spoVIC, a new sporulation locus in Bacillus subtilis affecting spore coats, germination and the rate of sporulation

A mutation near cysB on the Bacillus subtilis chromosome marks a new sporulation locus, spoVIC. It causes spores to germinate more slowly than those of the wild-type under all conditions and, from indirect evidence, it does not appear to alter the affinity for the germinant L-alanine. The mutant spores have some deficiency of coat proteins (particularly the alkalisoluble coat protein, Mr = 12 000) and the spore coat layers are disorganized. The mutant strain grows normally and sporulates normally until stage II, after which its sporulation is delayed by about 2 h compared to that of the wild-type. This delay results in the prolonged synthesis of some coat proteins and the late synthesis of others. The abnormal coat may be the cause of the germination deficiency. A double mutant strain carrying the spoVIC610 mutation together with gerE36 sporulates slowly. Its spores have very little coat protein, are sensitive to heat, lysozyme and organic solvents, but germinate as well as the strain carrying the spoVIC mutation alone. The role of the spore coat in germination is discussed in the light of these findings.     

Altered arrangement of proteins in the spore coat of a germination mutant of Bacillus subtilis

Spores produced by a mutant of Bacillus subtilis were slow to develop their resistance properties during sporulation, and were slower to germinate than were wild-type spores. The coat protein composition of the mutant spores, as analysed by SDS-PAGE, was similar to that of the wild-type spores. However, one of the proteins (mol. wt 12000) which is normally present in the outer-most layers of mature wild-type spores and which is surface-exposed, was assembled abnormally into the coat of the mutant spores and not surface-exposed. The mutation responsible for this phenotype (spo-520) has been mapped between pheA and leuB on the B. subtilis chromosome, and was 47% cotransformable with leuB16. This mutation, and three others closely linked to it, define a new sporulation locus, spoVIB, which is involved in spore coat assembly. The phenotype of the mutant(s) supports the contention that spore germination and resistance properties may be determined by the assembly of the coat.     

ExsY and CotY are required for the correct assembly of the exosporium and spore coat of Bacillus cereus

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.     

Localization of the Cortex Lytic Enzyme CwlJ in Spores of Bacillus subtilis

The enzyme CwlJ is involved in the depolymerization of cortex peptidoglycan during germination of spores of Bacillus subtilis. CwlJ with a C-terminal His tag was functional and was extracted from spores by procedures that remove spore coat proteins. However, this CwlJ was not extracted from disrupted spores by dilute buffer, high salt concentrations, Triton X-100, Ca(2+)-dipicolinic acid, dithiothreitol, or peptidoglycan digestion, disappeared during spore germination, and was not present in cotE spores in which the spore coat is aberrant. These findings indicate the following: (i) the reason decoated and cotE spores germinate poorly with dipicolinic acid is the absence of CwlJ from these spores; and (ii) CwlJ is located in the spore coat, presumably tightly associated with one or more other coat proteins.     

Amino acids in the Bacillus subtilis morphogenetic protein SpoIVA with roles in spore coat and cortex formation

Bacterial spores are protected from the environment by a proteinaceous coat and a layer of specialized peptidoglycan called the cortex. In Bacillus subtilis, the attachment of the coat to the spore surface and the synthesis of the cortex both depend on the spore protein SpoIVA. To identify functionally important amino acids of SpoIVA, we generated and characterized strains bearing random point mutations of spoIVA that result in defects in coat and cortex formation. One mutant resembles the null mutant, as sporulating cells of this strain lack the cortex and the coat forms a swirl in the surrounding cytoplasm instead of a shell around the spore. We identified a second class of six mutants with a partial defect in spore assembly. In sporulating cells of these strains, we frequently observed swirls of mislocalized coat in addition to a coat surrounding the spore, in the same cell. Using immunofluorescence microscopy, we found that in two of these mutants, SpoIVA fails to localize to the spore, whereas in the remaining strains, localization is largely normal. These mutations identify amino acids involved in targeting of SpoIVA to the spore and in attachment of the coat. We also isolated a large set of mutants producing spores that are unable to maintain the dehydrated state. Analysis of one mutant in this class suggests that spores of this strain accumulate reduced levels of peptidoglycan with an altered structure.     

