Dataset Information

A regulatory network involving Rpo, Gac and Rsm for nitrogen-fixing biofilm formation by Pseudomonas stutzeri.

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.

SUBMITTER: Shang L

PROVIDER: S-EPMC8249394 | biostudies-literature | 2021 Jul

REPOSITORIES: biostudies-literature

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A regulatory network involving Rpo, Gac and Rsm for nitrogen-fixing biofilm formation by Pseudomonas stutzeri.

Shang Liguo L Yan Yongliang Y Zhan Yuhua Y Ke Xiubin X Shao Yahui Y Liu Yaqun Y Yang Hua H Wang Shanshan S Dai Shuling S Lu Jiasi J Yan Ning N Yang Zhimin Z Lu Wei W Liu Zhu Z Chen Shanchun S Elmerich Claudine C Lin Min M

NPJ biofilms and microbiomes 20210701 1

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 nitrog ...[more]

PMID: 34210981

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Project description:The GacS/GacA two-component regulatory system activates the production of secondary metabolites including phenazines crucial for biological control activity in Pseudomonas chlororaphis 30-84. To better understand the role of the Gac system on phenazine regulation, transcriptomic analyses were conducted by comparing the wild-type strain to a gacA mutant. RNA-seq analysis identified 771 genes under GacA control, including many novel genes. Consistent with previous findings, phenazine biosynthetic genes were significantly downregulated in a gacA mutant. The transcript abundances of phenazine regulatory genes such as phzI, phzR, iopA, iopB, rpoS, and pip also were reduced. Moreover, the transcript abundance of three noncoding RNAs (ncRNAs) including rsmX, rsmY, and rsmZ was significantly decreased by gacA mutation consistent with the presence of consensus GacA-binding sites associated with their promoters. Our results also demonstrated that constitutive expression of rsmZ from a non-gac regulated promoter resulted in complete restoration of N-acyl-homoserine lactone (AHL) and phenazine production as well as the expression of other gac-dependent secondary metabolites in gac mutants. The role of RsmA and RsmE in phenazine production also was investigated. Overexpression of rsmE, but not rsmA, resulted in decreased AHL and phenazine production in P. chlororaphis, and only a mutation in rsmE bypassed the requirement for GacA in phenazine gene expression. In contrast, constitutive expression of the phzI/phzR quorum sensing system did not rescue phenazine production in the gacA mutant, indicating the direct posttranscriptional control by Gac on the phenazine biosynthetic genes. On the basis of these results, we propose a model to illustrate the hierarchic role of phenazine regulators modulated by Gac in the control of phenazine production. The transcriptomic analysis also was used to identify additional genes regulated by GacA that may contribute to the biological control capability of strain 30-84.

Project description:The biosynthesis of mupirocin, a clinically significant antibiotic produced by Pseudomonas sp. NCIMB 10586, is activated by the N-acyl homoserine lactone (AHL) MupR/I quorum sensing (QS) system. However, to date, limited research has focused on the influence of global regulators such as the GacS/A two-component system (TCS) on the MupR/I QS system or mupirocin biosynthesis. In this study, we characterized the regulatory components of the Gac/Rsm transduction system in the mupirocin-producing model strain NCIMB 10586 and investigated their interconnection with the MupR/I QS circuit and subsequent mupirocin biosynthesis. The production of mupirocin was hampered by either gacS inactivation, gacA inactivation, or the double-mutant of the sRNAs ( RsmY and RsmZ). Similarly, the expressions of mupR and mupI, and AHL synthesis significantly decreased in gacS, gacA, or rsmY/Z mutants, indicating that the GacS/A system stimulates mupirocin biosynthesis via the MupR/I QS system. Five CsrA family proteins, RsmA/E/I/F/N, were found in strain NCIMB 10586, and the single and multiple mutants of rsmA/E/I/F/N showed different phenotypes with respect to mupirocin production. Our results revealed that mupirocin biosynthesis was likely to be negatively regulated by RsmA/E/I, but positively regulated by RsmF. Additionally, the RsmF protein was shown to interact with the 5' leader of mupR mRNA. In summary, the Gac/Rsm system positively regulates the biosynthesis of mupirocin mainly through the MupR/I QS system, and the model of the regulatory mechanism is proposed. The elucidation of the Gac/Rsm-MupR/I regulatory pathway could help devise ways for improving mupirocin production through genetic engineering.IMPORTANCEThe Gac/Rsm regulatory system plays a global regulatory role in bacterial physiology and metabolism, including secondary metabolism. Mupirocin is a clinically important antibiotic, produced by Pseudomonas sp. NCIMB 10586, whose biosynthesis is activated by the MupR/I quorum sensing system. Global regulators have important impacts on the gene expression of secondary metabolic gene clusters and QS genes, and the GacS/A two-component system is one of the main regulators across Pseudomonas species, which significantly influences antibiotic production. Our study presented that the expressions of QS genes and mup gene cluster were downregulated in gacS, gacA, or rsmY/Z mutants compared to the wild-type. The inactivation of rsmA/E/I/F/N in NCIMB 10586, encoding CsrA family proteins, showed different regulatory traits of mupirocin production, in which the RsmF protein could interact with the 5' UTR region of mupR mRNA. These findings provide the understanding of the regulatory role of Gac/Rsm on mupirocin biosynthesis and mupR/I QS system and lay foundations for further improving mupirocin production.

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Dataset Information

A regulatory network involving Rpo, Gac and Rsm for nitrogen-fixing biofilm formation by Pseudomonas stutzeri.

Publications

A regulatory network involving Rpo, Gac and Rsm for nitrogen-fixing biofilm formation by Pseudomonas stutzeri.

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