The role of Fnr paralogs for controlling anaerobic metabolism in diazotrophPaenibacillus polymyxaWLY78

ABSTRACT Fnr is a transcriptional regulator that controls the expression of a variety of genes in response to oxygen limitation in bacteria. Genome sequencing revealed four genes ( fnr1 , fnr3 , fnr5 and fnr7 ) coding for Fnr proteins in Paenibacillus polymyxa WLY78. Fnr1 and Fnr3 showed more similarity to each other than to Fnr5 and Fnr7. Also, Fnr1 and Fnr3 exhibited high similarity with Bacillus cereus Fnr and Bacillus subtilis Fnr in sequence and structures. Deletion analysis showed that the four fnr genes, especially fnr1 and fnr3, have significant impacts on the growth and nitrogenase activity. Single deletion of fnr1 or fnr3 led to 50% reduction in nitrogenase activity and double deletion of fnr1 and fnr3 resulted to 90% reduction in activity. Both of the aerobically purified His-tagged Fnr1 and His-tagged Fnr3 in Escherichia coli could bind to the specific DNA promoter. Genome-wide transcription analysis showed that Fnr1 and Fnr3 indirectly activated expression of nif (nitrogen fixation) genes and Fe transport genes under anaerobic condition. Fnr1 and Fnr3 inhibited expression of the genes involved in aerobic respiratory chain and activated expression of genes responsible for anaerobic electron acceptor genes. IMPORTANCE Paenibacillus is a genus of Gram-positive, facultative anaerobic and endospore-forming bacteria. The members of nitrogen-fixing Paenibacillus have great potential use as a bacterial fertilizer in agriculture. However, the functions of fnr gene(s) in nitrogen fixation and other metabolisms in Paenibacillus spp. are not known. Here, we revealed that copy numbers vary largely among different Paenibacillus species and strains. Deletion and complementation analysis demonstrated that fnr1 and fnr3 have significant impacts on the growth and nitrogenase activity. Both of the aerobically purified His-tagged Fnr1 and His-tagged Fnr3 purified in Escherichia coli could bind to the specific DNA promoter as Bacillus cereus Fnr did. Fnr1 and Fnr3 indirectly activated nif expression under anaerobic condition. Fnr1 and Fnr3 directly or indirectly activated or inhibited expression of many important genes involved in respiration, energy metabolism, Fe uptake and potentially specific electron transport for nitrogenase under anaerobic condition. This study not only reveals the roles of fnr genes in nitrogen fixation and anaerobic metabolism, but also provides insight into the evolution and regulatory mechanisms of fnr in Paenibacillus .