Characterization of the yrbA gene of Bacillus subtilis, involved in resistance and germination of spores.
ABSTRACT: Insertional inactivation of the yrbA gene of Bacillus subtilis reduced the resistance of the mutant spores to lysozyme. The yrbA mutant spores lost their optical density at the same rate as the wild-type spores upon incubation with L-alanine but became only phase gray and did not swell. The response of the mutant spores to a combination of asparagine, glucose, fructose, and KCl was also extremely poor; in this medium yrbA spores exhibited only a small loss in optical density and gave a mixture of phase-bright, -gray, and -dark spores. Northern blot analysis of yrbA transcripts in various sig mutants indicated that yrbA was transcribed by RNA polymerase with sigma(E) beginning at 2 h after the start of sporulation. The yrbA promoter was localized by primer extension analysis, and the sequences of the -35 (TCATAAC) and -10 (CATATGT) regions were similar to the consensus sequences of genes recognized by sigma(E). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of proteins solubilized from intact yrbA mutant spores showed an alteration in the protein profile, as 31- and 36-kDa proteins, identified as YrbA and CotG, respectively, were absent, along with some other minor changes. Electron microscopic examination of yrbA spores revealed changes in the spore coat, including a reduction in the density and thickness of the outer layer and the appearance of an inner coat layer-like structure around the outside of the coat. This abnormal coat structure was also observed on the outside of the developing forespores of the yrbA mutant. These results suggest that YrbA is involved in assembly of some coat proteins which have roles in both spore lysozyme resistance and germination.
Project description:The outermost layer of spores of the Bacillus cereus family is a loose structure known as the exosporium. Spores of a library of Tn917-LTV1 transposon insertion mutants of B. cereus ATCC 10876 were partitioned into hexadecane; a less hydrophobic mutant that was isolated contained an insertion in the exsA promoter region. ExsA is the equivalent of SafA (YrbA) of Bacillus subtilis, which is also implicated in spore coat assembly; the gene organizations around both are identical, and both proteins contain a very conserved N-terminal cortex-binding domain of ca. 50 residues, although the rest of the sequence is much less conserved. In particular, unlike SafA, the ExsA protein contains multiple tandem oligopeptide repeats and is therefore likely to have an extended structure. The exsA gene is expressed in the mother cell during sporulation. Spores of an exsA mutant are extremely permeable to lysozyme and are blocked in late stages of germination, which require coat-associated functions. Two mutants expressing differently truncated versions of ExsA were constructed, and they showed the same gross defects in the attachment of exosporium and spore coat layers. The protein profile of the residual exosporium harvested from spores of the three mutants--two expressing truncated proteins and the mutant with the original transposon insertion in the promoter region--showed some differences from the wild type and from each other, but the major exosporium glycoproteins were retained. The exsA gene is extremely important for the normal assembly and anchoring of both the spore coat and exosporium layers in spores of B. cereus.
Project description:A major Bacillus anthracis spore coat protein of 13.4 kDa, designated Cot alpha, was found only in the Bacillus cereus group. A stable ca. 30-kDa dimer of this protein was also present in spore coat extracts. Cot alpha, which is encoded by a monocistronic gene, was first detected late in sporulation, consistent with a sigma(K)-regulated gene. On the basis of immunogold labeling, the protein is in the outer spore coat and absent from the exosporium. In addition, disruption of the gene encoding Cot alpha resulted in spores lacking a dark-staining outer spore coat in thin-section electron micrographs. The mutant spores were stable upon heating or storage, germinated at the same rate as the wild type, and were resistant to lysozyme. They were, however, more sensitive than the wild type to phenol, chloroform, and hypochlorite but more resistant to diethylpyrocarbonate. In all cases, resistance or sensitivity to these reagents was restored by introducing a clone of the cot alpha gene into the mutant. Since Cot alpha is an abundant outer spore coat protein of the B. cereus group with a prominent role in spore resistance and sensitivity, it is a promising target for the inactivation of B. anthracis spores.
Project description: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.
