Proteome profiling of Paenibacillus sonchi genomovar Riograndensis SBR5^T under conventional and alternative nitrogen...

Paenibacillus sonchi SBR5^T is a Gram-positive, endospore-forming facultative aerobic diazotrophic bacterium that can fix nitrogen via an alternative Fe-only nitrogenase (AnfHDGK). In several bacteria, this alternative system is expressed under molybdenum (Mo)-limiting conditions when the conventional Mo-dependent nitrogenase (NifHDK) production is impaired. The regulatory mechanisms, metabolic processes, and cellular functions of N₂ fixation by alternative and/or conventional systems are poorly understood in the Paenibacillus genus. We conducted a comparative proteomic profiling study of P. sonchi SBR5T grown under N₂-fixing conditions with and without Mo supply through an LC-MS/MS and label-free quantification analysis to address this gap. Protein abundances revealed overrepresented processes related to anaerobiosis growth adaption, Fe-S cluster biosynthesis, ammonia assimilation, electron transfer, and sporulation under N₂-fixing conditions compared to non-fixing control. Under Mo limitation, the Fe-only nitrogenase components were overrepresented together with the Mo-transporter system, while the dinitrogenase component (NifDK) of Mo‑nitrogenase was underrepresented. The dinitrogenase reductase component (NifH) and accessory proteins encoded by the nif operon had no significant differential expression, suggesting post-transcriptional regulation of nif gene products in this strain. Overall, this was the first comprehensive proteomic analysis of a diazotrophic strain from the Paenibacillaceae family, and it provided insights related to alternative N₂-fixation by Fe-only nitrogenase. SIGNIFICANCE: In this work, we try to understand how the alternative nitrogen fixation system, presented by some diazotrophic bacteria, works. For this, we used the SBR5 lineage of P. sonchi, which presents the alternative system in which the nitrogenase cofactor is composed only of iron. In addition, we tried to unravel the proteome of this strain in different situations of nitrogen fixation, since, for Gram-positive bacteria, these systems are little known. The results achieved, through LC-MS/MS and label-free quantitative analysis, showed an overrepresentation of proteins related to different processes involved with growth under stressful conditions in situations of nitrogen deficiency, in addition to suggesting that some encoded proteins by the nif operon may be regulated at post-transcriptional levels. Our findings represent important steps toward the elucidation of nitrogen fixation systems in Gram-positive diazotrophic bacteria.