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Transcriptomic Analysis Reveals the Role of tmRNA on Biofilm Formation in Bacillus subtilis.

ABSTRACT: Bacillus strains are widely distributed in terrestrial and marine environments, and some of them are used as biocontrol organisms for their biofilm-formation ability. In Bacillus subtilis, biofilm formation is fine-tuned by a complex network, a clear understanding of which still requires study. In bacteria, tmRNA, encoded by the ssrA gene, catalyzes trans-translation that can rescue ribosomes stalled on mRNA transcripts lacking a functional stop codon. tmRNA also affects physiological bioprocesses in some bacteria. In this study, we constructed a ssrA mutant in B. subtilis and found that the biofilm formation in the ssrA mutant was largely impaired. Moreover, we isolated a biofilm-formation suppressor of ssrA, in which the biofilm formation was restored to a level even stronger than that in the wild type. We further performed RNAseq assays with the wild type, ssrA mutant, and suppressor of ssrA for comparisons of their transcriptomes. By analyzing the transcriptomic data, we predicted the possible functions of some differentially expressed genes (DEGs) in the tmRNA regulation of biofilm formation in B. subtilis. Finally, we found that the overexpression of two DEGs, acoA and yhjR, could restore the biofilm formation in the ssrA mutant, indicating that AcoA and YhjR were immediate regulators involved in the tmRNA regulatory web controlling biofilm formation in B. subtilis. Our data can improve the knowledge about the molecular network involved in Bacillus biofilm formation and provide new targets for manipulation of Bacillus biofilms for future investigation.

SUBMITTER: Xu S

PROVIDER: S-EPMC9319509 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Bacillus subtilis forms robust biofilms in the presence of large amounts of carbon sources, such as glycerol. However, little is known about the importance of the metabolic systems, or the relationship between metabolic systems and regulatory systems, involved in biofilm formation. Glutamate synthase, encoded by gltAB, is an enzyme that converts 2-ketoglutarate (a tricarboxylic acid [TCA] cycle intermediate) and glutamine into glutamate, which is a general amino group donor in metabolism. Here, we show that a ?gltA mutant exhibited early arrest of biofilm formation in complex medium containing glycerol. This phenotype was not due to glutamate auxotrophy. Consistent with its biofilm formation phenotype, the ?gltA mutant exhibited an early decrease in expression of the epsA and tapA operons, which are responsible for production of biofilm matrix polymers. This resulted from decreased activity of their regulator, Spo0A, as evidenced by reduced expression of other Spo0A-regulated genes in the ?gltA mutant. The ?gltA mutation prevented biofilm formation only in the presence of large amounts of glycerol. Moreover, limited expression of citrate synthase (but not other TCA enzymes) restored biofilm-forming ability to the ?gltA mutant. These results indicate that the ?gltA mutant accumulates an inhibitory intermediate (citrate) in the TCA cycle in the presence of large amounts of glycerol. The ?gltA mutant formed biofilms when excess iron was added to the medium. Taken together, the data suggest that accumulation of citrate ions by the ?gltA mutant causes iron shortage due to chelation, which prevents activation of Spo0A and causes defective biofilm formation.IMPORTANCE Bacillus subtilis, a model organism for bacterial biofilm formation, forms robust biofilms in a medium-dependent manner. Although the regulatory network that controls biofilm formation has been well studied, the importance of the underlying metabolic systems remains to be elucidated. The present study demonstrates that a metabolic disorder in a well-conserved metabolic system causes accumulation of an inhibitory metabolic intermediate that prevents activation of the system that regulates biofilm formation. These findings increase our understanding of the coordination between cellular metabolic status and the regulatory networks governing biofilm formation.

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Transcriptomic Analysis Reveals the Role of tmRNA on Biofilm Formation in Bacillus subtilis.

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