Cloning and nucleotide sequencing of genes for small, acid-soluble spore proteins of Bacillus cereus, Bacillus stearothermophilus, and "Thermoactinomyces thalpophilus"

As found previously with other Bacillus species, spores of B. stearothermophilus and "Thermoactinomyces thalpophilus" contained significant levels of small, acid-soluble spore proteins (SASP) which were rapidly degraded during spore germination and which reacted with antibodies raised against B. megaterium SASP. Genes coding for a B. stearothermophilus and a "T. thalpophilus" SASP as well as for two B. cereus SASP were cloned, their nucleotide sequences were determined, and the amino acid sequences of the SASP coded for were compared. Strikingly, all of the amino acid residues previously found to be conserved in this group of SASP both within and between two other Bacillus species (B. megaterium and B. subtilis) were also conserved in the SASP coded for by the B. cereus genes as well as those coded for by the genes from the more distantly related organisms B. stearothermophilus and "T. thalpophilus." This finding strongly suggests that there is significant selective pressure to conserve SASP primary sequence and thus that these proteins serve some function other than simply amino acid storage.     

Effects of mutant small, acid-soluble spore proteins from Bacillus subtilis on DNA in vivo and in vitro.

alpha/beta-type small, acid-soluble spore proteins (SASP) of Bacillus subtilis bind to DNA and alter its conformation, topology, and photochemistry, and thereby spore resistance to UV light. Three mutations have been introduced into the B. subtilis sspC gene, which codes for the alpha/beta-type wild-type SASP, SspCwt. One mutation (SspCTyr) was a conservative change, as residue 29 (Leu) was changed to Tyr, an amino acid found at this position in other alpha/beta-type SASP. The other mutations changed residues conserved in all alpha/beta-type SASP. In one (SspCAla), residue 52 (Gly) was changed to Ala; in the second (SspCGln), residue 57 (Lys) was changed to Gln. The effects of the wild-type and mutant SspC on DNA properties were examined in vivo in B. subtilis spores and Escherichia coli as well as in vitro with use of purified protein. Both SspCwt and SspCTyr interacted similarly with DNA in vivo and in vitro, restoring much UV resistance to spores lacking major alpha/beta-type SASP, causing a large increase in plasmid negative supercoiling, and altering DNA UV photochemistry from cell type to spore type. In contrast, SspCAla had no detectable effect on DNA properties in vivo or in vitro, while SspCGln had effects intermediate between those of SspCAla and SspCwt. Strikingly, neither SspCAla nor SspCGln bound well to DNA in vitro. These results confirm the importance of the conserved primary sequence of alpha/beta-type SASP in the ability of these proteins to bind to spore DNA and cause spore UV resistance.     

Purification and amino acid sequence of two small, acid-soluble proteins from Clostridium bifermentans spores

Clostridium bifermentans spores contain two major small, acid-soluble, proteins (SASP) termed SASP-alpha and beta. The amino acid sequences of SASP-alpha and beta are almost identical, and are very similar to those of alpha/beta-type SASP from spores of C. perfringens and various Bacillus species. However, the C. bifermentans proteins contain an extra five amino acids in the middle of their sequence. Surprisingly, no gamma-type SASP were found in C. bifermentans or C. perfringens spores, although these are the most prominent SASP in spores of Bacillus species.     

Cloning and nucleotide sequence of the Bacillus megaterium gene coding for small, acid-soluble spore protein B.

The Bacillus megaterium gene coding for small, acid-soluble spore protein (SASP) B was cloned and its nucleotide sequence was determined. The amino acid sequence predicted from the DNA sequence was identical to that determined previously for SASP B, with the exception of the amino-terminal methionine predicted from the gene sequence which is presumably removed posttranslationally and an asparagine residue predicted at position 21 which was originally identified as an aspartate residue. The mRNA encoded by the SASP B gene is synthesized for only a discrete period midway in sporulation, in parallel with mRNAs coding for other SASPs. The small size of the SASP B mRNA (365 nucleotides) indicated that the mRNA is monocistronic. The SASP B gene itself hybridized strongly to only one band in Southern blots of restriction enzyme digests of B. megaterium DNA, suggesting that the SASP B gene is not a member of a highly conserved multigene family, as is the case for other SASP genes.     

