ExsY and CotY Are Required for the Correct Assembly of the Exosporium and Spore Coat of Bacillus cereus

Abstract

The exosporium-defective phenotype of a transposon insertion mutant of Bacillus cereus implicated ExsY, a homologue of B. subtilis cysteine-rich spore coat proteins CotY and CotZ, in assembly of an intact exosporium. Single and double mutants of B. cereus lacking ExsY and its paralogue, CotY, were constructed. The exsY mutant spores are not surrounded by an intact exosporium, though they often carry attached exosporium fragments. In contrast, the cotY mutant spores have an intact exosporium, although its overall shape is altered. The single mutants show altered, but different, spore coat properties. The exsY mutant spore coat is permeable to lysozyme, whereas the cotY mutant spores are less resistant to several organic solvents than is the case for the wild type. The exsY cotY double-mutant spores lack exosporium and have very thin coats that are permeable to lysozyme and are sensitive to chloroform, toluene, and phenol. These spore coat as well as exosporium defects suggest that ExsY and CotY are important to correct formation of both the exosporium and the spore coat in B. cereus. Both ExsY and CotY proteins were detected in Western blots of purified wild-type exosporium, in complexes of high molecular weight, and as monomers. Both exsY and cotY genes are expressed at late stages of sporulation. Endospores of the Bacillus cereus family, which includesBacillus anthracis and Bacillus thuringiensis, are enveloped by a large, balloon-like layer known as the exosporium (11,19). B. subtilis, the paradigm sporeformer, does not have this distinct layer, although the B. subtilis spore coat may have a tight-fitting outermost layer that can be visualized after extraction of coat material with urea or mercaptoethanol (23). Scanning electron microscopy has revealed that the exosporium is composed of two layers-a paracrystalline basal layer with hexagonal periodicity and a "hairy nap" outer layer (11). The exosporium is chemically complex and is composed of 53% protein, 20% amino and neutral polysaccharides, 18% lipids, and ϳ4% ash (17).Multiple proteins from the exosporium of both B. cereus and B. anthracis have been identified (22,24,31); the majority do not have homologues in B. subtilis. For example, the surface layers of B. cereus, B. anthracis, and B. thuringiensis spores all contain glycoproteins with characteristic collagen-like repeat regions (5,10,27). Of these, the B. anthracis glycoprotein BclA is an essential component of the hairy nap layer of the exosporium (27, 29).Analysis of genome sequence information from B. cereus ATCC 14579 and B. anthracis Ames identified a cluster of genes near bclA implicated in exosporium production, including the region whose genes were designated yjbX-exsYyjcA-yjcB-exsFA-cotY (31). The ExsY and CotY proteins are homologues (ca. 35% amino acid identity) of the B. subtilis coat proteins CotY and CotZ (37). ExsY and CotY proteins have both been detected in purified exosporium from B. anthracis endospores by N-terminal sequencing of separated peptides (22). ExsY and CotY are very sim...

Surface architecture of endospores of the Bacillus cereus/anthracis/thuringiensis family at the subnanometer scale

“…ExsFA and BclA are clearly not essential to hold the lattice together, because exosporium from ΔexsFA and ΔbclA, mutants maintains the crystal lattice (8,15). We speculate that ExsY and CotY could be good candidates for major components of the lattice because a ΔexsY strain of B. cereus makes only a small terminal cap of exosporium and a ΔexsY ΔcotY strain is completely devoid of exosporium (17,18,31). It is possible that the main bulk of the exosporium contains ExsY whereas the cap contains CotY, which is 85% identical to ExsY (17).…”

“…ExsFA/BxpB and its homolog ExsFB are required for attachment of BclA to the basal layer (15,16). BclA, ExsFA, and ExsY form high-molecular weight complexes, ExsY being required for the complete assembly of the exosporium (17)(18)(19). The exosporium is likely to contain a number of other structural proteins (many reviewed in ref.…”

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

“…The exosporium cap region is not firmly anchored to the spore and is lost, presumably due to fluid shear forces, when spores are prepared in a liquid medium (42,44). B. cereus mutants devoid of both ExsY and CotY lack an exosporium layer (42,44), whereas loss of ExsY results in spores with only fragments of exosporium-like material adherent to the spores. The absence of exosporium caps on B. cereus exsY mutant spores was likely the result of shear forces in place during preparation of the spores (44).…”

“…These have been identified through the isolation of mutants that fail to stably attach the exosporium and from which, as a consequence, the exosporium is released as sheets separate from the mature spores upon lysis of the mother cells. Proteins implicated in exosporium attachment include the spore coat protein CotE and the exosporium-or interspace-localized proteins CotY, ExsA, ExsB, ExsY, and ExsM (1,(41)(42)(43)(44)(45). Whether these proteins participate directly in exosporium attachment or indirectly, through proper positioning of proteins actually involved in the attachment, has not been established.…”

“…Loss of ExsY in B. anthracis results in the production of only the cap region of the exosporium, with no noncap region of the exosporium evident in either late-stage sporulating cells or released mature spores (42). The exosporium cap region is not firmly anchored to the spore and is lost, presumably due to fluid shear forces, when spores are prepared in a liquid medium (42,44). B. cereus mutants devoid of both ExsY and CotY lack an exosporium layer (42,44), whereas loss of ExsY results in spores with only fragments of exosporium-like material adherent to the spores.…”