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.     

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.     

Analysis of nucleoid morphology during germination and outgrowth of spores of Bacillus species

After a few minutes of germination, nucleoids in the great majority of spores of Bacillus subtilis and Bacillus megaterium were ring shaped. The major spore DNA binding proteins, the alpha/beta-type small, acid-soluble proteins (SASP), colocalized to these nucleoid rings early in spore germination, as did the B. megaterium homolog of the major B. subtilis chromosomal protein HBsu. The percentage of ring-shaped nucleoids was decreased in germinated spores with lower levels of alpha/beta-type SASP. As spore outgrowth proceeded, the ring-shaped nucleoids disappeared and the nucleoid became more compact. This change took place after degradation of most of the spores' pool of major alpha/beta-type SASP and was delayed when alpha/beta-type SASP degradation was delayed. Later in spore outgrowth, the shape of the nucleoid reverted to the diffuse lobular shape seen in growing cells.     

Role of the Nfo and ExoA apurinic/apyrimidinic endonucleases in radiation resistance and radiation-induced mutagenesis of Bacillus subtilis spores

The roles of DNA repair by apurinic/apyrimidinic (AP) endonucleases alone, and together with DNA protection by α/β-type small acid-soluble spore proteins (SASP), in Bacillus subtilis spore resistance to different types of radiation have been studied. Spores lacking both AP endonucleases (Nfo and ExoA) and major SASP were significantly more sensitive to 254-nm UV-C, environmental UV (>280 nm), X-ray exposure, and high-energy charged (HZE)-particle bombardment and had elevated mutation frequencies compared to those of wild-type spores and spores lacking only one or both AP endonucleases or major SASP. These findings further implicate AP endonucleases and α/β-type SASP in repair and protection, respectively, of spore DNA against effects of UV and ionizing radiation.     

Properties of Bacillus megaterium and Bacillus subtilis mutants which lack the protease that degrades small, acid-soluble proteins during spore germination.

During germination of spores of Bacillus species the degradation of the spore's pool of small, acid-soluble proteins (SASP) is initiated by a protease termed GPR, the product of the gpr gene. Bacillus megaterium and B. subtilis mutants with an inactivated gpr gene grew, sporulated, and triggered spore germination as did gpr+ strains. However, SASP degradation was very slow during germination of gpr mutant spores, and in rich media the time taken for spores to return to vegetative growth (defined as outgrowth) was much longer in gpr than in gpr+ spores. Not surprisingly, gpr spores had much lower rates of RNA and protein synthesis during outgrowth than did gpr+ spores, although both types of spores had similar levels of ATP. The rapid decrease in the number of negative supertwists in plasmid DNA seen during germination of gpr+ spores was also much slower in gpr spores. Additionally, UV irradiation of gpr B. subtilis spores early in germination generated significant amounts of spore photoproduct and only small amounts of thymine dimers (TT); in contrast UV irradiation of germinated gpr+ spores generated almost no spore photoproduct and three to four times more TT. Consequently, germinated gpr spores were more UV resistant than germinated gpr+ spores. Strikingly, the slow outgrowth phenotype of B. subtilis gpr spores was suppressed by the absence of major alpha/beta-type SASP. These data suggest that (i) alpha/beta-type SASP remain bound to much, although not all, of the chromosome in germinated gpr spores; (ii) the alpha/beta-type SASP bound to the chromosome in gpr spores alter this DNA's topology and UV photochemistry; and (iii) the presence of alpha/beta-type SASP on the chromosome is detrimental to normal spore outgrowth.     

Differentiation of Bacillus pumilus and Bacillus safensis Using MALDI-TOF-MS

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) despite being increasingly used as a method for microbial identification, still present limitations in which concerns the differentiation of closely related species. Bacillus pumillus and Bacillus safensis, are species of biotechnological and pharmaceutical significance, difficult to differentiate by conventional methodologies. In this study, using a well-characterized collection of B. pumillus and B. safensis isolates, we demonstrated the suitability of MALDI-TOF-MS combined with chemometrics to accurately and rapidly identify them. Moreover, characteristic species-specific ion masses were tentatively assigned, using UniProtKB/Swiss-Prot and UniProtKB/TrEMBL databases and primary literature. Delineation of B. pumilus (ions at m/z 5271 and 6122) and B. safensis (ions at m/z 5288, 5568 and 6413) species were supported by a congruent characteristic protein pattern. Moreover, using a chemometric approach, the score plot created by partial least square discriminant analysis (PLSDA) of mass spectra demonstrated the presence of two individualized clusters, each one enclosing isolates belonging to a species-specific spectral group. The generated pool of species-specific proteins comprised mostly ribosomal and SASPs proteins. Therefore, in B. pumilus the specific ion at m/z 5271 was associated with a small acid-soluble spore protein (SASP O) or with 50S protein L35, whereas in B. safensis specific ions at m/z 5288 and 5568 were associated with SASP J and P, respectively, and an ion at m/z 6413 with 50S protein L32. Thus, the resulting unique protein profile combined with chemometric analysis, proved to be valuable tools for B. pumilus and B. safensis discrimination, allowing their reliable, reproducible and rapid identification.     