SASP (Small, Acid-Soluble Spore Proteins) and Spore Properties in Bacillus thuringiensis israelensis and Bacillus sphaericus

Entomopathogenic bacilli B. thuringiensis israelensis (Bti) and B. sphaericus (Bf) exhibit low survival on field application. It was previously shown that their spores are very sensitive to different stress effectors (heat, UV light) and especially to osmotic variations. Since SASP (Small, Acid-Soluble Spore Proteins), α/β and γ type, are involved in spore tolerance to heat, UV light, peroxide, and salt, they were analyzed in Bti and Bf. The molecular weight, migration pattern and amino acid composition of different SASP were determined and compared with other bacilli, in particular to B. subtilis. A relation between spore osmotolerance, SASP content, and amino acid composition was shown. In addition, the absence of γ SASP in Bti and Bf is discussed. Received: 3 September 1997 / Accepted: 15 October 1997     

Small,acid-soluble proteins as biomarkers in mass spectrometry analysis of Bacillus spores

The use of 1 N HCl for extraction of small, acid-soluble proteins (SASP) from different Bacillus spore species was examined. The extracts were analyzed by high-performance liquid chromatography and matrix-assisted laser desorption mass spectrometry and were found to be both qualitatively and quantitatively superior to extraction by acetonitrile-5% trifluoroacetic acid (70:30, vol/vol). Both major and minor alpha/beta- and gamma-type SASP were characterized by their molecular masses or tryptic peptide maps and by searches of both protein and unannotated genome databases. For all but 1 pair (B. cereus T and B. thuringiensis subsp. Kurstaki) among the 11 variants studied the suites of SASP masses are distinctive, consistent with the use of these proteins as potential biomarkers for spore identification by mass spectrometry.     

DNA in dormant spores of Bacillus species is in an A-like conformation

The DNA in dormant spores of Bacillus species is associated with alpha/beta-type small, acid-soluble proteins (SASP), which are double-stranded DNA-binding proteins whose amino acid sequence has been highly conserved in evolution. In vitro these proteins bind most strongly to DNA which readily adopts an A-like conformation, as binding of alpha/beta-type SASP causes DNA to assume an A-like conformation. As predicted by this conformational change in DNA, binding of alpha/beta-type SASP to relaxed but covalently closed plasmid DNA results in the introduction of a large number of negative supercoils. Associated with the conformational change in DNA brought about by alpha/beta-type SASP binding is a change in its photochemistry such that ultraviolet irradiation does not generate pyrimidine dimers, but rather a thyminyl-thymine adduct termed spore photoproduct (SP). The latter two properties of DNA complexed with alpha/beta-type SASP in vitro are similar to those of DNA in dormant spores of Bacillus species in which: (i) plasmid DNA has a much higher number of negative supercoils than plasmid in growing cells; and (ii) ultraviolet irradiation produces SP and no pyrimidine dimers, while only pyrimidine dimers are formed in growing cells. During sporulation these changes in the properties of spore DNA take place in parallel with synthesis of alpha/beta-type SASP, and the magnitude of the changes is greatly reduced in mutants that make low amounts of these proteins. A straightforward interpretation of these data is that DNA in dormant spores of Bacillus species is in an A-like conformation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Studies on the mechanism of the osmoresistance of spores of Bacillus subtilis

