Genes involved in formation of structured multicellular communities by Bacillus subtilis

Abstract

The spore-forming bacterium Bacillus subtilis is capable of assembling multicellular communities (biofilms) that display a high degree of spatiotemporal organization. Wild strains that have not undergone domestication in the laboratory produce particularly robust biofilms with complex architectural features, such as fruiting-body-like aerial projections whose tips serve as preferential sites for sporulation. To discover genes involved in this multicellular behavior and to do so on a genome-wide basis, we took advantage of a large collection of mutants which have disruptions of most of the uncharacterized genes in the B. subtilis genome. This collection, which was generated with a laboratory strain, was screened for mutants that were impaired in biofilm formation. This subset of mutated genes was then introduced into the wild strain NCIB 3610 to study their effects on biofilm formation in liquid and solid media. In this way we identified six genes that are involved in the development of multicellular communities. These are yhxB (encoding a putative phosphohexomutase that may mediate exopolysaccharide synthesis), sipW (encoding a signal peptidase), ecsB (encoding an ABC transporter subunit), yqeK (encoding a putative phosphatase), ylbF (encoding a regulatory protein), and ymcA (a gene of unknown function). Further analysis revealed that these six genes play different roles in B. subtilis community development.

FIG. 1.

BFA mutants defective in B. subtilis community development. Mutants from the BFA collection were isolated on the basis of their failure to form wild-type pellicles. The mutant alleles were then reintroduced into parental strain 168 and also introduced into strain 3610. To assay pellicle formation (first and second columns), each mutant was inoculated at a low density into a standing culture consisting of 12 ml of MSgg plus MLS in a microtiter plate well, and the cultures were incubated at 30°C for 60 h without shaking. To assay colony development (third column), each mutant derived from 3610 was grown overnight in a rolled LB plus MLS culture, and a 3-μl sample of the culture was spotted onto MSgg agar and incubated at 30°C for 96 h. Bars = 5 mm.

FIG. 2.

Phenotype and complementation of mutant 3610 yhxBΔ. Pellicle and colony development were assayed as described in the legend to Fig. 1.

FIG. 3.

Behavior of individual cells during formation of wild-type and mutant pellicles. Strain 3610 and deletion mutants derived from it were grown in standing cultures, as described in the legend to Fig. 1. After incubation at 25°C for 36 h, samples were withdrawn from the air-medium interface and examined at a magnification of ×1,000X using phase-contrast microscopy.

FIG. 4.

Physical maps of the genes disrupted in BFA mutants of interest. Coding regions are represented as horizontal arrows, a canonical promoter element is shown as a small bent arrow, and canonical Rho-independent transcriptional terminators are shown as stem-loop symbols. Each insertional mutation is represented by an inverted triangle bearing the number of its corresponding BFA mutant. Chromosomal regions that were deleted and replaced by antibiotic resistance markers are represented as horizontal bars—grey if the deletion conferred a defect in community development, checkered if it didn't.

FIG. 5.

Phenotype of mutant 3610 yqxM-sipW-tasAΔ (sipWΔ). Pellicle and colony development were assayed as described in the legend to Fig. 1. The parental strain images (first column) are the same as in Fig. 2.

FIG. 6.

Phenotypes of mutants 3610 ecsBΔ and 3610 ecsCΔ. Pellicle and colony development were assayed as described in the legend to Fig. 1. The parental strain images (first column) are the same as in Fig. 2.

FIG. 7.

Phenotypes of mutants 3610 yqeKΔ and 3610 yqeLΔ. Pellicle and colony development were assayed as described in the legend to Fig. 1.

FIG. 8.

Phenotypes of mutants 3610 ylbFΔ, 3610 comKΔ, and 3610 ylbFΔ comKΔ and complementation of 3610 ylbFΔ. Pellicle and colony development were assayed as described in the legend to Fig. 1. The parental strain images (first column) are the same as in Fig. 2.

FIG. 9.

Phenotypes of mutants 3610 ymcAΔ and 3610 ymcAΔ ylbFΔ. Pellicle and colony development were assayed as described in the legend to Fig. 1. The parental strain images (first column) are the same as in Fig. 2, and the 3610 ylbFΔ images (third column) are the same as in Fig. 8.