Sporulation in Bacillus subtilis involves an asymmetric cell division followed by differentiation into two cell types, the endospore and the mother cell. The endospore coat is a multilayered shell that protects the bacterial genome during stress conditions and is composed of dozens of proteins. Recently, fluorescence microscopy coupled with high-resolution image analysis has been applied to the dynamic process of coat assembly and has shown that the coat is organized into at least four distinct layers. In this Review, we provide a brief summary of B. subtilis sporulation, describe the function of the spore surface layers and discuss the recent progress that has improved our understanding of the structure of the endospore coat and the mechanisms of coat assembly.

Spores of Bacillus subtilis with a mutation in spoVF cannot synthesize dipicolinic acid (DPA) and are too unstable to be purified and studied in detail. However, the spores of a strain lacking the three major germinant receptors (termed ⌬ger3), as well as spoVF, can be isolated, although they spontaneously germinate much more readily than ⌬ger3 spores. The ⌬ger3 spoVF spores lack DPA and have higher levels of core water than ⌬ger3 spores, although sporulation with DPA restores close to normal levels of DPA and core water to ⌬ger3 spoVF spores. The DPA-less spores have normal cortical and coat layers, as observed with an electron microscope, but their core region appears to be more hydrated than that of spores with DPA. The ⌬ger3 spoVF spores also contain minimal levels of the processed active form (termed P 41 ) of the germination protease, GPR, a finding consistent with the known requirement for DPA and dehydration for GPR autoprocessing. However, any P 41 formed in ⌬ger3 spoVF spores may be at least transiently active on one of this protease's small acid-soluble spore protein (SASP) substrates, SASP-␥. Analysis of the resistance of wild-type, ⌬ger3, and ⌬ger3 spoVF spores to various agents led to the following conclusions: (i) DPA and core water content play no role in spore resistance to dry heat, dessication, or glutaraldehyde; (ii) an elevated core water content is associated with decreased spore resistance to wet heat, hydrogen peroxide, formaldehyde, and the iodine-based disinfectant Betadine; (iii) the absence of DPA increases spore resistance to UV radiation; and (iv) wild-type spores are more resistant than ⌬ger3 spores to Betadine and glutaraldehyde. These results are discussed in view of current models of spore resistance and spore germination.Spores of Bacillus and Clostridium species normally contain Ն10% of their dry weight as pyridine-2,6-dicarboxylic acid (dipicolinic acid [DPA]) (21,22,39). This compound is synthesized late in sporulation in the mother cell compartment of the sporulating cell but accumulates only in the developing forespore (6, 36). The great majority of the spore's DPA is in the spore core, where it is most likely chelated with divalent cations, predominantly Ca 2ϩ , although there are also significant amounts of Mg 2ϩ and Mn 2ϩ , with smaller amounts of other divalent cations (21,22,37,39). In the first minutes of spore germination the DPA is excreted, along with the associated divalent cations (36, 37).Since DPA is found only in dormant spores of Bacillus and Clostridium species and since these spores differ in a number of properties from vegetative cells, in particular in their dormancy and heat resistance, it is not surprising that DPA and divalent cations have been suggested to be involved in some of the spore's unique properties. There is some evidence in support of this suggestion, since mutants whose spores do not accumulate DPA have been isolated in several Bacillus species, and often these DPA-less spores are heat sensitive (1,4,25,42,43). Unfortunately, for some of ...

Organisms from all domains of life use gene regulation networks to control cell growth, identity, function, and responses to environmental challenges. Although accurate global regulatory models would provide critical evolutionary and functional insights, they remain incomplete, even for the best studied organisms. Efforts to build comprehensive networks are confounded by challenges including network scale, degree of connectivity, complexity of organism–environment interactions, and difficulty of estimating the activity of regulatory factors. Taking advantage of the large number of known regulatory interactions in Bacillus subtilis and two transcriptomics datasets (including one with 38 separate experiments collected specifically for this study), we use a new combination of network component analysis and model selection to simultaneously estimate transcription factor activities and learn a substantially expanded transcriptional regulatory network for this bacterium. In total, we predict 2,258 novel regulatory interactions and recall 74% of the previously known interactions. We obtained experimental support for 391 (out of 635 evaluated) novel regulatory edges (62% accuracy), thus significantly increasing our understanding of various cell processes, such as spore formation.

SUMMARY In response to starvation, bacilli and clostridia undergo a specialized program of development that results in the production of a highly resistant dormant cell type known as the spore. A proteinacious shell, called the coat, encases the spore and plays a major role in spore survival. The coat is composed of over 25 polypeptide species, organized into several morphologically distinct layers. The mechanisms that guide coat assembly have been largely unknown until recently. We now know that proper formation of the coat relies on the genetic program that guides the synthesis of spore components during development as well as on morphogenetic proteins dedicated to coat assembly. Over 20 structural and morphogenetic genes have been cloned. In this review, we consider the contributions of the known coat and morphogenetic proteins to coat function and assembly. We present a model that describes how morphogenetic proteins direct coat assembly to the specific subcellular site of the nascent spore surface and how they establish the coat layers. We also discuss the importance of posttranslational processing of coat proteins in coat morphogenesis. Finally, we review some of the major outstanding questions in the field.

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