AIMS: To determine the reason that spores of Bacillus species, in particular Bacillus subtilis, are able to form colonies with high efficiency on media with very high salt concentrations. METHODS AND RESULTS: Spores of various Bacillus species have a significantly higher plating efficiency on media with high salt concentration (termed osmoresistance) than do log or stationary phase cells. This spore osmoresistance is higher on richer media. Bacillus subtilis spores lacking various small, acid-soluble spore proteins (SASP) were generally significantly less osmoresistant than were wild-type spores, as shown previously (Ruzal et al. 1994). Other results included: (a) spore osmoresistance varied significantly between species; (b) the osmoresistance of spores lacking SASP was not restored well by amino acid osmolytes added to plating media, but was completely restored by glucose; (c) the osmoresistance of spores lacking SASP was restored upon brief germination in the absence of salt in a process that did not require protein synthesis; (d) significant amounts of amino acids generated by SASP degradation were retained within spores upon germination in a medium with high but not low salt; (e) slowing but not abolishing SASP degradation by loss of the SASP-specific germination protease (GPR) did not affect spore osmoresistance; (f) sporulation at higher temperatures produced less osmoresistant spores; and (g) spore osmoresistance was not decreased markedly by the absence of the stress sigma factor for RNA polymerase, sigmaB. CONCLUSIONS: Spore osmoresistance appears as a result of three major factors: (1) specific characteristics of spores and cells of individual species; (2) the precise sporulation conditions that produce the spores; and (3) sufficient energy generation by the germinating and outgrowing spore to allow the spore to adapt to conditions of high osmotic strength; the substrates for this energy generation can come from either the endogenous generation of amino acids by SASP degradation or from the spore's environment, in the form of a readily taken up and metabolized energy source such as glucose. SIGNFICANCE AND IMPACT OF STUDY: These results provide information on the mechanisms of spore osmoresistance, a spore property that can be of major applied significance given the use of high osmotic strength with or without high salt as a means of food preservation.     

Small, Acid-Soluble Proteins as Biomarkers in Mass Spectrometry Analysis of Bacillus Spores

The use of 1 N HCl for extraction of small, acid-soluble proteins (SASP) from different Bacillus spore species was examined. The extracts were analyzed by high-performance liquid chromatography and matrix-assisted laser desorption mass spectrometry and were found to be both qualitatively and quantitatively superior to extraction by acetonitrile-5% trifluoroacetic acid (70:30, vol/vol). Both major and minor α/β- and γ-type SASP were characterized by their molecular masses or tryptic peptide maps and by searches of both protein and unannotated genome databases. For all but 1 pair (B. cereus T and B. thuringiensis subsp. Kurstaki) among the 11 variants studied the suites of SASP masses are distinctive, consistent with the use of these proteins as potential biomarkers for spore identification by mass spectrometry.     

A-type ATP binding consensus sequences are critical for the catalytic activity of the calmodulin-sensitive adenylyl cyclase from Bacillus anthracis

Analysis of the predicted amino acid sequence of Bacillus anthracis adenylyl cyclase revealed sequences with homology to consensus sequences for A- and B-type ATP binding domains found in many ATP binding proteins. Based on the analysis of nucleotide binding proteins, a conserved basic amino acid residue in the A-type consensus sequence and a conserved acidic amino acid residue in the B-type consensus sequence have been implicated in the binding of ATP. The putative ATP binding sequences in the B. anthracis adenylyl cyclase possess analogous lysine residues at positions 346 and 353 within two A-type consensus sequences and a glutamate residue at position 436 within a B-type consensus sequence. The two A-type consensus sequences overlap each other and have the opposite orientation. To determine whether Lys-346, Lys-353, or Glu-436 of the B. anthracis adenylyl cyclase are crucial for enzyme activity, Lys-346 and Lys-353 were replaced with methionine and Glu-436 with glutamine by oligonucleotide-directed mutagenesis. Furthermore, Lys-346 was also replaced with arginine. The genes encoding the wild type and mutant adenylyl cyclases were placed under the control of the lac promoter for expression in Escherichia coli, and extracts were assayed for adenylyl cyclase activity. In all cases, a 90-kDa polypeptide corresponding to the catalytic subunit of the enzyme was detected in E. coli extracts by rabbit polyclonal antibodies raised against the purified B. anthracis adenylyl cyclase. The proteins with the Lys-346 to methionine or arginine mutations exhibited no adenylyl cyclase activity, indicating that Lys-346 in the A-type ATP binding consensus sequence plays a critical role for enzyme catalysis. Furthermore, the enzyme with the Lys-353 to methionine mutation was also inactive, suggesting that Lys-353 may also directly contribute to enzyme catalysis. In contrast, the protein with the Glu-436 to glutamine mutation retained 75% of enzyme activity, suggesting that Glu-436 in the B-type ATP binding consensus sequence may not be directly involved in enzyme catalysis. It is concluded that Lys-346 and Lys-353 in B. anthracis adenylyl cyclase may interact directly with ATP and contribute to the binding of the nucleotide to the enzyme.     

