A novel small protein of Bacillus subtilis involved in spore germination and spore coat assembly

Two small genes named sscA (previously yhzE) and orf-62, located in the prsA-yhaK intergenic region of the Bacillus subtilis genome, were transcribed by SigK and GerE in the mother cells during the later stages of sporulation. The SscA-FLAG fusion protein was produced from T(5) of sporulation and incorporated into mature spores. sscA mutant spores exhibited poor germination, and Tricine-SDS-PAGE analysis showed that the coat protein profile of the mutant differed from that of the wild type. Bands corresponding to proteins at 59, 36, 5, and 3 kDa were reduced in the sscA null mutant. Western blot analysis of anti-CotB and anti-CotG antibodies showed reductions of the proteins at 59 kDa and 36 kDa in the sscA mutant spores. These proteins correspond to CotB and CotG. By immunoblot analysis of an anti-CotH antibody, we also observed that CotH was markedly reduced in the sscA mutant spores. It appears that SscA is a novel spore protein involved in the assembly of several components of the spore coat, including CotB, CotG, and CotH, and is associated with spore germination.     

Characterization of a major Bacillus anthracis spore coat protein and its role in spore inactivation

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.     

Mechanisms of Bacillus subtilis spore resistance to and killing by aqueous ozone

AIMS: To determine the mechanisms of Bacillus subtilis spore killing by and resistance to aqueous ozone. METHODS AND RESULTS: Killing of B. subtilis spores by aqueous ozone was not due to damage to the spore's DNA, as wild-type spores were not mutagenized by ozone and wild-type and recA spores exhibited very similar ozone sensitivity. Spores (termed alpha-beta-) lacking the two major DNA protective alpha/beta-type small, acid-soluble spore proteins exhibited decreased ozone resistance but were also not mutagenized by ozone, and alpha-beta- and alpha-beta-recA spores exhibited identical ozone sensitivity. Killing of spores by ozone was greatly increased if spores were chemically decoated or carried a mutation in a gene encoding a protein essential for assembly of the spore coat. Ozone killing did not cause release of the spore core's large depot of dipicolinic acid (DPA), but these killed spores released all of their DPA after a subsequent normally sublethal heat treatment and also released DPA much more readily when germinated in dodecylamine than did untreated spores. However, ozone-killed spores did not germinate with either nutrients or Ca(2+)-DPA and could not be recovered by lysozyme treatment. CONCLUSIONS: Ozone does not kill spores by DNA damage, and the major factor in spore resistance to this agent appears to be the spore coat. Spore killing by ozone seems to render the spores defective in germination, perhaps because of damage to the spore's inner membrane. SIGNIFICANCE AND IMPACT OF THE STUDY: These results provide information on the mechanisms of spore killing by and resistance to ozone.     

Temporal control of autoactivator synthesis and secretion during spontaneous spore germination in Dictyostelium discoideum

SG mutant and aged wild type spores of the cellular slime mold Dictyostelium discoideum germinate in the absence of an externally applied activation treatment. This type of germination is referred to as autoactivation. During the swelling stage of autoactivation, spores release a factor, the autoactivator, capable of stimulating germination in subsequent spore populations. The autoactivator was not present in the dormant spore, but it or a precursor was produced internally during the first hour of autoactivation. This production was sensitive to moderately high temperatures (+31° C) and was completely destroyed by heat activation (45° C for 30 min). Internal production of the autoactivator was not sensitive to protein synthesis inhibitors. However, the release of the activator from the spore appeared to be regulated by protein synthesis. Internal autoactivator was also produced in the aged wild type strain during the postautoactivation lag phase. The activator could not be directly isolated from within the germinating spore. Its activity on the rest of the spore population was dependent upon its release from the germinating spore. A model is presented integrating the effects of heat, cycloheximide, autoinhibitor and autoactivator on spores of D. discoideum.     

Expression of genes coding for GerA and GerK spore germination receptors is dependent on the protein phosphatase PrpE

The ability of Bacillus subtilis to form spores is a strategy for survival under unfavorable environmental conditions. It is equally crucial to break spore dormancy and return to vegetative growth at the appropriate time. Here we present data showing that the PrpE phosphatase is involved in the control of expression of genes coding for GerA receptors, which are necessary for L-alanine-induced spore germination. Moreover, PrpE is also involved in aspartic acid, glucose, fructose, and potassium (AGFK)-induced spore germination by controlling expression of genes coding for GerK receptors. In the absence of PrpE, the production of spores was essentially normal. However, L-alanine-induced spore germination and, to a lesser extent, the AGFK-induced pathway were abolished. In contrast, the germination pathway dependent on Ca2+-dipicolinate or dodecylamine remained intact. A protein phosphatase PrpE-green fluorescent protein fusion was localized to the prespore and to the dormant spore, consistent with a role in controlling expression of genes coding for GerA receptors. We propose that PrpE is an important element in a signal transduction pathway in Bacillus subtilis that controls the expression of genes coding for germination receptors.     

Germination properties of a spore coat-defective mutant of Bacillus subtilis.

The presence of the gerE36 mutation in strains of Bacillus subtilis 168 resulted in poor germination of their spores in a range of germinants, as measured by the fall in absorbance of spore suspensions. Although resistant to heat and organic solvents, spores were sensitive to lysozyme; electron microscopy revealed that their coat structure was incomplete. These spores responded to germinants by losing heat resistance and changing from phase bright to phase gray. The release of dipicolinic acid and the fall in absorbance of spore suspensions reached only 75 and 50% of wild-type levels, respectively, but followed the same time course as the loss of heat resistance. Although the germination response was incomplete, the concentration of L-alanine required to elicit it was the same for the mutant as for the wild type. The properties of mutant spores suggest that an intact spore coat is not required for the initial interaction between germinant and spore, but that the coat layers may contain molecules important in later stages of germination. In transduction with phage SPP1, the gerE36 mutation mapped between citF and ilvB and was 90% cotransduced with citF2. The gerE mutation identifies the location of a gene important for the progress of late stages of spore formation.     