PCR detection of potentially pathogenic aeromonads in raw and cold-smoked freshwater fish

AIMS: To determine the mechanism of killing of spores of Bacillus subtilis by ortho-phthalaldehyde (OPA), an aromatic dialdehyde currently in use as an antimicrobial agent. METHODS AND RESULTS: OPA is sporicidal, although spores are much more OPA resistant than are vegetative cells. Bacillus subtilis mutants deficient in DNA repair, spore DNA protection and spore coat assembly have been used to show that (i) the coat appears to be a major component of spore OPA resistance, which is acquired late in sporulation of B. subtilis at the time of spore coat maturation, and (ii) B. subtilis spores are not killed by OPA through DNA damage but by elimination of spore germination. Furthermore, OPA-treated spores that cannot germinate are not recovered by artificial germinants or by treatment with NaOH or lysozyme. CONCLUSIONS: OPA appears to kill spores by blocking the spore germination process. SIGNIFICANCE AND IMPACT OF THE STUDY: This work provides information on the mechanism of spore resistance to, and spore killing by, the disinfectant, OPA.     

Properties of Bacillus cereus temperature-sensitive mutants altered in spore coat formation.

Three conditional Bacillus cereus mutants altered in the assembly or formation of spore coat layers were analyzed. They all grew as well as the wild type in an enriched or minimal medium but produced lysozyme and octanol-sensitive spores at the nonpermissive temperature (35 to 38 degrees C). The spores also germinated slowly when produced at 35 degrees C. Temperature-shift experiments indicated that the defective protein or regulatory signal is expressed at the time of formation of the outer spore coat layers. Revertants regained all wild-type spore properties at frequencies consistent with initial point mutations. Spore coat defects were evident in thin sections and freeze-etch micrographs of mutant spores produced at 35 degrees C. In addition, one mutant contained an extra surface deposit, perhaps unprocessed spore coat precursor protein. A prevalent band of about 65,000 daltons (the same size as the presumptive precursor) was present in spore coat extracts of this mutant and may be incorrectly processed to mature spore coat polypeptides. Another class of mutants was defective in the late uptake of half-cystine residues into spore coats. Such a defect could lead to improper formation of the outer spore coat layers.     

CotL,a new morphogenetic spore coat protein of Clostridium difficile

The strict anaerobe Clostridium difficile is the most common cause of antibiotic-associated diarrhoea. The oxygen-resistant C. difficile spores play a central role in the infectious cycle, contributing to transmission, infection and recurrence. The spore surface layers, the coat and exosporium, enable the spores to resist physical and chemical stress. However, little is known about the mechanisms of their assembly. In this study, we characterized a new spore protein, CotL, which is required for the assembly of the spore coat. The cotL gene was expressed in the mother cell compartment under the dual control of the RNA polymerase sigma factors, σ^E and σ^K. CotL was localized in the spore coat, and the spores of the cotL mutant had a major morphologic defect at the level of the coat/exosporium layers. Therefore, the mutant spores contained a reduced amount of several coat/exosporium proteins and a defect in their localization in sporulating cells. Finally, cotL mutant spores were more sensitive to lysozyme and were impaired in germination, a phenotype likely to be associated with the structurally altered coat. Collectively, these results strongly suggest that CotL is a morphogenetic protein essential for the assembly of the spore coat in C. difficile.     

Changes of ultrastructure in spore coat of Bacillus thiaminolyticus during germination and outgrowth.

Electron microscopic observation showed that the spore coat of Bacillus thiaminolyticus consisted of at least four layers; a high electron dense outer spore coat layer with five prominent ridges, a middle spore coat layer including two layers of a high and a low electron density, and an inner spore coat layer composing six to seven laminated layers. Rapid breakdown of the cortex and swelling of the core occurred in spores which were allowed to germinate by L-alanine for 45 min, whereas no change of surface feature was observed by scanning electron microscopy. Germination and outgrowth of spores in nutrient broth proceeded, being accompanied by morphological changes, in three steps; the first is a rapid breakdown of the cortex and swelling of the core, the second degradation of the inner layer at prominent region of the spore coat, and the last rupture of the spore coat and emergence of a young vegetative cell.     

