www.nature.com/npjbiofilms ARTICLE OPEN A regulatory network involving Rpo, Gac and Rsm for nitrogen-fixing biofilm formation by Pseudomonas stutzeri Liguo Shang1,2,6, Yongliang Yan 1,6, Yuhua Zhan 1, Xiubin Ke1, Yahui Shao1, Yaqun Liu3, Hua Yang1, Shanshan Wang1, Shuling Dai1, ✉ Jiasi Lu1, Ning Yan1, Zhimin Yang1, Wei Lu1, Zhu Liu3, Shanchun Chen4, Claudine Elmerich5 and Min Lin 1,4 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. 1234567890():,; npj Biofilms and Microbiomes (2021) 7:54 ; https://doi.org/10.1038/s41522-021-00230-7 INTRODUCTION component system activates the transcription of one or several The term ‘biofilm’ can be defined as a community of microbes genes for Rsm ncRNAs, which contain multiple GGA motifs in 19 adhering to biotic or abiotic surfaces that is protected from exposed stem loops of their predicted secondary structures . The environmental stresses by a self-produced extracellular matrix1,2. GGA motifs allow Rsm ncRNAs to bind the RNA-binding proteins The extracellular matrix, often referred to as extracellular that act as global posttranscriptional repressors, e.g., CsrA (in polymeric substances, is composed of exopolysaccharides, pro- Escherichia coli) and RsmA (in P. aeruginosa), controlling important teins and extracellular DNA present in various concentrations cellular processes, such as secondary metabolism (e.g., metabo- depending on the bacterial species3,4. The biofilm state provides lism of pyocyanine or the QS signal N-butyryl-homoserine lactone 17,20 potential advantages over the planktonic state, including in P. aeruginosa), motility, and biofilm formation . RsmA increased resistance to antimicrobial agents, protection from specifically recognises and binds to conserved GGA motifs in environmental stresses, and improved adaptation to nutrient the 5′-untranslated region (5′-UTR) of target mRNAs, thereby deprivation5. Numerous investigations in recent decades have preventing ribosome access and protein translation17,21. RsmA demonstrated that bacterial biofilm formation is a sequential controls biofilm formation through direct repression of various process governed by complex regulatory networks that differ from target genes, such as pslA (involved in the synthesis of the one bacterial species to another1,6. It is now well accepted that exopolysaccharide Psl) and sadC (involved in c-di-GMP synth- microbial biofilms are the most widely distributed and predomi- esis)22,23. As a key biofilm regulatory molecule, the second nant mode of life on Earth, influencing our lives tremendously in messenger c-di-GMP is synthesised by diguanylate cyclases (DGCs) both positive and negative ways6–9. that bear a GGDEF domain and is degraded by phosphodies- In general, as established in the model bacterium Pseudomonas terases (PDEs) that harbour EAL or HD-GYP domains. P. aeruginosa aeruginosa, biofilm development usually begins with attachment encodes several DGCs and PDEs; for example, WspR/SadC/RoeA to a surface, followed by microcolony formation and production of (DGC) and RocR/BifA (PDE), are absent in the P. stutzeri A1501 the extracellular matrix responsible for the biofilm architecture10– genome, except for SadC and BifA, which modulate the level of c- 14. Biofilm formation has been studied intensively in the genus di-GMP and influence ‘surface-associated behaviours’ by control- – Pseudomonas, with an emphasis on genetic elements and ling polysaccharide syntheses16,24 27. The P. aeruginosa biofilm molecular mechanisms; Gac/Rsm, c-di-GMP signalling and matrix contains several polysaccharide components, including quorum-sensing (QS) pathways were reported as the main alginate, pellicle (Pel) and Psl exopolysaccharides28. It has been mechanisms leading to biofilm formation15,16. The Gac/Rsm shown that pslA is the first gene in the psl operon, which signalling pathway involves the GacS/GacA two-component comprises 15 cotranscribed genes that are involved in the regulatory system, the RNA-binding protein RmsA, and its cognate synthesis of Psl29. Although current data relating to the roles of regulatory non-coding RNAs (ncRNAs)17,18. The GacS/GacA two- Psl are limited, Psl is a critical component of the P. aeruginosa 1Biotechnology Research Institute/Key Laboratory of Agricultural Genomics (MOA), Chinese Academy of Agricultural Sciences, Beijing, China. 