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.

Spore-Associated Proteins Involved in c-di-GMP Synthesis and Degradation of Bacillus anthracis.

Bis-(3'-5')-cyclic-dimeric GMP (c-di-GMP) is an important bacterial regulatory signaling molecule affecting biofilm formation, toxin production, motility, and virulence. The genome of Bacillus anthracis, the causative agent of anthrax, is predicted to encode ten putative GGDEF/EAL/HD-GYP-domain containing proteins. Heterologous expression in Bacillus subtilis hosts indicated that there are five active GGDEF domain-containing proteins and four active EAL or HD-GYP domain-containing proteins. Using an mCherry gene fusion-Western blotting approach, the expression of the c-di-GMP-associated proteins was observed throughout the in vitro life cycle. Of the six c-di-GMP-associated proteins found to be present in sporulating cells, four (CdgA, CdgB, CdgD, and CdgG) contain active GGDEF domains. The six proteins expressed in sporulating cells are retained in spores in a CotE-independent manner and thus are not likely to be localized to the exosporium layer of the spores. Individual deletion mutations involving the nine GGDEF/EAL protein-encoding genes and one HD-GYP protein-encoding gene did not affect sporulation efficiency, the attachment of the exosporium glycoprotein BclA, or biofilm production. Notably, expression of anthrax toxin was not affected by deletion of any of the cdg determinants. Three determinants encoding proteins with active GGDEF domains were found to affect germination kinetics. This study reveals a spore association of cyclic-di-GMP regulatory proteins and a likely role for these proteins in the biology of the B. anthracis spore. IMPORTANCE The genus Bacillus is composed of Gram-positive, rod shaped, soil-dwelling bacteria. As a mechanism for survival in the harsh conditions in soil, the organisms undergo sporulation, and the resulting spores permit the organisms to survive harsh environmental conditions. Although most species are saprophytes, Bacillus cereus and Bacillus anthracis are human pathogens and Bacillus thuringiensis is an insect pathogen. The bacterial c-di-GMP regulatory system is an important control system affecting motility, biofilm formation, and toxin production. The role of c-di-GMP has been studied in the spore-forming bacilli Bacillus subtilis, Bacillus amyloliquefaciens, B. cereus, and B. thuringiensis. However, this regulatory system has not heretofore been examined in the high-consequence zoonotic pathogen of this genus, B. anthracis.

A Bacillus Spore-Based Display System for Bioremediation of Atrazine.

Owing to human activities, a large number of organic chemicals, including petroleum products, industrial solvents, pesticides, herbicides (including atrazine [ATR]), and pharmaceuticals, contaminate soil and aquatic environments. Remediation of these pollutants by conventional approaches is both technically and economically challenging. Bacillus endospores are highly resistant to most physical assaults and are capable of long-term persistence in soil. Spores can be engineered to express, on their surface, important enzymes for bioremediation purposes. We have developed a Bacillus thuringiensis spore platform system that can display a high density of proteins on the spore surface. The spore surface-tethered enzymes exhibit enhanced activity and stability relative to free enzymes in soil and water environments. In this study, we evaluated a B. thuringiensis spore display platform as a bioremediation tool against ATR. The Pseudomonas sp. strain ADP atzA determinant, an ATR chlorohydrolase important to the detoxification of ATR, was expressed as a fusion protein linked to the attachment domain of the BclA spore surface nap layer protein and expressed in B. thuringiensis Spores from this strain are decorated with AtzA N-terminally linked on the surface of the spores. The recombinant spores were assayed for ATR detoxification in liquid and soil environments, and enzyme kinetics and stability were assessed. We successfully demonstrated the utility of this spore-based enzyme display system to detoxify ATR in water and laboratory soil samples.IMPORTANCE Atrazine is one of the most widely applied herbicides in the U.S. midwestern states. The long environmental half-life of atrazine has contributed to the contamination of surface water and groundwater by atrazine and its chlorinated metabolites. The toxic properties of ATR have raised public health and ecological concerns. However, remediation of ATR by conventional approaches has proven to be costly and inefficient. We developed a novel B. thuringiensis spore platform system that is capable of long-term persistence in soil and can be engineered to surface express a high density of enzymes useful for bioremediation purposes. The enzymes are stably attached to the surface of the spore exosporium layer. The spore-based system will likely prove useful for remediation of other environmental pollutants as well.

