. sabinae T27 was isolated according to . Genome sequencing was performed by Tianjin Research Center for Functional Genomics and Biochip in China. The genome P. sabinae T27 was sequenced by using a hybrid sequencing approach that incorporates 454 pyrosequencing with Illumina Genome Analyzer. Sequencing by both methods was performed according to manufacturer’s instructions, Roche and Illumina.
The rRNA genes were identified with RNAmmer . Transfer RNA (tRNA) genes were identified by the program tRNAscan-SE . Genes coding for proteins with known functions were annotated by searches against KEGG Genes, Pfam, and SWISSPROT . The complete sequence has been assigned GenBank accession no. CP004078.
Construction of recombinant plasmid for expression of the complete nif cluster in E. coli
Genomic DNA of P. sabinae T27 was used as template for cloning nif genes. A 12 kb Xho I-Xho I DNA fragment containing the complete nif gene cluster (a 310 bp promoter region and the contiguous ten genes nifBHDKENXorf1hesAnifV and 194 bp downstream of the stop codon TAA of nifV) was PCR amplified with primers T-up and T-down (Additional file 11: Table S2). The PCR product was ligated to Xho I site of pVK100, yielding plasmid pKY100-27. Then the plasmid was transferred to E. coli JM109, yielding the recombinant E. coli 27 strain.
Construction of plasmids for complementation studies
In order to determine the function of nifH/nifH-like and nifD/nifD-like genes, overlap PCR was performed to fuse the coding regions of nifH1, nifH-like1, nifH-like2, nifD, nifD-like1, nifD-like2, nifD-like3, nifD-like4 and nifD-like5 of P. sabinae T27 with the nifH promoter of K. pneumoniae. The primers used in fusion were listed in Additional file 11: Table S2. The amplified PCR products were cloned to pVK100. The recombinant pVK100 were transformed to K. pneumoniae nifH mutant or K. pneumoniae nifD mutant for complementation.
Transcription start site identification
The 5′-RACE method was used to determine the transcription start site (TSS) using the SMARTer™ RACE cDNA Amplification Kit (Clontech). Gene-specific primers are listed in Additional file 11: Table S2. The PCR product was cloned into the pMD18-T Vector and then sequenced.
Overexpression and purification of σ70 from P. sabinae T27 in E. coli
A 1134 bp DNA fragment carrying the rpoD gene (encoding σ70 of P. sabinae T27) was PCR amplified with primers sigma A-F and sigma A-R (Additional file 11:Table S2). The PCR product was ligated to the pET-28b expression vector, yielding plasmid pET28-σ70. E. coli strain BL21 (DE3) was transformed with expression plasmid pET28-σ70 and utilized for protein expression. The bacterial cells were grown in LB medium to the end of log phase and then a final concentration of 1 mM IPTG (isopropyl-β-D-thiogalactopyranoside) was added to the culture and the cells were harvested after incubation for another 4 h at 16°C. The cells were then harvested and disrupted by sonication on ice. The protein was purified from the supernatant with Ni2+-NTA agarose (Qiagen) according to the manufacturer’s instructions.
Electrophoretic mobility shift assay (EMSA)
For the electrophoretic mobility shift assay (EMSA), a 50 bp nif promoter fragment (from −47 to +3 relative to the transcription start site of nifB in P. sabinae T27) was synthesized by Sangon Biotech Co., Ltd (Shanghai). To do this, two DNA fragments corresponding to the sequences of the first strand (5′- GGAGAAGTGAATTGACTGTATTTGTCCCTGTCTCTAAGA-TGTAATTATAT-3′) and the complementary DNA strand (5′- ATATAATTACATCTTAGAGAC-AGGGACAAATACAGTCAATTCACTTCTCC-3′) were synthesized. The two strands were annealed and then labeled with digoxin using the DIG Gel Shift Kit (Roche). The binding shift experiment of E. coli σ70-RNAP (RNA polymerase) (Epicentre) or σ70 of P. sabinae T27 to the nif promoter was carried out using a gel shift kit (Roche). At the same time, a scrambled 39 bp DNA fragment formed by annealing the following complementary oligonucleotides (5′- GTACGGAGTATCCAGCTCCGTAGCATGCAAATCCTCTGG-3′) and (5′-CCAGAGGATTTGCATGCTACGGAGCTGGATACTCCGTAC -3′) was used to assay non-specific binding.
RT-PCR and qRT-PCR analysis
For RT-PCR, P. sabinae T27 was grown in N2-fixing conditions (without NH4Cl and O2). For qRT-PCR, P. sabinae T27 was grown in N2-fixing conditions (without NH4Cl and O2) and non- N2-fixing conditions (100 mM ammonium and 21% O2). The culture was harvested by centrifugation at 4 °C, and total RNA was isolated using the PrimeScript® RT reagent Kit with gDNA Eraser (Takara Bio) according to the manufacturer’s instructions. The possibility of contamination of genomic DNA was eliminated by digestion with RNase-free DNase I (Takara Bio). The integrity and size distribution of the RNA was verified by agarose gel electrophoresis, and the concentration was determined spectrophotometrically. Synthesis of cDNA was carried out using RT Prime Mix according to the manufacturer’s specifications (Takara Bio). 0.8 μg of cDNA was used for RT-PCR. The nif and nif-like gene transcripts were detected by using an RT-PCR Kit with 16S rDNA as a control. Primers for nif, nif-like genes and 16S rDNA used for PCR are listed in (Additional file 11: Table S2).
Nitrogenase activity assays by acetylene reduction method
For nitrogenase activity assays, P. sabinae T27 and the recombinant E. coli 27 strain were grown in 5 mL of LD media (supplemented with antibiotics when necessary) in 50-ml flasks shaken at 250 rpm for 16 h at 30°C. Nitrogenase activity assays was performed according to Wang et al’s reports .
15 N2incorporation assay
P. sabinae T27 and the recombinant E. coli strain were grown overnight in LD medium. The cultures were collected and resuspended in 70 ml nitrogen-deficient medium containing 2 mM glutamate as nitrogen source to an OD600 of 0.4 in a 120 ml serum bottle. 15 N2 incorporation assay was performed according to Wang et al’s report .
This work was supported by funds from the National “973” Project (Grant No. 2010CB126504).
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