Rapid discrimination of Bacillus anthracis from other members of the B. cereus group by mass and sequence of "intact" small acid soluble proteins (SASPs) using mass spectrometry

The intentional contamination of buildings, e.g. anthrax in the bioterrorism attacks of 2001, demonstrated that the population can be affected rapidly and lethally if the appropriate treatment is not provided at the right time. Molecular approaches, primarily involving PCR, have proved useful in characterizing "white powders" used in these attacks as well as isolated organisms. However there is a need for a simpler approach, which does not involve temperamental reagents (e.g. enzymes and primers) which could potentially be used by first responders. It is demonstrated here that small acid-soluble proteins (SASPs), located in the core region of Bacillus spores, are reliable biomarkers for identification. The general strategy used in this study was to measure the molecular weight (MW) of an intact SASP by electrospray ionization mass spectrometry (ESI MS) followed by generation of sequence-specific information by ESI MS/MS (tandem mass spectrometry). A prominent SASP of mass 6679 was present in all B. anthracis strains. For B. cereus and B. thuringiensis strains the SASP had a mass of 6712. This represents a two amino acid substitution (serine to alanine; phenylalanine to tyrosine). The only SASP present in the B. anthracis genome consistent with this sequence is encoded by the gene ssB. This protein has a predicted mass of 6810, presumably post-translational processing leads to loss of methionine (mass 131) generating a SASP of mass 6679. This study showed that intact SASPs can be used as a biomarker for identification of B. anthracis; the protocol is simple and rapid. Extrapolation of this approach might prove important for real-time biodetection.     

Formaldehyde kills spores of Bacillus subtilis by DNA damage and small, acid-soluble spore proteins of the ααααααα/βββββββ-type protect spores against this DNA damage

Killing of wild-type spores of Bacillus subtilis with formaldehyde also caused significant mutagenesis; spores (termed α⁻β⁻) lacking the two major α/β-type small, acid-soluble spore proteins (SASP) were more sensitive to both formaldehyde killing and mutagenesis. A recA mutation sensitized both wild-type and α⁻β⁻ spores to formaldehyde treatment, which caused significant expression of a recA - lacZ fusion when the treated spores germinated. Formaldehyde also caused protein–DNA cross-linking in both wild-type and α⁻β⁻ spores. These results indicate that: (i) formaldehyde kills B. subtilis spores at least in part by DNA damage and (b) α/β-type SASP protect against spore killing by formaldehyde, presumably by protecting spore DNA.     

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

The nucleotide sequences of the single genes coding for the B-type small, acid-soluble spore proteins (SASP) of Bacillus cereus, B. stearothermophilus, and "Thermoactinomyces thalpophilus" were determined, and the amino acid sequences of all B-type SASP were compared. While this type of SASP showed significant sequence conservation around the two spore protease cleavage sites, alignment of these sequences required the introduction of gaps, and even then only 19 of the residues were conserved exactly in all five proteins. However, all five B-type SASP did contain a large (27 to 35-residue), rather well-conserved amino acid sequence repeat, and four of the five proteins had well-conserved regions of 14 to 17 amino acids which appeared three times.     

Protein Profile of <Emphasis Type="Italic">Bacillus subtilis</Emphasis> Spore

Natural wild-type strains of Bacillus subtilis spore is regarded as a non-pathogenic for both human and animal, and has been classified as a novel food which is currently being used as probiotics added in the consumption. To identify B. subtilis spore proteins, we have accomplished a preliminary proteomic analysis of B. subtilis spore, with a combination of two-dimensional electrophoretic separations and matrix-assisted laser desorption ionization tandem time of flight mass spectrometry (MALDI–TOF–MS). In this article, we presented a reference map of 158 B. subtilis spore proteins with an isoelectric point (pI) between 4 and 7. Followed by mass spectrometry (MS) analysis, we identified 71 B. subtilis spore proteins with high level of confidence. Database searches, combined with hydropathy analysis and GO analysis revealed that most of the B. subtilis spore proteins were hydrophilic proteins related to catalytic function. These results should accelerate efforts to understand the resistance of spore to harsh conditions.     

