The nifB genes of Paenibacillus genus
Here, the nitrogen fixation genes in the genomes of the 116 diazotrophic Paenibacillus strains taken from the RefSeq database were comparatively analyzed (Additional file 1: Table S1). A compact nif gene cluster composed of 9–10 genes (nifBHDKENX(orf1)hesAnifV) was conserved in all of the diazotrophic strains, in agreement with the previous studies [24]. In addition to the compact nif gene cluster encoding Mo-nitrogenase, 9 strains had additional anfHDGK encoding Fe-nitrogenase and 3 strains had additional vnfHDGKEN encoding V-nitrogenase.
A total of 138 NifB putative sequences were found in the 116 diazotrophic Paenibacillus strains. According to the nifB position and sequence similarity, the nifB genes were divided into 4 classes. The nifB1 was designated as the one that is the first gene in the compact nif gene cluster comprising 9–10 genes (nifB nifH nifD nifK nifE nifN nifX (orf1) hesA nifV). The nifB2 was linked to additional copies of nifENXorf(fer) genes preceding anfHDGK or additional copies of nifENXorforf genes preceding vnfHDGKEN or orforf preceding vnfHDGKEN. The nifB3 and nifB4 were scattered at different locations with sequence divergence.
Of the 116 diazotrophic Paenibacillus strains, 105 strains had only one nifB and 11 strains had 2–4 nifB genes. P. polymyxa WLY78 was a representative that has only a nifB1 located in the compact nif gene cluster consisting of 9 genes (nifBHDKENXhesAnifV) encoding Mo-nitrogenase (Fig. 1 and Additional file 1: Table S1). P. sabinae T27 was a representative strain with three nifB genes (nifB1, nifB3 and nifB4), but contained only Mo-nitrogenase. For the strains with both Mo- and V-nitrogenases, P. zanthoxyli JH29 had nifB1, nifB2 and nifB3, but P. durus ATCC 35681 had nifB2, nifB3 and 2 copies of nifB1: one being located in the compact nif cluster and the other being linked to another nifH. For the strains with both Mo- and Fe-nitrogenases, P. forsythiae T98 had three nifB genes (nifB1, nifB2 and nifB3), whereas P. sophorae S27 had four nifB genes (nifB2, nifB3, and 2 copies of nifB1. The other 4 strains (P. borealis FSL H70744, Paenibacillus sp. FSL H7-0357, Paenibacillus sp. HW567 and P. camerounensis G4) with both Mo- and Fe-nitrogenases possessed only one nifB gene. Organization of the nifB genes and other nitrogen fixation genes from 17 representatives of Paenibacillus strains was shown in Fig. 1.
Phylogeny and Structure of Paenibacillus NifB proteins
Here, 138 putative NifB sequences from 116 diazotrophic Paenibacillus strains were used to construct a phylogenetic tree, with 11 NifB sequences from 10 diazotrophs (A. vinelandii, K. oxytoca, Bradyrhizobium japonicum, Clostridium kluyveri, Dehalobacter sp., Kyrpidia spormannii, Methanosarcina acetivorans, Methanococcus maripaludis, Frankia sp. EAN1pec, Nostoc sp. PCC 7120) as control (Fig. 2 and Additional file 1: Table S1). The phylogenetic tree showed that all Paenibacillus putative NifB proteins form a large class which is separated from the NifB proteins from other diazotrophs. The data suggested that all Paenibacillus putative nifB genes had a common ancestor. The Paenibacillus putative NifB proteins were divided into 4 subclasses: NifB1, NifB2, NifB3 and NifB4, in agreement with the 4 nifB classes that were classified on basis of nifB sequence similarities and positions. Phylogeny analyses showed that the NifB1 protein was emerged firstly in the diazotrophic Paenibacillus species, and NifB2, NifB3 and NifB4 proteins may result from gene duplication.