Project description:The gerP1 transposon insertion mutation of Bacillus cereus is responsible for a defect in the germination response of spores to both L-alanine and inosine. The mutant is blocked at an early stage, before loss of heat resistance or release of dipicolinate, and the efficiency of colony formation on nutrient agar from spores is reduced fivefold. The protein profiles of alkaline-extracted spore coats and the spore cortex composition are unchanged in the mutant. Permeabilization of gerP mutant spores by coat extraction procedures removes the block in early stages of germination, although a consequence of the permeabilization procedure in both wild type and mutant is that late germination events are not complete. The complete hexacistronic operon that includes the site of insertion has been cloned and sequenced. Four small proteins encoded by the operon (GerPA, GerPD, GerPB, and GerPF) are related in sequence. A homologous operon (yisH-yisC) can be found in the Bacillus subtilis genome sequence; null mutations in yisD and yisF, constructed by integrational inactivation, result in a mutant phenotype similar to that seen in B. cereus, though somewhat less extreme and equally repairable by spore permeabilization. Normal rates of germination, as estimated by loss of heat resistance, are also restored to a gerP mutant by the introduction of a cotE mutation, which renders the spore coats permeable to lysozyme. The B. subtilis operon is expressed solely during sporulation, and is sigma K-inducible. We hypothesize that the GerP proteins are important as morphogenetic or structural components of the Bacillus spore, with a role in the establishment of normal spore coat structure and/or permeability, and that failure to synthesize these proteins during spore formation limits the opportunity for small hydrophilic organic molecules, like alanine or inosine, to gain access to their normal target, the germination receptor, in the spore.
Project description:During endospore formation in Bacillus subtilis, approximately a dozen proteins are synthesized and assembled around the prespore to form a protective coat. Little is known about the assembly process, but several of the genes encoding these coat proteins are expressed in the mother cell compartment, where the proteins accumulate on the outer side of the developing endospore. Transcription of these genes is directed by the mother cell-specific sigma factor, sigma K, during the later stages of endospore development. sigma E may direct expression of the genes that encode proteins that function in the earliest stages of coat assembly. By screening for sigma E-dependent promoters, we cloned a gene, designated spoVID, required for assembly of a normal spore coat. Expression of spoVID was initiated at about the second hour of sporulation and continued throughout development from a sigma E-dependent promoter. The spoVID gene was located on the B. subtilis chromosome just downstream of the previously characterized hemAXCDBL operon and is predicted to encode an extremely acidic protein with 575 residues. Insertion mutants of spoVID produced refractile spores that were resistant to heat and to chloroform but were sensitive to lysozyme. Electron microscopic examination of sporulating spoVID mutant cells revealed normal morphological development up to about the third hour of sporulation. However, during the later stages of development the coat proteins assembled into aberrant structures that occurred freely in the mother cell cytoplasm and that consisted of reiterations of the single inner and outer layers that normally make up the spore coat.
Project description:Bacillus spores are highly resistant to many environmental stresses, owing in part to the presence of multiple "extracellular" layers. Although the role of some of these extracellular layers in resistance to particular stresses is known, the function of one of the outermost layers, the spore coat, is not completely understood. This study sought to determine whether the spore coat plays a role in resistance to predation by the ciliated protozoan Tetrahymena, which uses phagocytosis to ingest and degrade other microorganisms. Wild-type dormant spores of Bacillus subtilis were efficiently ingested by the protozoan Tetrahymena thermophila but were neither digested nor killed. However, spores with various coat defects were killed and digested, leaving only an outer shell termed a rind, and supporting the growth of Tetrahymena. A similar rind was generated when coat-defective spores were treated with lysozyme alone. The sensitivity of spores with different coat defects to predation by T. thermophila paralleled the spores' sensitivities to lysozyme. Spore killing by T. thermophila was by means of lytic enzymes within the protozoal phagosome, not by initial spore germination followed by killing. These findings suggest that a major function of the coat of spores of Bacillus species is to protect spores against predation. We also found that indigestible rinds were generated even from spores in which cross-linking of coat proteins was greatly reduced, implying the existence of a coat structure that is highly resistant to degradative enzymes.
Project description:During endospore formation in Bacillus subtilis, over two dozen polypeptides are assembled into a multilayered structure known as the spore coat, which protects the cortex peptidoglycan (PG) and permits efficient germination. In the initial stages of coat assembly a protein known as CotE forms a ring around the forespore. A second morphogenetic protein, SpoVID, is required for maintenance of the CotE ring during the later stages, when most of proteins are assembled into the coat. Here, we report on a protein that appears to associate with SpoVID during the early stage of coat assembly. This protein, which we call SafA for SpoVID-associated factor A, is encoded by a locus previously known as yrbA. We confirmed the results of a previous study that showed safA mutant spores have defective coats which are missing several proteins. We have extended these studies with the finding that SafA and SpoVID were coimmunoprecipitated by anti-SafA or anti-SpoVID antiserum from whole-cell extracts 3 and 4 h after the onset of sporulation. Therefore, SafA may associate with SpoVID during the early stage of coat assembly. We used immunogold electron microscopy to localize SafA and found it in the cortex, near the interface with the coat in mature spores. SafA appears to have a modular design. The C-terminal region of SafA is similar to those of several inner spore coat proteins. The N-terminal region contains a sequence that is conserved among proteins that associate with the cell wall. This motif in the N-terminal region may target SafA to the PG-containing regions of the developing spore.
