Biological nitrogen fixation (BNF), performed by diazotrophic prokaryotes, is responsible for reducing dinitrogen (N₂) present in the biosphere into biologically available forms of nitrogen. Paenibacillus brasilensis PB24 is a diazotrophic Gram-positive bacterium and is considered ecologically and industrially important because it is able to produce antimicrobial substances and 2,3-butanediol. However, the genetics and regulation of its nitrogen fixing (nif) genes have never been assessed so far. Therefore, the present study aimed to (i) identify the structural and regulatory genes related to BNF in the PB24 genome, (ii) perform comparative genomics analysis of the nif operon among different Paenibacillus species and (iii) study the expression of these genes in the presence and absence of NH₄. Strain PB24 showed a nif operon composed of nine genes (nifBHDKENXhesAV), with a conserved synteny (with small variations) among the Paenibacillus species evaluated. BNF regulatory genes, glnK and amtB (encoding GlnK signal transduction protein and AmtB transmembrane protein, respectively) and glnR and glnA genes (encoding the transcription factor GlnR and glutamine synthetase) were found in the PB24 genome. Primers were designed for qPCR amplification of the nitrogenase structural (nifH, nifD and nifK) and regulatory (glnA and amtB) BNF genes. The structural gene expression in PB24 was up- and downregulated in the absence and presence of NH₄, respectively. The gene expression levels indicated a GlnR-mediated repression of genes associated with ammonium import (amtBglnK) and BNF (nif genes). Additionally, the regulatory mechanism of GlnR in P. brasilensis PB24 differed from the other Paenibacillus evaluated, considering the different distribution of binding sites recognized by GlnR.

重氮营养原核生物进行的生物固氮（BNF）负责将生物圈中存在的二氮（N ₂）还原为可生物利用的氮形式。巴西芽孢杆菌PB24是重氮革兰氏阳性细菌，被认为在生态和工业上都很重要，因为它能够产生抗菌物质和2,3-丁二醇。然而，遗传学及其固氮的调节（NIF）基因从未到目前为止评估。因此，本研究的目的是（i）确认在PB24基因组相关的BNF的结构和调节基因，（ⅱ）进行比较基因组学的分析NIF不同间操纵子类芽孢杆菌和（iii）研究这些基因在NH ₄存在和不存在下的表达。PB24菌株显示出由9个基因（nifBHDKENXhesAV）组成的nif操纵子，在所评估的Paenibacillus物种中具有保守的同义性（变异很小）。在PB24基因组中发现了BNF调节基因glnK和amtB（分别编码GlnK信号转导蛋白和AmtB跨膜蛋白）以及glnR和glnA基因（编码转录因子GlnR和谷氨酰胺合成酶）。设计引物用于qPCR扩增固氮酶结构（nifH，nifD和nifK）和调控性（glnA和amtB）BNF基因。在不存在和存在NH ₄的情况下，PB24中的结构基因表达分别被上调和下调。基因表达水平表明，GlnR介导的与铵输入（amtBglnK）和BNF（nif基因）相关的基因被抑制。此外，考虑到GlnR识别的结合位点的不同分布，巴西假单胞菌PB24中GlnR的调控机制与其他被评估的Paenibacillus不同。