Dataset Information

Functional analysis of the protein Veg, which stimulates biofilm formation in Bacillus subtilis.

ABSTRACT: Biofilm is a complex aggregate of cells that adhere to each other and produce an extracellular matrix. In Bacillus subtilis, an extracellular polysaccharide (EPS) and amyloid fiber (TasA), synthesized by the epsA-epsO and tapA-sipW-tasA operons, respectively, are the primary components of the extracellular matrix. In the current study, we investigated the functional role of the previously uncharacterized veg gene in B. subtilis. Overproduction of Veg, a small protein highly conserved among Gram-positive bacteria, stimulated biofilm formation via inducing transcription of the tapA-sipW-tasA operon. Moreover, overproduced Veg restored the impairment of biofilm formation in mutants carrying a deletion of of sinI, slrA, or slrR, encoding an antirepressor of SinR that acts as the master regulator of biofilm formation, while biofilm morphology in the absence of SinR was not affected by either additional veg deletion or overproduction, indicating that Veg negatively regulates SinR activity independently of the known antirepressors. Expression of sinR was not affected in Veg-overproducing cells, and amounts of SinR were similar in cells expressing different levels of Veg, strongly suggesting that Veg modulates the repressor activity of SinR. Interestingly, the results of in vivo pulldown assays of the SinR complex indicate that Veg inhibits the interactions between SinR and SlrR. Based on these findings, we propose that Veg or a Veg-induced protein acts as an antirepressor of SinR to regulate biofilm formation.

SUBMITTER: Lei Y

PROVIDER: S-EPMC3624561 | biostudies-literature | 2013 Apr

REPOSITORIES: biostudies-literature

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Functional analysis of the protein Veg, which stimulates biofilm formation in Bacillus subtilis.

Lei Ying Y Oshima Taku T Ogasawara Naotake N Ishikawa Shu S

Journal of bacteriology 20130201 8

Biofilm is a complex aggregate of cells that adhere to each other and produce an extracellular matrix. In Bacillus subtilis, an extracellular polysaccharide (EPS) and amyloid fiber (TasA), synthesized by the epsA-epsO and tapA-sipW-tasA operons, respectively, are the primary components of the extracellular matrix. In the current study, we investigated the functional role of the previously uncharacterized veg gene in B. subtilis. Overproduction of Veg, a small protein highly conserved among Gram- ...[more]

PMID: 23378512

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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.

Project description:Iron (Fe) is the most important metal in biology. Despite its abundance, Fe is mostly present as a ferric form in soils, strongly limiting its bioavailability. To overcome the challenge of Fe acquisition, many microorganisms produce siderophores to retrieve Fe from natural sources. Another ubiquitous feature of bacteria in natural environments is biofilm formation. Previous studies showed that external Fe strongly influenced biofilm formation in several bacteria, suggesting that this microenvironment plays a mechanistic role in micronutrient acquisition for bacteria. Here, we applied a complementary set of analytical methods and deletion mutants to evaluate the role of biofilm formation, siderophore production, and their interaction in Fe homeostasis in Bacillus subtilis We observed that Fe homeostasis, i.e., active growth at a constant intracellular Fe concentration, requires both siderophore production and biofilm formation. Also, we report that in B. subtilis, both biofilm formation and siderophore production are required to achieve active Fe acquisition from the medium and to sustain normal growth. Furthermore, we provide evidence that the formation of biofilm slightly enhances the kinetics of Fe complexation by catechol siderophores and markedly improves siderophore use efficiency. These results provide new perspectives on the mechanism underlying siderophore-based acquisition of Fe in biofilm-forming bacteria.IMPORTANCE Iron acquisition is of fundamental importance for microorganisms, since this metal is generally poorly bioavailable under natural conditions. In the environment, most bacteria are found tightly packed within multicellular communities named biofilms. Here, using the soil Gram-positive bacterium Bacillus subtilis, we show that biofilm formation and the production of siderophores, i.e., small molecules specifically binding metals, are both essential to ensure Fe uptake from the medium and maintain cellular Fe homeostasis. The biofilm matrix appears to play an important role favoring the efficient usage of siderophores. Taken together, our results demonstrate a close link between biofilm formation and iron acquisition in B. subtilis, allowing a better comprehension of how bacteria can cope with metal limitation under environmental conditions.

Dataset Information

Functional analysis of the protein Veg, which stimulates biofilm formation in Bacillus subtilis.

Publications

Functional analysis of the protein Veg, which stimulates biofilm formation in Bacillus subtilis.

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