Assembly of the outermost spore layer: pieces of the puzzle are coming together.

Certain endospore-forming soil dwelling bacteria are important human, animal or insect pathogens. These organisms produce spores containing an outer layer, the exosporium. The exosporium is the site of interactions between the spore and the soil environment and between the spore and the infected host during the initial stages of infection. The composition and assembly process of the exosporium are poorly understood. This is partly due to the extreme stability of the exosporium that has proven to be refractive to existing methods to deconstruct the intact structure into its component parts. Although more than 20 proteins have been identified as exosporium-associated, their abundance, relationship to other proteins and the processes by which they are assembled to create the exosporium are largely unknown. In this issue of Molecular Microbiology, Terry, Jiang, and colleagues in Per Bullough's laboratory show that the ExsY protein is a major structural protein of the exosporium basal layer of B. cereus family spores and that it can self-assemble into complex structures that possess many of the structural features characteristic of the exosporium basal layer. The authors refined a model for exosporium assembly. Their findings may have implications for exosporium formation in other spore forming bacteria, including Clostridium species.

Assembly of the BclB glycoprotein into the exosporium and evidence for its role in the formation of the exosporium 'cap' structure in Bacillus anthracis.

The outermost layer of the Bacillus anthracis spore consists of an exosporium comprised of an outer hair-like nap layer and an internal basal layer. A major component of the hair-like nap is the glycosylated collagen-like protein BclA. A second collagen-like protein, BclB, is also present in the exosporium. BclB possesses an N-terminal sequence that targets it to the exosporium and is similar in sequence to a cognate targeting region in BclA. BclB lacks, however, sequence similarity to the region of BclA thought to mediate attachment to the basal layer via covalent interactions with the basal layer protein BxpB. Here we demonstrate that BxpB is critical for correct localization of BclB during spore formation and that the N-terminal domains of the BclA and BclB proteins compete for BxpB-controlled assembly sites. We found that BclB is located principally in a region of the exosporium that excludes a short arc on one side of the exosporium (the so-called bottle-cap region). We also found that in bclB mutant spores, the distribution of exosporium proteins CotY and BxpB is altered, suggesting that BclB has roles in exosporium assembly. In bclB mutant spores, the distance between the exosporium and the coat, the interspace, is reduced.

Does environmental replication contribute to Bacillus anthracis spore persistence and infectivity in soil?

Bacillus anthracis is the zoonotic causal agent of anthrax. Its infectious form is the spore, which can persist in soil. Herbivores usually acquire the disease from grazing in spore-contaminated sites. There are two schools of thought regarding B. anthracis activities in soil. One contends the bacteria are obligate animal parasites and soil-based spores remain inert until taken up by another animal host. Others contend that spores can germinate in soil and the bacteria replicate and re-sporulate to maintain and/or increase spore numbers. This review discusses whether soil replication of B. anthracis is an important part of its life cycle.

Quantification and characterization of biological activities of glansreginin A in black walnuts (Juglans nigra).

Glansreginin A has been reported to be an indicator of the quality of walnuts (Juglans spp.). However, bioactive properties of glansreginin A have not been adequately explored. In the present study, we quantified concentrations of glansreginin A in black walnuts (Juglans nigra) using high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) and performed an array of in vitro bioassays to characterize biological activities (e.g., antibacterial, antioxidant, anticancer capacities) of this compound. Results from HPLC-MS/MS analysis indicated that glansreginin A was presented in all 12 black cultivars examined and its contents were variable among black walnut cultivars, ranged from 6.8 mg/kg (Jackson) to 47.0 mg/kg (Hay). Glansreginin A possessed moderate antibacterial activities against Gram-positive pathogens (Staphylococcus aureus and Bacillus anthracis). This compound exhibited no antioxidant activities, did not induce the activity of antioxidant response element signaling pathways, and exerted no antiproliferative effects on tumorigenic alveolar epithelial cells and non-tumorigenic lung fibroblast cells.

The co-dependence of BxpB/ExsFA and BclA for proper incorporation into the exosporium of Bacillus anthracis.