Project description:How organisms develop into specific shapes is a central question in biology. The maintenance of bacterial shape is connected to the assembly and remodelling of the cell envelope. In endospore-forming bacteria, the pre-spore compartment (the forespore) undergoes morphological changes that result in a spore of defined shape, with a complex, multi-layered cell envelope. However, the mechanisms that govern spore shape remain poorly understood. Here, using a combination of fluorescence microscopy, quantitative image analysis, molecular genetics and transmission electron microscopy, we show that SsdC (formerly YdcC), a poorly-characterized new member of the MucB / RseB family of proteins that bind lipopolysaccharide in diderm bacteria, influences spore shape in the monoderm Bacillus subtilis. Sporulating cells lacking SsdC fail to adopt the typical oblong shape of wild-type forespores and are instead rounder. 2D and 3D-fluorescence microscopy suggest that SsdC forms a discontinuous, dynamic ring-like structure in the peripheral membrane of the mother cell, near the mother cell proximal pole of the forespore. A synthetic sporulation screen identified genetic relationships between ssdC and genes involved in the assembly of the spore coat. Phenotypic characterization of these mutants revealed that spore shape, and SsdC localization, depend on the coat basement layer proteins SpoVM and SpoIVA, the encasement protein SpoVID and the inner coat protein SafA. Importantly, we found that the ?ssdC mutant produces spores with an abnormal-looking cortex, and abolishing cortex synthesis in the mutant largely suppresses its shape defects. Thus, SsdC appears to play a role in the proper assembly of the spore cortex, through connections to the spore coat. Collectively, our data suggest functional diversification of the MucB / RseB protein domain between diderm and monoderm bacteria and identify SsdC as an important factor in spore shape development.
Project description:Endospores of Bacillus subtilis are enclosed in a proteinaceous coat which can be differentiated into a thick, striated outer layer and a thinner, lamellar inner layer. We found that the N-terminal sequence of a 25-kDa protein present in a preparation of spore coat proteins matched that of the Mn-dependent superoxide dismutase (SOD) encoded by the sod4 locus. sod4 is transcribed throughout the growth and sporulation of a wild-type strain and is responsible for the SOD activity detected in total cell extracts prepared from B. subtilis. Disruption of the sod4 locus produced a mutant that lacked any detectable SOD activity during vegetative growth and sporulation. The sodA mutant was not impaired in the ability to form heat- or lysozyme-resistant spores. However, examination of the coat layers of sodA mutant spores revealed increased extractability of the tyrosine-rich outer coat protein CotG. We showed that this condition was not accompanied by augmented transcription of the cotG gene in sporulating cells of the sodA mutant. We conclude that SodA is required for the assembly of CotG into the insoluble matrix of the spore and suggest that CotG is covalently cross-linked into the insoluble matrix by an oxidative reaction dependent on SodA. Ultrastructural analysis revealed that the inner coat formed by a sodA mutant was incomplete. Moreover, the outer coat lacked the characteristic striated appearance of wild-type spores, a pattern that was accentuated in a cotG mutant. These observations suggest that the SodA-dependent formation of the insoluble matrix containing CotG is largely responsible for the striated appearance of this coat layer.
Project description:We cloned and characterized a gene, cotM, that resides in the 173 degrees region of the Bacillus subtilis chromosome and is involved in spore outer coat assembly. We found that expression of the cotM gene is induced during development under sigma K control and is negatively regulated by the GerE transcription factor. Disruption of the cotM gene resulted in spores with an abnormal pattern of coat proteins. Electron microscopy revealed that the outer coat in cotM mutant spores had lost its multilayered type of organization, presenting a diffuse appearance. In particular, significant amounts of material were absent from the outer coat layers, which in some areas had a lamellar structure more typical of the inner coat. Occasionally, a pattern of closely spaced ridges protruding from its surface was observed. No deficiency associated with the inner coat or any other spore structure was found. CotM is related to the alpha-crystallin family of low-molecular-weight heat shock proteins, members of which can be substrates for transglutaminase-mediated protein cross-linking. CotM was not detected among the extractable spore coat proteins. These observations are consistent with a model according to which CotM is part of a cross-linked insoluble skeleton that surrounds the spore, serves as a matrix for the assembly of additional outer coat material, and confers structural stability to the final structure.