Patrick Eichenberger

The Spo0A regulon of Bacillus subtilis

The master regulator for entry into sporulation in Bacillus subtilis is the DNA‐binding protein Spo0A, which has been found to influence, directly or indirectly, the expression of over 500 genes during the early stages of development. To search on a genome‐wide basis for genes under the direct control of Spo0A, we used chromatin immunoprecipitation in combination with gene microarray analysis to identify regions of the chromosome at which an activated form of Spo0A binds in vivo. This information in combination with transcriptional profiling using gene microarrays, gel electrophoretic mobility shift assays, using the DNA‐binding domain of Spo0A, and bioinformatics enabled us to assign 103 genes to the Spo0A regulon in addition to 18 previously known members. Thus, in total, 121 genes, which are organized as 30 single‐gene units and 24 operons, are likely to be under the direct control of Spo0A. Forty of these genes are under the positive control of Spo0A, and 81 are under its negative control. Among newly identified members of the regulon with transcription that was stimulated by Spo0A are genes for metabolic enzymes and genes for efflux pumps. Among  members  with  transcription  that  was in‐hibited by Spo0A are genes encoding components of the DNA replication machinery and genes that govern flagellum biosynthesis and chemotaxis. Also in‐cluded in the regulon are many (25) genes with products that are direct or indirect regulators of gene transcription. Spo0A is a master regulator for sporulation, but many of its effects on the global pattern of gene transcription are likely to be mediated indirectly by regulatory genes under its control.

The Program of Gene Transcription for a Single Differentiating Cell Type during Sporulation in Bacillus subtilis

Asymmetric division during sporulation by Bacillus subtilis generates a mother cell that undergoes a 5-h program of differentiation. The program is governed by a hierarchical cascade consisting of the transcription factors: σE, σK, GerE, GerR, and SpoIIID. The program consists of the activation and repression of 383 genes. The σE factor turns on 262 genes, including those for GerR and SpoIIID. These DNA-binding proteins downregulate almost half of the genes in the σE regulon. In addition, SpoIIID turns on ten genes, including genes involved in the appearance of σK . Next, σK activates 75 additional genes, including that for GerE. This DNA-binding protein, in turn, represses half of the genes that had been activated by σK while switching on a final set of 36 genes. Evidence is presented that repression and activation contribute to proper morphogenesis. The program of gene expression is driven forward by its hierarchical organization and by the repressive effects of the DNA-binding proteins. The logic of the program is that of a linked series of feed-forward loops, which generate successive pulses of gene transcription. Similar regulatory circuits could be a common feature of other systems of cellular differentiation.

Genome-Wide Analysis of the Stationary-Phase Sigma Factor (Sigma-H) Regulon of Bacillus subtilis

Sigma-H is an alternative RNA polymerase sigma factor that directs the transcription of many genes that function at the transition from exponential growth to stationary phase in Bacillus subtilis. Twenty-three promoters, which drive transcription of 33 genes, are known to be recognized by sigma-H-containing RNA polymerase. To identify additional genes under the control of sigma-H on a genome-wide basis, we carried out transcriptional profiling experiments using a DNA microarray containing >99% of the annotated B. subtilis open reading frames. In addition, we used a bioinformatics-based approach aimed at the identification of promoters recognized by RNA polymerase containing sigma-H. This combination of approaches was successful in confirming most of the previously described sigma-H-controlled genes. In addition, we identified 26 putative promoters that drive expression of 54 genes not previously known to be under the direct control of sigma-H. Based on the known or inferred function of most of these genes, we conclude that, in addition to its previously known roles in sporulation and competence, sigma-H controls genes involved in many physiological processes associated with the transition to stationary phase, including cytochrome biogenesis, generation of potential nutrient sources, transport, and cell wall metabolism.

The Bacillus subtilis endospore: Assembly and functions of the multilayered coat

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.

The σE regulon and the identification of additional sporulation genes in Bacillus subtilis

We report the identification and characterization on a genome-wide basis of genes under the control of the developmental transcription factor sigma(E) in Bacillus subtilis. The sigma(E) factor governs gene expression in the larger of the two cellular compartments (the mother cell) created by polar division during the developmental process of sporulation. Using transcriptional profiling and bioinformatics we show that 253 genes (organized in 157 operons) appear to be controlled by sigma(E). Among these, 181 genes (organized in 121 operons) had not been previously described as members of this regulon. Promoters for many of the newly identified genes were located by transcription start site mapping. To assess the role of these genes in sporulation, we created null mutations in 98 of the newly identified genes and operons. Of the resulting mutants, 12 (in prkA, ybaN, yhbH, ykvV, ylbJ, ypjB, yqfC, yqfD, ytrH, ytrI, ytvI and yunB) exhibited defects in spore formation. In addition, subcellular localization studies were carried out using in-frame fusions of several of the genes to the coding sequence for GFP. A majority of the fusion proteins localized either to the membrane surrounding the developing spore or to specific layers of the spore coat, although some fusions showed a uniform distribution in the mother cell cytoplasm. Finally, we used comparative genomics to determine that 46 of the sigma(E)-controlled genes in B.subtilis were present in all of the Gram-positive endospore-forming bacteria whose genome has been sequenced, but absent from the genome of the closely related but not endospore-forming bacterium Listeria monocytogenes, thereby defining a core of conserved sporulation genes of probable common ancestral origin. Our findings set the stage for a comprehensive understanding of the contribution of a cell-specific transcription factor to development and morphogenesis.