The outermost layer of the Bacillus anthracis spore consists of an exosporium comprised of two distinct layers, an outer hair-like nap layer and an internal basal layer. The hair-like nap is primarily comprised of the glycosylated collagen-like protein BclA. BclA is found in a trimeric form in close association with many other exosporium proteins in high-molecular weight complexes. We previously had characterized an N-terminal sequence of BclA that is sufficient for incorporation into the exosporium. Here we utilized site-directed mutagenesis to identify BclA residues critical to two steps in this process, positioning of the protein at the site of the developing exosporium basal layer and stable incorporation which includes a proteolytic cleavage of BclA after residue 19. The BxpB (ExsFA) protein is known to be important for proper incorporation of BclA onto the exosporium. BxpB and BclA were found to be expressed at the same time in sporulating cells of B. anthracis and immediately colocalize to high-molecular weight complexes. The BxpB protein was found to be in close proximity to the BclA NTD. BxpB and BclA are co-dependent for exosporium incorporation, with the BclA NTD being sufficient to deliver BxpB to the exosporium.

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.

Localization and assembly of the novel exosporium protein BetA of Bacillus anthracis.

The exosporium of Bacillus anthracis is comprised of two distinct layers: a basal layer and a hair-like nap that covers the basal layer. The hair-like nap contains the glycoproteins BclA and, most likely, BclB. BclA and BclB are directed to assemble into the exosporium by motifs in their N-terminal domains. Here, we identify a previously uncharacterized putative gene encoding this motif, which we have named betA (Bacillus exosporium-targeted protein of B. anthracis). Like bclA, betA encodes a putative collagenlike repeat region. betA is present in several genomes of exosporium-producing Bacillus species but, so far, not in any others. Using fluorescence microscopic localization of a BetA-enhanced green fluorescent protein (eGFP) fusion protein and immunofluorescence microscopy with anti-BetA antibodies, we showed that BetA resides in the exosporium basal layer, likely underneath BclA. BetA assembles at the spore surface at around hour 5 of sporulation and under the control of BxpB, similar to the control of deposition of BclA. We suggest a model in which BclA and BetA are incorporated into the exosporium by a mechanism that depends on their similar N termini. These data suggest that BetA is a member of a growing family of exosporium proteins that assemble under the control of targeting sequences in their N termini.

Targeting of the BclA and BclB proteins to the Bacillus anthracis spore surface.

The exosporium is the outermost layer of the Bacillus anthracis spore. The predominant protein on the exosporium surface is BclA, a collagen-like glycoprotein. BclA is incorporated on the spore surface late in the B. anthracis sporulation pathway. A second collagen-like protein, BclB, has been shown to be surface-exposed on B. anthracis spores. We have identified sequences near the N-terminus of the BclA and BclB glycoproteins responsible for the incorporation of these proteins into the exosporium layer of the spore and used these targeting domains to incorporate reporter fluorescent proteins onto the spore surface. The BclA and BclB proteins are expressed in the mother cell cytoplasm and become spore-associated in a two-step process involving first association of the protein with the spore surface followed by attachment of the protein in a process that involves a proteolytic cleavage event. Protein domains associated with each of these events have been identified. This novel targeting system can be exploited to incorporate foreign proteins into the exosporium of inactivated, spores resulting in the surface display of recombinant immunogens for use as a potential vaccine delivery system.

Regulation of Rot expression in Staphylococcus aureus.

Repressor of toxins (Rot) is known to be a global regulator of virulence gene expression in Staphylococcus aureus. The function of Rot, but not the transcription of rot, is regulated by the staphylococcal accessory gene regulator (Agr) quorum-sensing system. In addition, the alternative sigma factor (sigma(B)) has a repressive effect on rot expression during the postexponential phase of growth. The transcriptional profiles of Rot in sigma(B)-positive and sigma(B)-negative strains in the postexponential and stationary phases of growth were compared. An upregulation of rot expression was observed during the stationary phase of growth, and this upregulation occurred in a sigma(B)-dependent manner. The effects of other staphylococcal transcriptional factors were also investigated. Electrophoretic mobility shift assays revealed that proteins present in staphylococcal lysates retarded the mobility of the rot promoter fragment and that the effect was reduced, but not eliminated, with lysates from strains lacking a functional SarS protein. A modest upregulation of rot expression was also observed in sarS-negative strains. Affinity purification of proteins binding to the rot promoter fragment, followed by N-terminal protein sequencing, identified the SarA and SarR proteins. Primer extension analysis of the rot promoter revealed a number of discreet products. However, these RNA species were not associated with identifiable promoter activity and likely represented RNA breakdown products. Loss of Rot function during the postexponential phase of growth likely involves degradation of the rot mRNA but not the inhibition of rot transcription.

