Abstract
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.
Highlights
The term ‘biofilm’ can be defined as a community of microbes adhering to biotic or abiotic surfaces that is protected from environmental stresses by a self-produced extracellular matrix[1,2].The extracellular matrix, often referred to as extracellular polymeric substances, is composed of exopolysaccharides, proteins and extracellular DNA present in various concentrations depending on the bacterial species[3,4]
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 ecological advantages and thereby facilitate their physiological and metabolic adaptation to successfully survive in the rhizosphere, a nitrogen-limited environment
We found that the rpoS mutant exhibited increased biofilm production compared to that of A1501 under nitrogen-sufficient conditions (Table 1), consistent with results previously described for the P. aeruginosa rpoS mutant[32] but different from the E. coli rpoS mutant showing decreased biofilm production[31]
Summary
The term ‘biofilm’ can be defined as a community of microbes adhering to biotic or abiotic surfaces that is protected from environmental stresses by a self-produced extracellular matrix[1,2].The extracellular matrix, often referred to as extracellular polymeric substances, is composed of exopolysaccharides, proteins and extracellular DNA present in various concentrations depending on the bacterial species[3,4]. The term ‘biofilm’ can be defined as a community of microbes adhering to biotic or abiotic surfaces that is protected from environmental stresses by a self-produced extracellular matrix[1,2]. As established in the model bacterium Pseudomonas aeruginosa, biofilm development usually begins with attachment to a surface, followed by microcolony formation and production of the extracellular matrix responsible for the biofilm architecture[10,11,12,13,14]. Biofilm formation has been studied intensively in the genus Pseudomonas, with an emphasis on genetic elements and molecular mechanisms; Gac/Rsm, c-di-GMP signalling and quorum-sensing (QS) pathways were reported as the main mechanisms leading to biofilm formation[15,16]. The Gac/Rsm signalling pathway involves the GacS/GacA two-component regulatory system, the RNA-binding protein RmsA, and its cognate regulatory non-coding RNAs (ncRNAs)[17,18].
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