Modification of GerQ reveals a functional relationship between Tgl and YabG in the coat of Bacillus subtilis spores

Here we describe the functional relationship between YabG and transglutaminase (Tgl), enzymes that modify the spore coat proteins of Bacillus subtilis. In wild-type spores at 37 degrees C, Tgl mediates the crosslinking of GerQ into higher molecular mass forms; however, some GerQ multimers are found in tgl mutant spores, indicating that Tgl is not essential. Immunoblotting showed that spores isolated from a yabG mutant after sporulation at 37 degrees C contain only very low levels of GerQ multimers. Heat treatment for 20 min at 60 degrees C, which maximally activates the enzymatic activity of Tgl, caused crosslinking of GerQ in isolated yabG spores but not in tgl/yabG double-mutant spores. In addition, the germination frequency of the tgl/yabG spores in the presence of l-alanine with or without heat activation at 60 degrees C was lower than that of wild-type spores. These findings suggest that Tgl cooperates with YabG to mediate the temperature-dependent modification of the coat proteins, a process associated with spore germination in B. subtilis.     

The Clostridium botulinum GerAB germination protein is located in the inner membrane of spores

Clostridium botulinum dormant spores germinate in presence of l-alanine via a specific receptor composed of GerAA, GerAB and GerAC proteins. In Bacillus subtilis spores, GerAA and GerAC proteins were located in the inner membrane of the spore. We studied the location of the GerAB protein in C. botulinum spore fractions by Western-blot analysis, using an antipeptidic antibody. The protein GerAB was in vitro translated and used to confirm the specificity of the antibodies. GerAB was not present in a coat and spore outer membrane fraction but was present in a fraction of decoated spores containing inner membrane. These results strongly suggest that the protein GerAB is located in the inner membrane of the spore.     

Localization of a germinant receptor protein (GerBA) to the inner membrane of Bacillus subtilis spores

Dormant Bacillus subtilis spores germinate in response to specific nutrients called germinants, which are recognized by multisubunit receptor complexes encoded by members of the gerA family of operons, of which the gerB operon is a member. The germinant receptors are expected to be membrane associated, but there is some debate about whether they are located in the inner or outer spore membrane. In this study we have used Western blot analysis to determine the precise location of GerBA, a gerB-encoded receptor protein, in various spore fractions. GerBA was not extracted from spores by a decoating treatment that removes the coat and outer membrane but was present in lysates from decoated spores and in the insoluble fraction (termed P100) from such lysates that contained inner-membrane vesicles. GerBA was also solubilized from the P100 fraction with detergent but not with high salt. These findings suggest that GerBA is an integral membrane protein located in the spore's inner membrane. Consistent with this idea, GerBA was present in the cell membrane of the outgrowing spore, a membrane that is derived from the dormant spore's inner membrane. Based on these observations we propose that GerBA and probably the entire GerB germinant receptor are located in the inner membrane of the dormant spore. We also estimated that there are only 24 to 40 molecules of GerBA per spore, a number that is consistent with the previously reported low level of gerB operon expression and with the putative receptor function of the proteins encoded by the gerB operon.     

Mutants of Bacillus subtilis affected in spore outgrowth.

Six mutants of Bacillus subtilis 168 that are temperature-sensitive in spore outgrowth were isolated. The outgrowth process proceeds normally at 35 degrees C, but at the non-permissive temperature (47 degrees C) it is arrested at a specific stage characteristic for each mutant strain. The mutants are not altered in vegetative growth whether at 35 degrees C or at 47 degrees C. They were characterized for their ability to synthesize RNA, proteins and DNA during outgrowth. A mutant defective in spore germination was also isolated; less than 5% of its spores can germinate at any of the temperatures tested. The mutations were mapped by means of transduction and transformation. The isolation of a number of outgrowth mutants which map at different loci and which affect outgrowth at different times is discussed in relation to the regulation of this process.     

Spore activation by acetate, propionate and heat in Phycomyces mutants

Summary Exposure to heat, acetate, or propionate activates the spores of the fungus Phycomyces blakesleeanus and allows them to germinate. Using counterselection with the antibiotic N-glycosyl-polyfungin, seven mutants were isolated on the basis of decreased spore activation by acetate and two on the basis of decreased spore activation by propionate. The nine mutants showed decreased activation by both chemicals and by heat, increased heat lethality, and altered patterns of trehalase activation. These and other observations indicate that spore activation by the three agents and spore death by heat are mediated by the same cellular component(s), which is probably involved in the regulation of cyclic AMP concentration.     

