Roles and Organization of BxpB (ExsFA) and ExsFB in the Exosporium Outer Basal Layer of Bacillus anthracis.

BxpB (also known as ExsFA) and ExsFB are an exosporium basal layer structural protein and a putative interspace protein of Bacillus anthracis that are known to be required for proper incorporation of the BclA collagen-like glycoprotein on the spore surface. Despite extensive similarity of the two proteins, their distribution in the spore is markedly different. We utilized a fluorescent fusion approach to examine features of the two genes that affect spore localization. The timing of expression of the bxpB and exsFB genes and their distinct N-terminal sequences were both found to be important for proper assembly into the exosporium basal layer. Results of this study provided evidence that the BclA nap glycoprotein is not covalently attached to BxpB protein despite the key role that the latter plays in BclA incorporation. Assembly of the BxpB- and ExsFB-containing outer basal layer appears not to be completely abolished in mutants lacking the ExsY and CotY basal layer structural proteins despite these spores lacking a visible exosporium. The BxpB and, to a lesser extent, the ExsFB proteins, were found to be capable of self-assembly in vitro into higher-molecular-weight forms that are stable to boiling in SDS under reducing conditions. IMPORTANCE The genus Bacillus consists of spore-forming bacteria. Some species of this genus, especially those that are pathogens of animals or insects, contain an outermost spore layer called the exosporium. The zoonotic pathogen B. anthracis is an example of this group. The exosporium likely contributes to virulence and environmental persistence of these pathogens. This work provides important new insights into the exosporium assembly process and the interplay between BclA and BxpB in this process.

ExsY, CotY, and CotE Effects on Bacillus anthracis Outer Spore Layer Architecture.

Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis are the major pathogens of the spore-forming genus Bacillus and possess an outer spore layer, the exosporium, not found in many of the nonpathogenic species. The exosporium consists of a basal layer with the ExsY, CotY, and BxpB proteins being the major structural components and an exterior nap layer containing the BclA glycoprotein. During the assembly process, the nascent exosporium basal layer is attached to the spore coat by a protein linker that includes the CotO and CotE proteins. Using transmission electron microscopy, Western blotting, immunofluorescence, and fluorescent fusion protein approaches, we examined the impact of single, double, and triple mutants of the major exosporium proteins on exosporium protein content and distribution. Plasmid-based expression of exsY and cotE resulted in increased production of exosporium lacking spores, and the former also resulted in outer spore coat disruptions. The exosporium bottlecap produced by exsY null spores was found to be more stable than previously reported, and its spore association was partially dependent on CotE. Deletion mutants of five putative spore genes (bas1131, bas1142, bas1143, bas2277, and bas3594) were created and shown not to have obvious effects on spore morphology or BclA and BxpB content. The BclC collagen-like glycoprotein was found to be present in the spore and possibly localized to the interspace region. IMPORTANCE B. anthracis is an important zoonotic animal pathogen causing sporadic outbreaks of anthrax worldwide. Spores are the infectious form of the bacterium and can persist in soil for prolonged periods of time. The outermost B. anthracis spore layer is the exosporium, a protein shell that is the site of interactions with both the soil and with the innate immune system of infected hosts. Although much is known regarding the sporulation process among members of the genus Bacillus, significant gaps in our understanding of the exosporium assembly process exist. This study provides evidence for the properties of key exosporium basal layer structural proteins. The results of this work will guide future studies on exosporium protein-protein interactions during the assembly process.

Localization of the CotY and ExsY proteins to the exosporium basal layer of Bacillus anthracis.

Spores are an infectious form of the zoonotic bacterial pathogen, Bacillus anthracis. The outermost spore layer is the exosporium, comprised of a basal layer and an external glycoprotein nap layer. The major structural proteins of the inner basal layer are CotY (at the mother cell central pole or bottlecap) and ExsY around the rest of the spore. The basis for the cap or noncap specificity of the CotY and ExsY proteins is currently unknown. We investigated the role of sequence differences between these proteins in localization during exosporium assembly. We found that sequence differences were less important than the timing of expression of the respective genes in the positioning of these inner basal layer structural proteins. Fusion constructs with the fluorescent protein fused at the N-terminus resulted in poor incorporation whereas fusions at the carboxy terminus of CotY or ExsY resulted in good incorporation. However, complementation studies revealed that fusion constructs, although accurate indicators of protein localization, were not fully functional. A model is presented that explains the localization patterns observed. Bacterial two-hybrid studies in Escherichia coli hosts were used to examine protein-protein interactions with full-length and truncated proteins. The N-terminus amino acid sequences of ExsY and CotY appear to be recognized by spore proteins located in the spore interspace, consistent with interactions seen with ExsY and CotY with the interspace proteins CotE and CotO, known to be involved with exosporium attachment.

FtsZ ring stability: of bundles, tubules, crosslinks, and curves.