Correlation between spore structure and spore properties in Bacillus megaterium

The spores of six strains of Bacillus megaterium were divided into two distinct groups on the basis of germination. Three of the strains germinated in a mixture of l-alanine and inosine (AL type spores), and three strains germinated in a mixture of glucose and potassium nitrate (GN type spores); recriprocal germination in the respective solutions did not occur. The AL spores and the GN spores were morphologically distinct. Other differences between the two spore groups included germination inhibition characteristics, dipicolinic acid content, hexosamine content, phosphorus and magnesium content, spore coat features, ion exchange properties, and heat resistance. A correlation appears to exist between spore morphology and certain other spore properties in strains of B. megaterium.     

Isolation and properties of Streptomyces spore membranes.

A simple procedure for the isolation of membranes from Streptomyces spores is described which produces about 12 mg of membrane protein per g of dry weight. The membrane fractions were contaminated by low levels of DNA, RNA, and hexosamines. The functional integrity of the membrane is conserved through the isolation procedure, as evaluated by the presence of several activities of the membrane-bound electron transport chain. This isolation procedure allowed the determination of the biosynthesis of proteins and phospholipids of the membrane. Both biosynthetic processes started in the first 5 min of germination and increased progressively during spore germination. A stable mRNA fraction of the dormant spore encoded 44% of the membrane proteins synthesized early in germination, but most of the phospholipid biosynthesis was not dependent on this fraction.     

Bacterial spore germination and protein mobility

Fluorescence recovery after photobleaching (FRAP) of green fluorescent protein (GFP) has been used to report on protein mobility in single spores. Proteins found in dormant Bacillus spores are not mobile; however, mobility is restored when germination occurs and the core rehydrates. Spores of a cwlD mutant, in which the cortex is resistant to hydrolysis, are able to complete the earliest stages of germination in response to a specific germinant stimulus; in these circumstances, the protein in the spore remains immobile. Therefore, the earliest stages of spore germination, including loss of resistance to extreme heat and the complete release of the spore component dipicolinic acid, are achieved without the restoration of protein mobility.     

Properties of spores of Bacillus subtilis strains which lack the major small, acid-soluble protein.

Bacillus subtilis strains containing a deletion in the gene coding for the major small, acid-soluble, spore protein (SASP-gamma) grew and sporulated, and their spores initiated germination normally, but outgrowth of SASP-gamma- spores was significantly slower than that of wild-type spores. The absence of SASP-gamma had no effect on spore protoplast density or spore resistance to heat or radiation. Consequently, SASP-gamma has a different function in spores than do the other major small, acid-soluble proteins.     

The Bacillus subtilis dacB gene, encoding penicillin-binding protein 5*, is part of a three-gene operon required for proper spore cortex synthesis and spore core dehydration.

Studies of gene expression using fusions to lacZ demonstrated that the Bacillus subtilis dacB gene, encoding penicillin-binding protein 5*, is in an operon with two downstream genes, spmA and spmB. Mutations affecting any one of these three genes resulted in the production of spores with reduced heat resistance. The cortex peptidoglycan in dacB mutant spores had more peptide side chains, a higher degree of peptide cross-linking, and possibly less muramic acid lactam than that of wild-type spores. These cortex structure parameters were normal in spmA and spmB mutant spores, but these spores did not attain normal spore core dehydration. This defect in spore core dehydration was exaggerated by the additional loss of dacB expression. However, loss of dacB alone did not alter the spore core water content. Spores produced by spmA and spmB mutants germinated faster than did those of the wild type. Spores produced by dacB mutants germinated normally but were delayed in spore outgrowth. Electron microscopy revealed a drastically altered appearance of the cortex in dacB mutants and a minor alteration in an spmA mutant. Measurements of electron micrographs indicate that the ratio of the spore protoplast volume to the sporoplast (protoplast-plus-cortex) volume was increased in dacB and spmA mutants. These results are consistent with spore core water content being the major determinant of spore heat resistance. The idea that loosely cross-linked, flexible cortex peptidoglycan has a mechanical activity involved in achieving spore core dehydration is not consistent with normal core dehydration in spores lacking only dacB.     

Isolation and characterization of Bacillus megaterium mutants containing decreased levels of spore protease.

A proteolytic activity present in spores of Bacillus megaterium has previously been implicated in the initiation of hydrolysis of the A, B, and C proteins which are degraded during spore germination. Four mutants of B. megaterium containing 20 to 30% of the normal level of spore proteolytic activity have been isolated. Partial purification of the protease from wild-type spores by a reviewed procedure resulted in the resolution of spore protease activity on the A, B, and C proteins into two peaks--a major one (protease II) and a minor one (protease I). The protease mutants tested lacked active protease II. All of the mutants exhibited a decreased rate of degradation of the A, B, and C proteins during spore germination at 30 degrees C, but degradation of the proteins did occur. Degradation of the A, B, and C proteins during germination of the mutant spores was decreased neither by blockade of ATP production nor by germination at 44 degrees C. Initiation of spore germination was normal in all four mutants, and all four mutants went through outgrowth, grew, and sporulated normally in rich medium. Similarly, outgrowth of spores of two of the four mutants was normal in minimal medium at 30 degrees C. In the two mutants studied, the kinetics of loss of spore heat resistance and spore UV light resistance during germination were identical to those of wild-type spores. This indicates that the A, B, and C proteins alone are not sufficient to account for the heat or UV light resistance of the dormant spore.