Protein structure analysis showed that Paenibacillus NifB1, NifB2 and NifB4 proteins had the same structure composed of an N-terminal SAM-radical domain and a C-terminal NifX-like domain. Most NifB3 proteins possessed the two domains. But the NifB3 proteins from the 2 strains (P. zanthoxyli JH29 and P. durus DSM 1735) had only a SAM-radical domain. The Paenibacillus NifB1, NifB2, NifB3 and NifB4 proteins that possessed both domains were composed of 427–505 amino acids (Additional file 1: Table S1) and had similarity (> 57%) at amino acid levels. These proteins had a number of conserved motifs in the SAM-radical domain, including HPC motif, Cx3Cx2C motif, ExRP motif, AGPG motif, TxTxN motif and Cx2CRxDAxG (Fig. 2). However, NifB3 proteins of P. zanthoxyli JH29 and P. durus DSM 1735 had only a SAM-radical domain that lacks the Cx2CRxDAxG motif. Sequence alignment of 13 NifB proteins including NifB1, NifB2, NifB3 and NifB4 from 4 representatives of Paenibacillus strains (P. polymyxa WLY78, P. sabinae T27, P. forsythia T98 and P. zanthoxyli JH29) was shown in Additional file 2: Figure S1.
Transcription analysis of multiple nifB genes in medium containing only Mo or Fe or V
As described above, P. sabinae T27 with only Mo-nitrogenase had NifB1, NifB3 and NifB4, P. zanthoxyli JH29 with both Mo- and V-nitrogenases had NifB1, NifB2 and NifB3 and P. forsythiae T98 with both Mo- and Fe-nitrogenases possessed NifB1, NifB2 and NifB3. Here, the three species P. sabinae T27, P. forsythia T98 and P. zanthoxyli JH29 were used to investigate the transcriptions of the multiple nifB genes under different conditions by RT-qPCR. P. sabinae T27 was cultivated in Mo-dependent nitrogen fixation conditions, while P. forsythia T98 and P. zanthoxyli JH29 were cultivated in Mo-dependent and Fe-dependent or V-dependent nitrogen fixation conditions, respectively, with non-nitrogen fixing conditions of N-rich (LD medium) cultures as negative controls (Fig. 3). For P. sabinae T27 under Mo-dependent condition, nifB1 was significantly transcribed, but the other two genes nifB3 and nifB4 were nearly not expressed (Fig. 3a). For P. forsythia T98 under both Mo-dependent and Fe-dependent conditions, both nifB1 and nifB2 genes were transcribed, but nifB3 was nearly not expressed. The transcript level of nifB1 was much higher in Mo-dependent condition than in Fe-dependent condition, while the transcript level of nifB2 was higher in Fe-dependent condition than in Mo-dependent condition (Fig. 3b). For P. zanthoxyli JH29 under both Mo-dependent and V-dependent conditions, both nifB1 and nifB2 genes were transcribed, but nifB3 was nearly not detected. The transcript level of nifB1 was higher in Mo-dependent condition than in V-dependent condition, while the transcript level of nifB2 was higher in V-dependent condition than in Mo-dependent condition (Fig. 3c). These results indicated that the nifB1 and nifB2 may be selectively expressed according to metal availability.
Functional analysis of multiple nifB genes in synthesis of Mo-nitrogenase
The nifB deletion mutant (∆nifB) of P. polymyxa WLY78 was here constructed by using recombination method as described in materials and methods. The P. polymyxa ∆nifB mutant nearly completely lost the nitrogenase activity and its nifB gene carried in plasmid can restore the nitrogenase activity (Fig. 4). Thus, P. polymyxa ∆nifB mutant was used as a host for complementation to investigate the functionality of the multiple nifB genes. Each nifB gene from P. sabinae T27, P. forsythia T98 and P. zanthoxyli JH29 was cloned into a low-copy plasmid pRN5101[27, 28], in which the expression of these nifB genes were driven under the control of the nifB promoter of P. polymyxa (details are provided in materials and methods). Among the 3 nifB genes of P. sabinae T27, only the nifB1 can effectively restore the nitrogenase activity of the P. polymyxa ∆nifB mutant, showing the same result with transcription data that only nifB1 gene was upregulated under nitrogen fixation condition. Both nifB1 and nifB2 from P. forsythia T98 or P. zanthoxyli JH29 can effectively restore nitrogenase activity of the P. polymyxa ∆nifB mutant, but the nifB3 from P. forsythia T98 or P. zanthoxyli JH29 can not restore activity, in agreement with the transcription data and suggesting that both nifB1 and nifB2 were functional in synthesis of Mo-nitrogenase.