The first step in bacterial cytokinesis is the assembly of a stable but dynamic cytokinetic ring made up of the essential tubulin homolog FtsZ at the future site of division. Although FtsZ and its role in cytokinesis have been studied extensively, the precise architecture of the in vivo medial FtsZ ring (Z ring) is not well understood. Recent advances in superresolution imaging suggest that the Z ring comprises short, discontinuous, and loosely bundled FtsZ polymers, some of which are tethered to the membrane. A diverse array of regulatory proteins modulate the assembly, stability, and disassembly of the Z ring via direct interactions with FtsZ. Negative regulators of FtsZ play a critical role in ensuring the accurate positioning of FtsZ at the future site of division and in maintaining Z ring dynamics by controlling FtsZ polymer assembly/disassembly processes. Positive regulators of FtsZ are essential for tethering FtsZ polymers to the membrane and promoting the formation of stabilizing lateral interactions, permitting assembly of a mature Z ring. The past decade has seen the identification of several factors that promote FtsZ assembly, presumably through a variety of distinct molecular mechanisms. While a few of these proteins are broadly conserved, many positive regulators of FtsZ assembly are limited to small groups of closely related organisms, suggesting that FtsZ assembly is differentially modulated across bacterial species. In this review, we focus on the roles of positive regulators in Z ring assembly and in maintaining the integrity of the cytokinetic ring during the early stages of division.

Identification of ZapD as a cell division factor that promotes the assembly of FtsZ in Escherichia coli.

The tubulin homolog FtsZ forms a polymeric membrane-associated ring structure (Z ring) at midcell that establishes the site of division and provides an essential framework for the localization of a multiprotein molecular machine that promotes division in Escherichia coli. A number of regulatory proteins interact with FtsZ and modulate FtsZ assembly/disassembly processes, ensuring the spatiotemporal integrity of cytokinesis. The Z-associated proteins (ZapA, ZapB, and ZapC) belong to a group of FtsZ-regulatory proteins that exhibit functionally redundant roles in stabilizing FtsZ-ring assembly by binding and bundling polymeric FtsZ at midcell. In this study, we report the identification of ZapD (YacF) as a member of the E. coli midcell division machinery. Genetics and cell biological evidence indicate that ZapD requires FtsZ but not other downstream division proteins for localizing to midcell, where it promotes FtsZ-ring assembly via molecular mechanisms that overlap with ZapA. Biochemical evidence indicates that ZapD directly interacts with FtsZ and promotes bundling of FtsZ protofilaments. Similarly to ZapA, ZapB, and ZapC, ZapD is dispensable for division and therefore belongs to the growing group of FtsZ-associated proteins in E. coli that aid in the overall fitness of the division process.

Identification and characterization of ZapC, a stabilizer of the FtsZ ring in Escherichia coli.

In Escherichia coli, spatiotemporal control of cell division occurs at the level of the assembly/disassembly process of the essential cytoskeletal protein FtsZ. A number of regulators interact with FtsZ and modulate the dynamics of the assembled FtsZ ring at the midcell division site. In this article, we report the identification of an FtsZ stabilizer, ZapC (Z-associated protein C), in a protein localization screen conducted with E. coli. ZapC colocalizes with FtsZ at midcell and interacts directly with FtsZ, as determined by a protein-protein interaction assay in yeast. Cells lacking or overexpressing ZapC are slightly elongated and have aberrant FtsZ ring morphologies indicative of a role for ZapC in FtsZ regulation. We also demonstrate the ability of purified ZapC to promote lateral bundling of FtsZ in a sedimentation reaction visualized by transmission electron microscopy. While ZapC lacks sequence similarity with other nonessential FtsZ regulators, ZapA and ZapB, all three Zap proteins appear to play an important role in FtsZ regulation during rapid growth. Taken together, our results suggest a key role for lateral bundling of the midcell FtsZ polymers in maintaining FtsZ ring stability during division.

Binding characteristics and regulatory mechanisms of the transcription factors controlling oleate-responsive genes in Saccharomyces cerevisiae.

Transcriptional activation of many genes involved in peroxisome-related functions is regulated by the Oaf1p, Pip2p, and Adr1p transcription factors in Saccharomyces cerevisiae. We have analyzed the in vivo binding characteristics of Oaf1p-Pip2p and found that this complex is recruited to its target oleate-response element (ORE) under all growth conditions tested. In addition, this complex also binds to ORE-containing genes that do not appear to be regulated by these proteins, as well as to some genes lacking conventional OREs. The recruitment of the Oaf1p-Pip2p complex was greatly increased upon glucose derepression, possibly due to Oaf1p phosphorylation with only moderate increases upon oleate induction. Thus, this complex may receive a nutritional cue while it is already bound to DNA, suggesting that, in addition to the increase in Oaf1p-Pip2p binding, other mechanism(s) such as enhanced Adr1p association may drive the expression of highly inducible fatty acid-responsive genes. Adr1p binds to target genes in an oleate-dependent fashion and is involved in Oaf1p-Pip2p binding. In turn, the Oaf1p-Pip2p complex appears to be important for Adr1p binding to a subset of oleate-responsive genes. Adr1p is a positive regulator of ORE-containing genes, but it also acts as a negative factor in expression of some of these genes. Finally, we have also shown that Adr1p is directly involved in mediating oleate induction of Oaf1p-Pip2p target genes.