Functional analysis of multiple nifB genes of Paenibacillus strains in synthesis of Mo-, Fe- and V-nitrogenases

Abstract

Background Biological nitrogen fixation is catalyzed by Mo-, V- and Fe-nitrogenases that are encoded by nif, vnf and anf genes, respectively. NifB is the key protein in synthesis of the cofactors of all nitrogenases. Most diazotrophic Paenibacillus strains have only one nifB gene located in a compact nif gene cluster (nifBHDKENX(orf1)hesAnifV). But some Paenibacillus strains have multiple nifB genes and their functions are not known. Results A total of 138 nifB genes are found in the 116 diazotrophic Paenibacillus strains. Phylogeny analysis shows that these nifB genes fall into 4 classes: nifBI class including the genes (named as nifB1 genes) that are the first gene within the compact nif gene cluster, nifBII class including the genes (named as nifB2 genes) that are adjacent to anf or vnf genes, nifBIII class whose members are designated as nifB3 genes and nifBIV class whose members are named as nifB4 genes are scattered on genomes. Functional analysis by complementation of the ∆nifB mutant of P. polymyxa which has only one nifB gene has shown that both nifB1 and nifB2 are active in synthesis of Mo-nitrogenase, while nifB3 and nifB4 genes are not. Deletion analysis also has revealed that nifB1 of Paenibacillus sabinae T27 is involved in synthesis of Mo-nitrogenase, while nifB3 and nifB4 genes are not. Complementation of the P. polymyxa ∆nifBHDK mutant with the four reconstituted operons: nifB1anfHDGK, nifB2anfHDGK, nifB1vnfHDGK and nifB2vnfHDGK, has shown both that nifB1 and nifB2 were able to support synthesis of Fe- or V-nitrogenases. Transcriptional results obtained in the original Paenibacillus strains are consistent with the complementation results. Conclusions The multiple nifB genes of the diazotrophic Paenibacillus strains are divided into 4 classes. The nifB1 located in a compact nif gene cluster (nifBHDKENX(orf1)hesAnifV) and the nifB2 genes being adjacent to nif or anf or vnf genes are active in synthesis of Mo-, Fe and V-nitrogenases, but nifB3 and nifB4 are not. The reconstituted anf system comprising 8 genes (nifBanfHDGK and nifXhesAnifV) and vnf system comprising 10 genes (nifBvnfHDGKEN and nifXhesAnifV) support synthesis of Fe-nitrogenase and V-nitrogenase in Paenibacillus background, respectively.

Mineral-Bound Trace Metals as Cofactors for Anaerobic Biological Nitrogen Fixation

“…One possible explanation is that some low levels of all three nitrogenase proteins are required for the expression of a dominant functional nitrogenase. For example, nifH , in addition to providing electrons to NifDK, also serves multiple roles in the maturation of nitrogenases, and nifB is the key protein in the synthesis of the cofactors of all nitrogenases . Furthermore, simultaneous activation of three nitrogenases has been observed under different conditions. , For instance, the number of nifD transcripts in A.…”

“…For example, nif H, in addition to providing electrons to NifDK, also serves multiple roles in the maturation of nitrogenases, 85 and nif B is the key protein in the synthesis of the cofactors of all nitrogenases. 86 Furthermore, simultaneous activation of three nitrogenases has been observed under different conditions. 55,87 For instance, the number of nif D transcripts in A. vinelandii cells remained high irrespective of Mo limitation.…”

Isotopic evidence for increased carbon and nitrogen exchanges between peatland plants and their symbiotic microbes with rising atmospheric CO2 concentrations since 15,000 cal. year BP

“…Therefore, it is possible that Anf gene (encoding Fe‐containing nitrogenase) was present in Sphagnum mosses. Previous studies have found that α‐proteobacterium Rhodobacter capsulatus (Rhodobacteraceae) (Demtröder et al, 2020) and Paenibacillus (Paenibacillaceae) (Li et al, 2021) all have Anf genes, and further evidence indicates their presence with Sphagnum in bogs (Tveit et al, 2020; Warren et al, 2017). Because we have limited information on the rate of N deposition during the Pleistocene epoch, we could not rule out the possibility that the changes in Sphagnum δ 15 N were caused by N deposition instead of N 2 ‐fixation.…”