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Bacillus subtilis is a non-pathogenic soil bacterium that has been extensively studied as a model organism for the molecular biology and physiology of Gram positive bacteria. When B. subtilis cells are starved, they initiate a developmental program that culminates in the formation of highly resistant spores. Hence, the sporulation process constitutes a relatively simple system in which the generation of distinct cell types can be investigated, both genetically and biochemically. Our research is oriented along two axes: 1) We are studying the mechanisms directing the formation of the spore coat, a multi-layered protein assembly that constitutes the envelope of Bacillus spores. Our experimental design integrates genetics, fluorescence microscopy, yeast two-hybrid and biochemical approaches. 2) In collaboration with the group of computational biologist Richard Bonneau, our goal is to infer the B. subtilis transcriptional regulatory network. To achieve this goal, we are collecting dynamic gene expression data over various conditions and using a genome-wide mutant library for B. subtilis that is currently being generated as a result of a concerted effort of the B. subtilis research community in the USA.

1. Spore coat assembly in B. subtilis

The coat is a proteinaceous shell surrounding the spore and is made of more than seventy different proteins in B. subtilis. Spore coat assembly is a dynamic process that can be studied by fluorescence microscopy. Using a combination of genetic and fluorescence microscopy analyses, we systematically mapped a network of genetic dependencies among 40 coat protein-GFP fusions and placed them in one of three genetic dependency clusters, each group corresponding to a spatially distinct layer of the coat.  We directly measured the localization of 17 individual proteins in the coat using PSICIC, a Matlab-based image-analysis toolbox capable of extracting sub-pixel information from fluorescence microscopy images. Our investigation revealed a previously uncharacterized outermost layer of the B. subtilis spore coat that we named the spore crust (Fig.1). In parallel, we have been collecting yeast two-hybrid data and performed biochemical experiments to analyze if the genetic dependencies that were identified above were caused by direct interactions between pairs of coat proteins. Our most immediate goal is to further characterize the structure of the spore crust.

Figure 1: An electron micrograph of a Bacillus subtilis spore. The material surrounding the spore is the newly defined spore crust, a protective layer presumably made of glycoproteins that is only visible in the presence of Ruthenium red stain. The center of the spore is the core, which contains the bacterial genome. The white layer surrounding the core is the spore cortex. The lightly stained lamellar layer is the inner coat. The darkly stained layer is the outer coat. For details see McKenney et al. 2010.

2. The transcriptional regulatory network in B. subtilis

To model the transcriptional regulatory network of B. subtilis, we are collecting dynamic global gene expression data over various conditions. Specifically, we are performing time-series profiling during two types of experiments: 1) using an inducible promoter to trigger overexpression of selected transcription factors, and 2) exposure of B. subtilis to chemical and environmental perturbations. The B. subtilis transcriptional regulatory network will be inferred in a manner similar to that of the halophilic archaeon Halobacterium salinarium (Bonneau et al., 2007, Cell 131:1354-65).

Research is supported by grants from NIH.