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Comparative Study
. 2014 Mar 20;10(3):e1004231.
doi: 10.1371/journal.pgen.1004231. eCollection 2014 Mar.

Comparative Genomic Analysis of N2-fixing and non-N2-fixing Paenibacillus Spp.: Organization, Evolution and Expression of the Nitrogen Fixation Genes

Free PMC article
Comparative Study

Comparative Genomic Analysis of N2-fixing and non-N2-fixing Paenibacillus Spp.: Organization, Evolution and Expression of the Nitrogen Fixation Genes

Jian-Bo Xie et al. PLoS Genet. .
Free PMC article


We provide here a comparative genome analysis of 31 strains within the genus Paenibacillus including 11 new genomic sequences of N2-fixing strains. The heterogeneity of the 31 genomes (15 N2-fixing and 16 non-N2-fixing Paenibacillus strains) was reflected in the large size of the shell genome, which makes up approximately 65.2% of the genes in pan genome. Large numbers of transposable elements might be related to the heterogeneity. We discovered that a minimal and compact nif cluster comprising nine genes nifB, nifH, nifD, nifK, nifE, nifN, nifX, hesA and nifV encoding Mo-nitrogenase is conserved in the 15 N2-fixing strains. The nif cluster is under control of a σ(70)-depedent promoter and possesses a GlnR/TnrA-binding site in the promoter. Suf system encoding [Fe-S] cluster is highly conserved in N2-fixing and non-N2-fixing strains. Furthermore, we demonstrate that the nif cluster enabled Escherichia coli JM109 to fix nitrogen. Phylogeny of the concatenated NifHDK sequences indicates that Paenibacillus and Frankia are sister groups. Phylogeny of the concatenated 275 single-copy core genes suggests that the ancestral Paenibacillus did not fix nitrogen. The N2-fixing Paenibacillus strains were generated by acquiring the nif cluster via horizontal gene transfer (HGT) from a source related to Frankia. During the history of evolution, the nif cluster was lost, producing some non-N2-fixing strains, and vnf encoding V-nitrogenase or anf encoding Fe-nitrogenase was acquired, causing further diversification of some strains. In addition, some N2-fixing strains have additional nif and nif-like genes which may result from gene duplications. The evolution of nitrogen fixation in Paenibacillus involves a mix of gain, loss, HGT and duplication of nif/anf/vnf genes. This study not only reveals the organization and distribution of nitrogen fixation genes in Paenibacillus, but also provides insight into the complex evolutionary history of nitrogen fixation.

Conflict of interest statement

The authors have declared that no competing interests exist.


Figure 1
Figure 1. Genomic diversity of strains in the genus Paenibacillus.
Each strain is represented by an oval that is colored: N2-fixing strains (red), non- N2-fixing strains (purple). The number of orthologous coding sequences (CDSs) shared by all strains (i.e., the core genome) is in the center. Overlapping regions show the number of CDSs conserved only within the specified genomes. Numbers in non-overlapping portions of each oval show the number of CDSs unique to each strain. The total number of protein coding genes within each genome is listed below the strain name.
Figure 2
Figure 2. Functional classification of gene content of the 31 Paenibacillus strains.
(A) Profiles of Cluster of Orthologous Groups (COG) showing percentage of genes in each category out of total annotated genes. Taxa from inside of circle to outside of circle are from Paenibacillus sp. JDR 2 (top in the strain list) to P. sabinae T27 (down in the strain list). (B) Profiles of COG showing function categories for genes in core genomes. Taxa from inside of circle to outside of circle are from Paenibacillus sp. JDR 2 (top in the strain list) to P. sabinae T27 (down in the strain list).
Figure 3
Figure 3. Organization of nif, vnf, anf and nif-like genes in N2-fixing Paenibacillus strains.
nif, vnf, anf and nif-like genes are marked with different colors. The 9 nif genes nifBHDKENXhesAnifV are contiguous within Sub-group I and there is a orf between nifX and hesA within Sub-group II.
Figure 4
Figure 4. Comparison of G+C contents of the nif clusters with those of the average of the chromosomal genomes.
Figure 5
Figure 5. Synteny of the chromosomal regions flanking the nif gene cluster among each sub-group.
(A) nif clusters of Sub-group I. (B) The chromosomal regions of non-N2-fixing strains corresponding to those flanking the nif gene cluster of Sub-group I. (C) nif clusters of Sub-group II.
Figure 6
Figure 6. Bayesian inferred phylogenetic tree of concatenated NifHDK homologs.
The interior node values of the tree are clade credibility values, values lower than 100% are indicated. Branches are colored blue (Mo-nitrogenase, Nif), green (V-nitrogenase, Vnf), purple (Fe-nitrogenase, Anf), light blue (uncharacterized homolog), dark yellow (uncharacterized nitrogenase). The text colored red was Paenibacillus.
Figure 7
Figure 7. Maximum-likelihood phylogenetic tree of Paenibacillus strains and the 4 possible evolutionary pathways of nitrogen fixation in Paenibacillus.
The tree was constructed based on 275 single-copy core proteins shared by the 31 Paenibacillus genomes and the rooting strain B. subtilis 168. Four likely pathways are marked with number 1–4.
Figure 8
Figure 8. Maximum-likelihood phylogenetic tree of Mo-, Fe- and V-nitrogenases and nitrogenase-like sequences.
Nif/Vnf/AnfH, Nif/Vnf/AnfD, Nif/Vnf/AnfK, Nif/Vnf/AnfH-like, Nif/Vnf/AnfD-like and Nif/Vnf/AnfK sequences were derived from the 15 N2-fixing Paenibacillus strains and other representative species.

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Grant support

This work was supported by funds from the National “973” Project (Grant No. 2010CB126504) and the National Nature Science Foundation of China (Grant No. 31270129). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.