Human Fusobacterium necrophorum strains have a leukotoxin gene and exhibit leukotoxic activity.

Fusobacterium necrophorum, a Gram-negative anaerobe, causes a variety of necrotic infections in humans and animals. There are two subspecies: subsp. necrophorum and subsp. funduliforme. In cattle, subsp. necrophorum is more prevalent and production of leukotoxin is a major virulence factor. The leukotoxin operon (lktBAC) consists of three genes, lktB, lktA and lktC, of which lktA is the structural toxin gene. The subspecies identity of human F. necrophorum is less certain and it is not known whether human strains possess the leukotoxin gene or leukotoxin activity. Therefore, the objective of this study was to identify the subspecies status of four human clinical strains of F. necrophorum and determine whether they have the leukotoxin gene or leukotoxin activity. Phenotypic and genotypic characteristics suggested that the four strains belonged to subsp. funduliforme, which was confirmed by sequencing the 16S rRNA gene. Analysis of the four strains by PCR revealed the presence of the leukotoxin operon. Partial DNA sequencing identified one human strain with full-length lktA, whereas the others exhibited considerable heterogeneity in size. All strains had a leukotoxin operon promoter-containing intergenic region similar to that of bovine subsp. funduliforme strains, which was confirmed by DNA sequencing and Southern blotting. Despite variations in the lktA gene, all strains secreted leukotoxin as demonstrated by Western blotting. Flow cytometry assays revealed that the leukotoxin was toxic to human white blood cells. In conclusion, the human strains examined contained a leukotoxin gene whose gene product was biologically active. The importance of leukotoxin as a virulence factor in human fusobacterial infections needs further evaluation.

Fusobacterium equinum possesses a leukotoxin gene and exhibits leukotoxin activity.

Fusobacterium equinum, a gram negative, rod-shaped and an obligate anaerobic bacterium is a newly described species. The organism is associated with necrotic infections of the respiratory tract in horses that include necrotizing pneumonia, pleuritis and paraoral infections. The species is closely related to F. necrophorum that causes liver abscesses in cattle and sheep, calf-diphtheria in cattle, and foot-rot in sheep and cattle. Leukotoxin, an exotoxin, is an important virulence factor in bovine strains of F. necrophorum. Our objective was to examine strains (n=10) of F. equinum for leukotoxin (lktA) gene and its toxic effects on equine leukocytes. Southern hybridization and partial DNA sequencing revealed that all the 10 strains had the lktA gene with greater similarities to F. necrophorum subsp. necrophorum. The secreted leukotoxin was detected in the culture supernatant and its biological activity was determined by viability assays with equine polymorphonuclear cells (PMNs) using flow cytometry. While culture supernatants of four strains (E1, E7, E9, and E10) were highly toxic to equine PMNs; strain E5 was moderately toxic and the remaining strains (E2, E3, E4, E6, and E8) were only mildly toxic. Our data indicated that F. equinum isolates had lktA gene and its product was toxic to equine leukocytes. Therefore, leukotoxin may be an important virulence factor in F. equinum infections.

Identifying Antibacterial Compounds in Black Walnuts (Juglans nigra) Using a Metabolomics Approach.

Black walnut (Juglans nigra L.) is one of the most economically valuable hardwood species and a high value tree for edible nut production in the United States. Although consumption of black walnut has been linked to multiple health-promoting effects (e.g., antioxidant, antimicrobial, anti-inflammatory), the bioactive compounds have not been systematically characterized. In addition, the associations between different black walnut cultivars and their health-promoting compounds have not been well established. In this study, the kernels of twenty-two black walnut cultivars selected for nut production by the University of Missouri Center for Agroforestry (Columbia, MO, USA) were evaluated for their antibacterial activities using agar-well diffusion assay. Among the selected cultivars, four black walnut cultivars (i.e., Mystry, Surprise, D.34, and A.36) exhibited antibacterial activity against a Gram-positive bacterium (Staphylococcus aureus), whereas other cultivars showed no effect on the inhibition of this bacterium. The antibacterial compounds showing the strongest activity were isolated with bioassay-guided purification and identified using a metabolomics approach. Six antibacterial bioactive compounds responsible for antimicrobial activity were successfully identified. Glansreginin A, azelaic acid, quercetin, and eriodictyol-7-O-glucoside are novel antibacterial compounds identified in the kernels of black walnuts. The metabolomics approach provides a simple and cost-effective tool for bioactive compound identification.