Involvement of calcium in germination of coat-modified spores of Bacillus cereus T.

The effect of calcium on germination of coat-modified Bacillus cereus T spores was investigated. Coat-modified spores produced either by chemical extraction (SDS-DTT-treated spores) or by mutagenesis (10LD mutant spores) were unable to germinate in response to inosine. While SDS-DTT-treated spores could germinate slowly in the presence of L-alanine, 10LD mutant spores could not germinate at all. The lost or reduced germinability of coat-modified spores was restored when exogenous Ca2+ was supplemented to the germination media. The calcium requirement of coat-modified spores for germination was fairly specific. The simultaneous presence of germinant with Ca2+ was also required for germination of coat-modified spores. The optimal recovery of germinability was observed in the presence of 1.0 mM of calcium acetate. The calcium requirement itself was remarkably diminished under the condition in which L-alanine and a certain purine nucleoside analog, adenosine or inosine, coexisted. The lost or diminished germinability observed in SDS-DTT-treated spores or 10LD mutant spores may be attributed to the loss of calcium associated with the spore integuments.     

Changes of Ultrastructure in Spore Coat of Bacillus thiaminolyticus during Germination and Outgrowth

Electron microscopic observation showed that the spore coat of Bacillus thiaminolyticus consisted of at least four layers; a high electron dense outer spore coat layer with five prominent ridges, a middle spore coat layer including two layers of a high and a low electron density, and an inner spore coat layer composing six to seven laminated layers. Rapid breakdown of the cortex and swelling of the core occurred in spores which were allowed to germinate by L -alanine for 45 min, whereas no change of surface feature was observed by scanning electron microscopy. Germination and outgrowth of spores in nutrient broth proceeded, being accompanied by morphological changes, in three steps; the first is a rapid breakdown of the cortex and swelling of the core, the second degradation of the inner layer at a prominent region of the spore coat, and the last rupture of the spore coat and emergence of a young vegetative cell.     

Spore Dormancy Mutants of Phycomyces

Rivero, F., and Cerdá-Olmedo, E. 1994. Spore dormancy mutants of Phycomyces. Experimental Mycology 18, 221-229. The spores of the Zygomycete Phycomyces blakesleeanus are called dormant because few of them germinate when placed in a medium that sustains mycelial growth and development. Nearly all the spores germinate after activation, that is, exposure to heat or certain chemicals. We have looked for mutants whose spores would not need activation. Nine mutants formed authentic, but transient spores, which germinated spontaneously in the sporangium. Mutant mycelia had lower alcohol and aldehyde dehydrogenase activities and less glycogen than wild-type mycelia. The spontaneous germination and the metabolic alterations are attributed to the same recessive mutations. No differences were found between mutants and wild type in the cyclic AMP and fructose 2,6-bisphosphate concentrations in immature sporangia and the trehalase activity in the mycelia. In another mutant the spore primordia did not form spores, but remained viable for some time in the sporangium. The mutants were difficult to keep in the laboratory (except as lyophils); this stresses the importance of preventing spore germination in the sporangium.     

Comparison of coats and surface-dependent properties of Bacillus cereus T prepared in two sporulation environments

The surface or coat-associated properties of Bacillus cereus T spores produced from modified G medium (MGM) and fortified nutrient agar (FNA) were compared. The two populations appeared structurally similar by transmission electron microscopy. Spores prepared on FNA were more susceptible to ozone inactivation than MGM-prepared spores. When activated by heating for 15 min at 70–85°C, FNA-prepared spores were optimally activated at 85°C and did not become hydrophilic on heat activation while MGM spores were optimally activated at 70°C and became hydrophilic on activation. Susceptibility to removal of coat and outer membrane by chemical and enzymatic extraction treatments was measured by monitoring reduced ability to germinate in nutrients and acquired ability to germinate in the presence of lysozyme. Bacillus cereus T MGM-prepared spores germinated in lysozyme upon<1 h exposure to sodium dodecyl sulphate-dithiothreitol. FNA-prepared spores were lysozyme sensitive after > 2 h treatment. Thus, B. cereus T FNA spore coats and outer membranes were more resistant to these denaturing agents. Transmission electron micrographs revealed no change in appearance of extracted spores. Sporulation environment must be considered when laboratory-prepared spores are used to assess or predict the effect of control procedures on spores present in nature.