Identification of AbrB-regulated genes involved in biofilm formation by Bacillus subtilis

Abstract

Bacillus subtilis is a ubiquitous soil bacterium that forms biofilms in a process that is negatively controlled by the transcription factor AbrB. To identify the AbrB-regulated genes required for biofilm formation by B. subtilis, genome-wide expression profiling studies of biofilms formed by spo0A abrB and sigH abrB mutant strains were performed. These data, in concert with previously published DNA microarray analysis of spo0A and sigH mutant strains, led to the identification of 39 operons that appear to be repressed by AbrB. Eight of these operons had previously been shown to be repressed by AbrB, and we confirmed AbrB repression for a further six operons by reverse transcription-PCR. The AbrB-repressed genes identified in this study are involved in processes known to be regulated by AbrB, such as extracellular degradative enzyme production and amino acid metabolism, and processes not previously known to be regulated by AbrB, such as membrane bioenergetics and cell wall functions. To determine whether any of these AbrB-regulated genes had a role in biofilm formation, we tested 23 mutants, each with a disruption in a different AbrB-regulated operon, for the ability to form biofilms. Two mutants had a greater than twofold defect in biofilm formation. A yoaW mutant exhibited a biofilm structure with reduced depth, and a sipW mutant exhibited only surface-attached cells and did not form a mature biofilm. YoaW is a putative secreted protein, and SipW is a signal peptidase. This is the first evidence that secreted proteins have a role in biofilm formation by Bacillus subtilis.

Figure 1

Model of the genetic network regulating biofilm formation in Bacillus subtilis. The arrow indicates positive regulation, and the perpendicular lines indicate negative regulation.

Figure 2

Diagram of the genetic organization of the chromosomal regions surrounding yoaW (A) and yqxM (B). Gene direction is indicated by an arrow below the genes, terminators are indicated with boxes, and the yqxM promoter is indicated by a large arrow above the gene.

Figure 3

Microtiter plate assay of biofilm formation by yqxM operon mutants. All strains were assayed after 24 hours of growth under biofilm formation conditions. The error bars indicate the standard error of the mean.

Figure 4

CSLM analysis of wild-type, tasA yqxM, sipW, and yoaW mutant strains of B. subtilis. Biofilms of cells expressing the green fluorescent protein from a chromosomal locus were grown on the surface of glass coverslides and then analyzed by CSLM. Shown are representative images of those obtained on at least three independent occasions. Top images are single sections through the X-Y plane, and the bottom images are single sections through the X-Z plane. Panel A shows BAL835, Panel B shows BAL1062, Panel C shows BAL1061, and Panel D shows BAL1946.