TLR sensing of bacterial spore-associated RNA triggers host immune responses with detrimental effects.

The spores of pathogenic bacteria are involved in host entry and the initial encounter with the host immune system. How bacterial spores interact with host immunity, however, remains poorly understood. Here, we show that the spores of Bacillus anthracis (BA), the etiologic agent of anthrax, possess an intrinsic ability to induce host immune responses. This immunostimulatory activity is attributable to high amounts of RNA present in the spore surface layer. RNA-sensing TLRs, TLR7, and TLR13 in mice and their human counterparts, are responsible for detecting and triggering the host cell response to BA spores, whereas TLR2 mediates the sensing of vegetative BA. BA spores, but not vegetative BA, induce type I IFN (IFN-I) production. Although TLR signaling in itself affords protection against BA, spore RNA-induced IFN-I signaling is disruptive to BA clearance. Our study suggests a role for bacterial spore-associated RNA in microbial pathogenesis and illustrates a little known aspect of interactions between the host and spore-forming bacteria.

The two major subspecies of Fusobacterium necrophorum have distinct leukotoxin operon promoter regions.

Fusobacterium necrophorum, a gram-negative, non-spore-forming anaerobe, is a normal inhabitant of the alimentary tract of animals and humans. Two types of F. necrophorum, subspecies necrophorum (biotype A) and funduliforme (biotype B), have been recognized, which differ morphologically, biochemically and biologically. The organism is an opportunistic pathogen that causes numerous necrotic conditions (necrobacillosis) such as bovine hepatic abscesses and ruminant foot abscesses. Subspecies necrophorum strains are considered to be more virulent for cattle and have been shown to produce greater amounts of leukotoxin than subspecies funduliforme strains. The leukotoxin operon of F. necrophorum consists of three genes (lktBAC) of which the leukotoxin structural gene (lktA) is the second gene in the operon. In this study, the promoter regions of the leukotoxin operons from the two subspecies were identified and their nucleotide sequence compared. The promoter regions were found to differ in sequence, in length of the sequence between the upstream determinant (oppF) and the first gene of the leukotoxin operon (lktB), and in promoter strength as assayed in Escherichia coli host cells.

The Exosporium Layer of Bacterial Spores: a Connection to the Environment and the Infected Host.

Much of what we know regarding bacterial spore structure and function has been learned from studies of the genetically well-characterized bacterium Bacillus subtilis. Molecular aspects of spore structure, assembly, and function are well defined. However, certain bacteria produce spores with an outer spore layer, the exosporium, which is not present on B. subtilis spores. Our understanding of the composition and biological functions of the exosporium layer is much more limited than that of other aspects of the spore. Because the bacterial spore surface is important for the spore's interactions with the environment, as well as being the site of interaction of the spore with the host's innate immune system in the case of spore-forming bacterial pathogens, the exosporium is worthy of continued investigation. Recent exosporium studies have focused largely on members of the Bacillus cereus family, principally Bacillus anthracis and Bacillus cereus. Our understanding of the composition of the exosporium, the pathway of its assembly, and its role in spore biology is now coming into sharper focus. This review expands on a 2007 review of spore surface layers which provided an excellent conceptual framework of exosporium structure and function (A. O. Henriques and C. P. Moran, Jr., Annu Rev Microbiol 61:555-588, 2007, http://dx.doi.org/10.1146/annurev.micro.61.080706.093224). That review began a process of considering outer spore layers as an integrated, multilayered structure rather than simply regarding the outer spore components as independent parts.

Sequence Analysis of Staphylococcus hyicus ATCC 11249T, an Etiological Agent of Exudative Epidermitis in Swine, Reveals a Type VII Secretion System Locus and a Novel 116-Kilobase Genomic Island Harboring Toxin-Encoding Genes.

Staphylococcus hyicus is the primary etiological agent of exudative epidermitis in swine. Analysis of the complete genome sequence of the type strain revealed a locus encoding a type VII secretion system and a large chromosomal island harboring the genes encoding exfoliative toxin ExhA and an EDIN toxin homolog.