The Bacillus subtilis spore coat protein interaction network

Bacterial spores are surrounded by a morphologically complex, mechanically flexible protein coat, which protects the spore from toxic molecules. The interactions among the over 50 proteins that make up the coat remain poorly understood. We have used cell biological and protein biochemical approaches to identify novel coat proteins in Bacillus subtilis and describe the network of their interactions, in order to understand coat assembly and the molecular basis of its protective functions and mechanical properties. Our analysis characterizes the interactions between 32 coat proteins. This detailed view reveals a complex interaction network. A key feature of the network is the importance of a small subset of proteins that direct the assembly of most of the coat. From an analysis of the network topology, we propose a model in which low‐affinity interactions are abundant in the coat and account, to a significant degree, for the coats mechanical properties as well as structural variation between spores.

Hierarchical Evolution of the Bacterial Sporulation Network

Genome sequencing of multiple species makes it possible to understand the main principles behind the evolution of developmental regulatory networks. It is especially interesting to analyze the evolution of well-defined model systems in which conservation patterns can be directly correlated with the functional roles of various network components. Endospore formation (sporulation), extensively studied in Bacillus subtilis, is driven by such a model bacterial network of cellular development and differentiation. In this review, we analyze the evolution of the sporulation network in multiple endospore-forming bacteria. Importantly, the network evolution is not random but primarily follows the hierarchical organization and functional logic of the sporulation process. Specifically, the sporulation sigma factors and the master regulator of sporulation, Spo0A, are conserved in all considered spore-formers. The sequential activation of these global regulators is also strongly conserved. The feed-forward loops, which are likely used to fine-tune waves of gene expression within regulatory modules, show an intermediate level of conservation. These loops are less conserved than the sigma factors but significantly more than the structural sporulation genes, which form the lowest level in the functional and evolutionary hierarchy of the sporulation network. Interestingly, in spore-forming bacteria, gene regulation is more conserved than gene presence for sporulation genes, while the opposite is true for non-sporulation genes. The observed patterns suggest that, by understanding the functional organization of a developmental network in a model organism, it is possible to understand the logic behind the evolution of this network in multiple related species.

Identification of a New Gene Essential for Germination of Bacillus subtilis Spores with Ca2+-Dipicolinate

Bacillus subtilis spores can germinate with a 1:1 chelate of Ca(2+) and dipicolinic acid (DPA), a compound present at high levels in the spore core. Using a genetic screen to identify genes encoding proteins that are specifically involved in spore germination by Ca(2+)-DPA, three mutations were identified. One was in the gene encoding the cortex lytic enzyme, CwlJ, that was previously shown to be essential for spore germination by Ca(2+)-DPA. The other two were mapped to an open reading frame, ywdL, encoding a protein of unknown function. Analysis of ywdL expression showed that the gene is expressed during sporulation in the mother cell compartment of the sporulating cell and that its transcription is sigma(E) dependent. Functional characterization of YwdL demonstrated that it is a new spore coat protein that is essential for the presence of CwlJ in the spore coat. Assembly of YwdL itself into the spore coat is dependent on the coat morphogenetic proteins CotE and SpoIVA. However, other than lacking CwlJ, ywdL spores have no obvious defect in their spore coat. Because of the role for YwdL in a part of the spore germination process, we propose renaming ywdL as a spore germination gene, gerQ.

A Distance-Weighted Interaction Map Reveals a Previously Uncharacterized Layer of the Bacillus subtilis Spore Coat

Bacillus subtilis spores are encased in a protein assembly called the spore coat that is made up of at least 70 different proteins. Conventional electron microscopy shows the coat to be organized into two distinct layers. Because the coat is about as wide as the theoretical limit of light microscopy, quantitatively measuring the localization of individual coat proteins within the coat is challenging. We used fusions of coat proteins to green fluorescent protein to map genetic dependencies for coat assembly and to define three independent subnetworks of coat proteins. To complement the genetic data, we measured coat protein localization at subpixel resolution and integrated these two data sets to produce a distance-weighted genetic interaction map. Using these data, we predict that the coat comprises at least four spatially distinct layers, including a previously uncharacterized glycoprotein outermost layer that we name the spore crust. We found that crust assembly depends on proteins we predicted to localize to the crust. The crust may be conserved in all Bacillus spores and may play critical functions in the environment.

A three-protein inhibitor of polar septation during sporulation in Bacillus subtilis

We present evidence for a three‐protein inhibitor of polar division that locks in asymmetry after the formation of a polar septum during sporulation in Bacillus subtilis. Asymmetric division involves the formation of cytokinetic Z‐rings near both poles of the developing cell. Next, a septum is formed at one of the two polar Z‐rings, thereby generating a small, forespore cell and a mother cell. Gene expression under the control of the mother‐cell transcription factor σE is needed to block cytokinesis at the pole distal to the newly formed septum. We report that this block in polar cytokinesis is mediated partly by σE‐directed transcription of spoIID, spoIIM and spoIIP, sporulation genes that were known to be involved in the subsequent process of forespore engulfment. We find that a spoIID, spoIIM and spoIIP triple mutant substantially mimicked the bipolar division phenotype of a σE mutant and that cells engineered to produce SpoIID, SpoIIM and SpoIIP prematurely were inhibited in septum formation at both poles. Consistent with the hypothesis that SpoIID, SpoIIM and SpoIIP function at both poles of the sporangium, a GFP–SpoIIM fusion localized to the membrane that surrounds the engulfed forespore and to the potential division site at the distal pole.