Effect of a small, acid-soluble spore protein from Clostridium perfringens on the resistance properties of Bacillus subtilis spores

Alpha/beta-type small, acid-soluble spore proteins (SASP) are essential for the resistance of DNA in spores of Bacillus species to damage. An alpha/beta-type SASP, Ssp2, from Clostridium perfringens was expressed at significant levels in B. subtilis spores lacking one or both major alpha/beta-type SASP (alpha- and alpha- beta- strains, respectively). Ssp2 restored some of the resistance of alpha- beta- spores to UV and nitrous acid and of alpha- spores to dry heat. Ssp2 also restored much of the resistance of alpha- spores to nitrous acid and restored full resistance of alpha- spores to UV and moist heat. These results further indicate the interchangeability of alpha/beta-type SASP in DNA protection in spores.     

Properties of Bacillus subtilis small, acid-soluble spore proteins with changes in the sequence recognized by their specific protease.

Alpha/beta-type small, acid-soluble proteins (SASP) of dormant spores of Bacillus subtilis bind to DNA and increase its resistance to a variety of damaging agents both in vivo and in vitro. When spores germinate, degradation of alpha/beta-type SASP is rapidly initiated by a sequence-specific protease, which is termed GPR. Three mutations have been introduced into the B. subtilis sspC gene, which codes for the wild-type alpha/beta-type SASP SspCwt; all three mutations change residues in the highly conserved sequence recognized by GPR. In one mutant protein (SspCV), residue 33 (Ser) was changed to Val; in the second (SspCDL), residues 30 and 31 (Glu and Ile) were changed to Asp and Leu, respectively; and in the third mutant protein (SspCDLV), residues 30, 31, and 33 were changed to Asp, Leu, and Val. All three mutant proteins were rapidly degraded by GPR during spore germination, and SspCDL and SspCDLV were degraded by GPR in vitro at rates 8 to 9% of that for SspCwt, although not exclusively at the single site cleaved by GPR in SspCwt. These results indicate (i) that the sequence specificity of GPR is broader than originally imagined and (ii) that GPR can cleave the sequence in SspCDLV. Since the latter sequence is identical to that cleaved during the proteolytic activation of GPR, this result further supports an autoprocessing model for GPR activation during sporulation. The properties of these mutant proteins were also examined, both in vivo in B. subtilis spores and in Escherichia coli and in vitro with purified protein. SspC(v) interacted with DNA similarly to SspC(wt) in vivo, resorting UV and heat resistance to spores lacking major alpha/beta-type SASP to the same extent as SspC(wt). In contrasst, SspC(DL) had much less effect on DNA properties in vivo and bound strongly only to poly(dG) . poly(dC) in vitro; SspC(DLV) exhibited only weak binding to poly(dG).poly(dC) in vitro. These results confirm the importance of the conserved primary sequence of alpha/beta-type SASP in the binding of these proteins to spore DNA and alteration of DNA properties and show further that the GRP recognition region in alpha/beta-type SASP plays some role in DNA binding.     

Equilibrium and kinetic binding interactions between DNA and a group of novel, nonspecific DNA-binding proteins from spores of Bacillus and Clostridium species

Binding of alpha/beta-type small acid-soluble spore proteins (SASP) is the major determinant of DNA resistance to damage caused by UV radiation, heat, and oxidizing agents in spores of Bacillus and Clostridium species. Analysis of several alpha/beta-type SASP showed that these proteins have essentially no secondary structure in the absence of DNA, but become significantly alpha-helical upon binding to double-stranded DNAs or oligonucleotides. Folding of alpha/beta-type SASP induced by a variety of DNAs and oligonucleotides was measured by CD spectroscopy, and this allowed determination of a DNA binding site size of 4 base pairs as well as equilibrium binding parameters of the alpha/beta-type SASP-DNA interaction. Analysis of the equilibrium binding data further allowed determination of both intrinsic binding constants (K) and cooperativity factors (omega), as the alpha/beta-type SASP-DNA interaction was significantly cooperative, with the degree of cooperativity depending on both the bound DNA and the salt concentration. Kinetic analysis of the interaction of one alpha/beta-type SASP, SspC(Tyr), with DNA indicated that each binding event involves the dimerization of SspC(Tyr) monomers at a DNA binding site. The implications of these findings for the structure of the alpha/beta-type SASP.DNA complex and the physiology of alpha/beta-type SASP degradation during spore germination are discussed.     

Analysis of deamidation of small, acid-soluble spore proteins from Bacillus subtilis in vitro and in vivo.

