Biofilm and nitrogen fixation are two competitive strategies used by many plant-associated bacteria; however, the mechanisms underlying the formation of nitrogen-fixing biofilms remain largely unknown. Here, we examined the roles of multiple signalling systems in the regulation of biofilm formation by root-associated diazotrophic P. stutzeri A1501. Physiological analysis, construction of mutant strains and microscale thermophoresis experiments showed that RpoN is a regulatory hub coupling nitrogen fixation and biofilm formation by directly activating the transcription of pslA, a major gene involved in the synthesis of the Psl exopolysaccharide component of the biofilm matrix and nifA, the transcriptional activator of nif gene expression. Genetic complementation studies and determination of the copy number of transcripts by droplet digital PCR confirmed that the regulatory ncRNA RsmZ serves as a signal amplifier to trigger biofilm formation by sequestering the translational repressor protein RsmA away from pslA and sadC mRNAs, the latter of which encodes a diguanylate cyclase that synthesises c-di-GMP. Moreover, RpoS exerts a braking effect on biofilm formation by transcriptionally downregulating RsmZ expression, while RpoS expression is repressed posttranscriptionally by RsmA. These findings provide mechanistic insights into how the Rpo/Gac/Rsm regulatory networks fine-tune nitrogen-fixing biofilm formation in response to the availability of nutrients.

生物膜和固氮是许多植物相关细菌使用的两种竞争策略；然而，固氮生物膜形成的潜在机制在很大程度上仍然未知。在这里，我们检查了多个信号系统在根相关重氮营养P. stutzeri A1501 调节生物膜形成中的作用。生理分析、突变菌株的构建和微型热泳实验表明，RpoN 是通过直接激活pslA的转录来耦合固氮和生物膜形成的调节中枢，pslA 是参与生物膜基质和nifA的 Psl 胞外多糖成分合成的主要基因, nif的转录激活因子基因表达。遗传互补研究和通过液滴数字 PCR 确定转录本拷贝数证实，调节性 ncRNA RsmZ 作为信号放大器通过将翻译抑制蛋白 RsmA 与pslA和sadC mRNA 隔离来触发生物膜形成，后者编码合成 c-di-GMP 的二鸟苷酸环化酶。此外，RpoS 通过转录下调 RsmZ 表达对生物膜形成产生制动作用，而 RpoS 表达在转录后被 RsmA 抑制。这些发现提供了关于 Rpo/Gac/Rsm 调节网络如何根据营养物质的可用性微调固氮生物膜形成的机制见解。