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J. Bacteriol. doi:10.1128/JB.00157-07
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Bacillus subtilis pellicle formation proceeds through genetically defined morphological changes

Graduate School of Information Science, Nara Institute of Science and Technology, Takayama 8916-5, Ikoma, Nara 630-0192, Japan

^* To whom correspondence should be addressed. Email: kazuok{at}bs.naist.jp.

	Abstract

Biofilms are structured multi-cellular communities of bacteria that form through a developmental process. In standing culture, undomesticated strains of B. subtilis produce a floating biofilm, pellicle, with a distinct macroscopic architecture. Here, we report on a comprehensive analysis of B. subtilis pellicle formation, with a focus on transcriptional regulators and morphological changes. To date, 288 known or putative transcriptional regulators in the B. subtilis genome have been identified or assigned based on similarity to other known proteins. The genes encoding these regulators were systematically disrupted and the effect of the mutations on pellicle formation was examined, resulting in identification of 19 regulators involved in pellicle formation. In addition, morphological analysis revealed that pellicle formation begins with formation of cell chains, which is followed by clustering and degradation of cell chains. Genetic and morphological evidence showed that each stage of morphological change can be defined genetically based on the mutations of transcriptional regulators, each of which blocks pellicle formation at a specific morphological stage. Formation and degradation of cell chains are controlled by down- and up-regulation of ^D- and ^H-dependent autolysins expressed at specific stages during pellicle formation. Transcriptional analysis revealed that the transcriptional activation of sigH depends on formation of cell clusters, which in turn activates transcription of ^H-dependent autolysin in cell clusters. Taken together, our results reveal relationships between transcriptional regulators and morphological development during pellicle formation by B. subtilis.

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