Functional analysis of nifB1 and nifB2 genes in synthesis of Fe- and V-nitrogenases
In order to investigate whether the nifB1 and nifB2 from P. forsythia T98 and P. zanthoxyli JH29 were active in synthesis of Fe-nitrogenase and V-nitrogenases, the ΔnifBHDK and ΔnifBHDKEN mutants of P. polymyxa WLY78 which lost the ability to synthesize Mo-nitrogenase were constructed. As shown in Fig. 5, the nifBHDK and nifBHDKEN of P. polymyxa WLY78 carried in plasmid could restore the nitrogenase activity to 90% wild-type level in the complementary strain (ΔnifBHDK/nifBHDK) and (ΔnifBHDKEN/nifBHDKEN), suggesting that the mutants can be used as a host for complementation study of alternative nitrogenases.
Two new operons nifB1anfHDGK and nifB2anfHDGK of P. forsythia T98 under the control of the P. polymyxa WLY78 nifB promoter were constructed (Fig. 5). Each of the reconstituted nifB1anfHDGK and nifB2anfHDGK operons of P. forsythia T98 carried in the recombinant plasmids can enable P. polymyxa ΔnifBHDK mutant to have nitrogenase activity in medium containing Fe and lacking Mo. The data suggest that either nifB1 or nifB2 together with anfHDGK of P. forsythia can support synthesis of Fe-nitrogenase in the heterologous host P. polymyxa which originally has only Mo-nitrogenase system. Furthermore, in order to investigate whether nifE and nifN genes (designed nifE2 and nifN2 genes) preceding anfHDGK of P. forsythia T98 were functional, another new operon nifB2E2N2anfHDGK of P. forsythia T98 was constructed (Fig. 5). Then, nifB2E2N2anfHDGK and nifB2anfHDGK carried in the recombinant plasmids are individually used to complement ΔnifBHDKEN mutant of P. polymyxa WLY78. As shown in Fig. 5, either nifB2E2N2anfHDGK or nifB2anfHDGK can support ΔnifBHDKEN mutant of P. polymyxa WLY78 to have nitrogenase activity in medium containing Fe and lacking Mo. Like the P. forsythia T98 that was capable of diazotrophic growth, the reconstituted nifB/anf-complemented strains can grow in liquid media with dinitrogen as the sole nitrogen source (Fig. S2). The results indicated that that nifEN is not necessary for the biosynthesis and the reconstituted anf system composed of 8 genes (nifBanfHDGK of P. forsythia T98 and nifXhesAnifV of P. polymyxa WLY78) can support synthesis of Fe-nitrogenase to fix nitrogen.
Similarly, two new operons nifB1vnfHDGK and nifB2vnfHDGK of P. zanthoxyli JH29 under the control of the nifB promoter of P. polymyxa WLY78 were constructed (Fig. 5a). Each of the nifB1vnfHDGK and nifB2vnfHDGK operons of P. zanthoxyli JH29 carried in the recombinant plasmids can enable P. polymyxa ΔnifBHDK mutant to have nitrogenase activity in medium containing V and lacking Mo (Fig. 5b). The data suggest that either of nifB1 or nifB2 together with vnfHDGK of P. zanthoxyli JH29 can support synthesis of V-nitrogenase. Furthermore, a new operon comprising nifB2 and vnfHDGKEN under the control of the nifB promoter of P. polymyxa WLY78 was constructed. The reconstituted operons nifB2vnfHDGKEN and nifB2vnfHDGK of P. zanthoxyli JH29 are individually used to complement ΔnifBHDKEN mutant of P. polymyxa WLY78. The operon nifB2vnfHDGKEN can effectively enable ΔnifBHDKEN mutant of P. polymyxa WLY78 to synthesize V-nitrogenase (Fig. 5). Our data demonstrate that the reconstituted vnf system with vnfEN exhibited higher nitrogenase activity compared to the reconstituted vnf system with nifEN. However, the nifB2vnfHDGK operon of P. zanthoxyli JH29 can not complement the ΔnifBHDKEN mutant of P. polymyxa WLY78, suggesting that the vnfEN or nifEN was required for the biosynthesis of VFe-co. The diazotrophic growth tests showed that all the reconstituted nifB/vnf-complemented strains excluding ΔnifBHDKEN/nifB2vnfHDGK strain grew as well as the P. zanthoxyli JH29 (Additional file 3: Figure S2). The results indicated that the reconstituted vnf system composed of 10 genes (nifBvnfHDGK of P. zanthoxyli JH29 and nifENXhesAnifV of P. polymyxa WLY78 or nifBvnfHDGKEN of P. zanthoxyli JH29 and nifXhesAnifV of P. polymyxa WLY78) can support synthesis of V-nitrogenase to fix nitrogen.