Cloning, nucleotide sequencing, and genetic mapping of the gene for small, acid-soluble spore protein gamma of Bacillus subtilis.

The Bacillus subtilis gene (sspE) which codes for small acid-soluble spore protein gamma (SASP-gamma) was cloned, and its chromosomal location (65 degrees, linked to glpD) and nucleotide sequence were determined. The amino acid sequence of SASP-gamma is similar to that of SASP-B of Bacillus megaterium, but these sequences are not as highly conserved across species as are those of other SASPs. The SASP-gamma gene is transcribed only in sporulation in parallel with other SASP genes and gives a single mRNA that is approximately 340 nucleotides long. The results of hybridization of an sspE gene probe to Southern blots of B. subtilis DNA suggested that there is only a single gene coding for the SASP-gamma type of protein in B. subtilis. This was confirmed by introducing a deletion mutation into the cloned sspE gene and transferring the deletion into the B. subtilis chromosome, with concomitant loss of the wild-type gene. This sspE deletion strain sporulated well, but lacked the SASP-gamma type of protein.     

Site-directed mutagenesis and structural studies suggest that the germination protease, GPR, in spores of Bacillus species is an atypical aspartic acid protease

Germination protease (GPR) initiates the degradation of small, acid-soluble spore proteins (SASP) during germination of spores of Bacillus and Clostridium species. The GPR amino acid sequence is not homologous to members of the major protease families, and previous work has not identified residues involved in GPR catalysis. The current work has focused on identifying catalytically essential amino acids by mutagenesis of Bacillus megaterium gpr. A residue was selected for alteration if it (i) was conserved among spore-forming bacteria, (ii) was a potential nucleophile, and (iii) had not been ruled out as inessential for catalysis. GPR variants were overexpressed in Escherichia coli, and the active form (P41) was assayed for activity against SASP and the zymogen form (P46) was assayed for the ability to autoprocess to P41. Variants inactive against SASP and unable to autoprocess were analyzed by circular dichroism spectroscopy and multi-angle laser light scattering to determine whether the variant's inactivity was due to loss of secondary or quaternary structure, respectively. Variation of D127 and D193, but no other residues, resulted in inactive P46 and P41, while variants of each form were well structured and tetrameric, suggesting that D127 and D193 are essential for activity and autoprocessing. Mapping these two aspartate residues and a highly conserved lysine onto the B. megaterium P46 crystal structure revealed a striking similarity to the catalytic residues and propeptide lysine of aspartic acid proteases. These data indicate that GPR is an atypical aspartic acid protease.     

Proteolytic processing of the protease which initiates degradation of small, acid-soluble proteins during germination of Bacillus subtilis spores.

Degradation of small, acid-soluble spore proteins during germination of Bacillus subtilis spores is initiated by a sequence-specific protease called GPR. Western blot (immunoblot) analysis of either Bacillus megaterium or B. subtilis GPR expressed in B. subtilis showed that GPR is synthesized at about the third hour of sporulation in a precursor form and is processed to an approximately 2- to 5-kDa-smaller species 2 to 3 h later, at or slightly before the time of accumulation of dipicolinic acid by the forespore. This was found with both normal levels of expression of B. subtilis and B. megaterium GPR in B. subtilis, as well as when either protein was overexpressed up to 100-fold. The sporulation-specific processing of GPR was blocked in all spoIII, -IV, and -V mutants tested (none of which accumulated dipicolinic acid), but not in a spoVI mutant which accumulated dipicolinic acid. The amino-terminal sequences of the B. megaterium and B. subtilis GPR initially synthesized in sporulation were identical to those predicted from the coding genes' sequences. However, the processed form generated in sporulation lacked 15 (B. megaterium) or 16 (B. subtilis) amino-terminal residues. The amino acid sequence surrounding this proteolytic cleavage site was very homologous to the consensus sequence recognized and cleaved by GPR in its small, acid-soluble spore protein substrates. This observation, plus the efficient processing of overproduced GPR during sporulation, suggests that the GPR precursor may autoproteolyze itself during sporulation. During spore germination, the GPR from either species expressed in B. subtilis was further processed by removal of one additional amino-terminal amino acid (leucine), generating the mature protease which acts during spore germination.     