Deamidation of one specific asparagine residue in an alpha/beta-type small, acid-soluble spore protein (SASP) of Bacillus subtilis took place readily in vitro (time for 50% deamidation [t(1/2)], approximately 1 h at 70 degrees C), and the deamidated SASP no longer bound to DNA effectively. However, DNA binding protected against this deamidation in vitro. A mutant alpha/beta-type SASP in which the reactive asparagine was changed to aspartate also failed to bind to DNA in vitro, and this protein did not restore UV radiation and heat resistance to spores lacking the majority of their alpha/beta-type SASP. When expressed in Escherichia coli, where it is bound to DNA, the alpha/beta-type SASP deamidated with a t(1/2) of 2 to 3 h at 95 degrees C. However, the alpha/beta-type SASP was extremely resistant to deamidation within spores (t(1/2), >50 h at 95 degrees C). A gamma-type SASP of B. subtilis also deamidated readily in vitro (t(1/2) for one net deamidation, approximately 1 h at 70 degrees C), but this protein (which is not associated with DNA) deamidated fairly readily in spores (t(1/2), approximately 1 h at 95 degrees C). Total spore core protein also deamidated in vivo, although the rate was two- to threefold slower than that of deamidation of total protein in heated vegetative cells. These data indicate that protein deamidation is slowed significantly in spores, presumably due to the spore's environment. However, alpha/beta-type SASP are even more strongly protected against deamidation in vivo, presumably by their binding to spore DNA. Thus, not only do alpha/beta-type SASP protect spore DNA from damage; DNA also protects alpha/beta-type SASP.     

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

Three mutant forms of the protease (GPR) that initiates degradation of small, acid-soluble spore proteins (SASP) during germination of spores of Bacillus species have been generated. In one variant (GPR delta), the putative pro sequence removed in conversion of the GPR zymogen (termed P46) to the active enzyme (termed P41) was deleted. GPR delta was expressed in both Escherichia coli and Bacillus subtilis as a polypeptide of 41 kDa (P41) which was active both in vivo and in vitro. The other two variants had changes in the sequence around the site where the pro sequence is removed, making this sequence even more like that recognized and cleaved by GPR in its SASP substrates. One of these variants (GPRS) was synthesized as P46S in both B. subtilis and E. coli, but P46S was processed to P41S earlier in B. subtilis sporulation than was wild-type P46. The second variant (GPREI) was made as P46EI but underwent extremely rapid processing to P41EI in both E. coli and B. subtilis. Expression of elevated (> 100-fold) levels of GPR delta or GPREI blocked sporulation at the time of synthesis of glucose dehydrogenase. Expression of elevated levels of GPRS or low levels (< 20% of the wild-type level) of GPR delta or GPREI did not retard sporulation, but the SASP level in the resultant spores was greatly reduced. Prolonged incubation of P41 delta, P41EI, or wild-type P41, either in vivo or with purified proteins in vitro, resulted in a second self-cleavage event generating a 39-kDa polypeptide termed P39. The sequence in the P(41)-->P(39) cleavage site was also quite similar to that recognized and cleaved by GPR in SASP. Together, these results strongly support a model in which activation of GPR during sporulation by conversion of P(46) to P(41) is a self-processing event triggered by a change in the spore core environment (i.e., dehydration) which precludes attack of the active P(41) on its SASP substrates. However, in the first minutes of spore germination, rapid spore core hydration allows rapid attack of active GPR on SASP.     

Proteomic analysis of Bacillus thuringiensis at different growth phases by using an automated online two-dimensional liquid chromatography-tandem mass spectrometry strategy

The proteome of a new Bacillus thuringiensis subsp. kurstaki strain, 4.0718, from the middle vegetative (T(1)), early sporulation (T(2)), and late sporulation (T(3)) phases was analyzed using an integrated liquid chromatography (LC)-based protein identification system. The system comprised two-dimensional (2D) LC coupled with nanoscale electrospray ionization (ESI) tandem mass spectrometry (MS/MS) on a high-resolution hybrid mass spectrometer with an automated data analysis system. After deletion of redundant proteins from the different batches and B. thuringiensis subspecies, 918, 703, and 778 proteins were identified in the respective three phases. Their molecular masses ranged from 4.6 Da to 477.4 Da, and their isoelectric points ranged from 4.01 to 11.84. Function clustering revealed that most of the proteins in the three phases were functional metabolic proteins, followed by proteins participating in cell processes. Small molecular and macromolecular metabolic proteins were further classified according to the Kyoto Encyclopedia of Genes and Genome and BioCyc metabolic pathway database. Three protoxins (Cry2Aa, Cry1Aa, and Cry1Ac) as well as a series of potential intracellular active factors were detected. Many significant proteins related to spore and crystal formation, including sporulation proteins, help proteins, chaperones, and so on, were identified. The expression patterns of two identified proteins, CotJc and glutamine synthetase, were validated by Western blot analysis, which further confirmed the MS results. This study is the first to use shotgun technology to research the proteome of B. thuringiensis. Valuable experimental data are provided regarding the methodology of analyzing the B. thuringiensis proteome (which can be used to produce insecticidal crystal proteins) and have been added to the related protein database.