2School of Basic Medicine, Guangxi University of Chinese Medicine, Nanning, China. 3Key Laboratory of Tropical Biological Resources of Ministry of Education, School of Life and Pharmaceutical Sciences, Hainan University, Haikou, China. 4Citrus Research Institute, Southwest University/Chinese Academy of Agricultural Sciences, Chongqing, China. 5Institut Pasteur, Paris, France. 6*These ✉ authors contributed equally: Liguo Shang, Yongliang Yan. email: [email protected] Published in partnership with Nanyang Technological University L. Shang et al. 2 biofilm matrix, which functions as a scaffold, holding biofilm cells involved in biofilm formation and mutant construction led to the together to initiate biofilm development30. In addition, evidence identification of a complex regulatory circuitry involving the demonstrates that biofilm formation is controlled positively by alternative sigma factors RpoN and RpoS and the Gac/Rsm RpoN but negatively by RpoS, suggesting global antagonism regulators, and to the proposal of a model that integrates multiple between RpoN and RpoS, although there are contradictory levels of positive and negative regulation. reports31–35. Microbial biofilms are common on plant surfaces and have been associated with phytopathogenic infections and colonisation by RESULTS nitrogen-fixing rhizobacteria36,37. Because of dynamically fluctuat- Effect of carbon and nitrogen sources on biofilm formation ing conditions in the rhizosphere, the ability of diazotrophic and biofilm-based nitrogenase activity bacteria to form nitrogen-fixing biofilms may confer many It was previously shown that when lactate was the sole carbon ecological advantages and thereby facilitate their physiological source, P. stutzeri A1501 tended to form biofilms rather than and metabolic adaptation to successfully survive in the rhizo- maintain a planktonic state under nitrogen-deficient conditions41. sphere, a nitrogen-limited environment. An early study compared To further examine this behaviour, the ability of A1501 to form biofilm formation by a nitrogen-fixing strain of Klebsiella mature biofilms and fix nitrogen was assayed 48 h after pneumoniae with that of two other members of Enterobacter- inoculation using carbon sources other than lactate and different iaceae, Salmonella enteritidis and E. coli, and showed that the concentrations of NH Cl. Among the carbon substrates tested at fi 4 nitrogen- xing strain formed the densest and most metabolically 50 mM, lactate was the best for both biofilm formation and fi 38 fi active bio lms . Many nitrogen- xing bacteria, such as those of nitrogen fixation (Fig. 1a, b). The ability of A1501 to form biofilms the genera Rhizobium, Gluconacetobacter and Azospirillum, pro- gradually decreased with increasing NH + concentration (Fig. 1c) fi 39–42 4 duce bio lms containing various exopolysaccharides . For but was enhanced with increasing lactate concentration (Fig. 1d). instance, Sinorhizobium meliloti produces two symbiosis- In addition, ~35% of the maximum nitrogenase activity was promoting exopolysaccharides, succinoglycan and galactoglucan, observed in planktonic growth at a low lactate concentration fi which function in host speci city and participate in early stages of (1.0 mM), but very low nitrogenase activity was detected in biofilm a host plant infection, biofilm formation, and, most importantly, fi 43–45 growth (Fig. 1e), indicating that bio lm cells were incapable of protection from environmental stresses . Azospirillum cells are fixing nitrogen unless supplied with an adequately available also capable of forming biofilms on both abiotic surfaces and in fi fi 46 carbon source. These results indicated that nitrogen- xing bio lm 1234567890():,; association with host plants . Previous studies have demon- growth requires a sufficient supply of carbon sources, as both strated that two response regulator