A conserved ClpP‐like protease involved in spore outgrowth in Bacillus subtilis

Germination and outgrowth of endospores of the Gram‐positive bacterium Bacillus subtilis involves the degradation and conversion to free amino acids of abundant proteins located in the spore core known as small acid‐soluble proteins (SASP). This degradation is mediated primarily by the germination protease Gpr. Here we show that YmfB, a distant homologue of ClpP serine proteases that is highly conserved among endospore‐forming bacteria, contributes to SASP degradation but that its function is normally masked by Gpr. Spores from a ymfB gpr double mutant were more delayed in spore outgrowth and more impaired in SASP degradation than were spores from a gpr single mutant. The activity of YmfB relied on three putative active‐site residues as well as on the product of a small gene ylzJ located immediately downstream of, and overlapping with, ymfB. We propose that YmfB is an orphan ClpP protease that is dedicated to the degradation of a specialized family of small protein substrates.     

Spores of Bacillus subtilis: their resistance to and killing by radiation, heat and chemicals

A number of mechanisms are responsible for the resistance of spores of Bacillus species to heat, radiation and chemicals and for spore killing by these agents. Spore resistance to wet heat is determined largely by the water content of spore core, which is much lower than that in the growing cell protoplast. A lower core water content generally gives more wet heat-resistant spores. The level and type of spore core mineral ions and the intrinsic stability of total spore proteins also play a role in spore wet heat resistance, and the saturation of spore DNA with alpha/beta-type small, acid-soluble spore proteins (SASP) protects DNA against wet heat damage. However, how wet heat kills spores is not clear, although it is not through DNA damage. The alpha/beta-type SASP are also important in spore resistance to dry heat, as is DNA repair in spore outgrowth, as Bacillus subtilis spores are killed by dry heat via DNA damage. Both UV and gamma-radiation also kill spores via DNA damage. The mechanism of spore resistance to gamma-radiation is not well understood, although the alpha/beta-type SASP are not involved. In contrast, spore UV resistance is due largely to an alteration in spore DNA photochemistry caused by the binding of alpha/beta-type SASP to the DNA, and to a lesser extent to the photosensitizing action of the spore core's large pool of dipicolinic acid. UV irradiation of spores at 254 nm does not generate the cyclobutane dimers (CPDs) and (6-4)-photoproducts (64PPs) formed between adjacent pyrimidines in growing cells, but rather a thymidyl-thymidine adduct termed spore photoproduct (SP). While SP is formed in spores with approximately the same quantum efficiency as that for generation of CPDs and 64PPs in growing cells, SP is repaired rapidly and efficiently in spore outgrowth by a number of repair systems, at least one of which is specific for SP. Some chemicals (e.g. nitrous acid, formaldehyde) again kill spores by DNA damage, while others, in particular oxidizing agents, appear to damage the spore's inner membrane so that this membrane ruptures upon spore germination and outgrowth. There are also other agents such as glutaraldehyde for which the mechanism of spore killing is unclear. Factors important in spore chemical resistance vary with the chemical, but include: (i) the spore coat proteins that likely react with and detoxify chemical agents; (ii) the relative impermeability of the spore's inner membrane that restricts access of exogenous chemicals to the spore core; (iii) the protection of spore DNA by its saturation with alpha/beta-type SASP; and (iv) DNA repair for agents that kill spores via DNA damage. Given the importance of the killing of spores of Bacillus species in the food and medical products industry, a deeper understanding of the mechanisms of spore resistance and killing may lead to improved methods for spore destruction.