Small Acid-soluble Proteins Research Articles

Effect of Small, Acid-Soluble Proteins on Spore Resistance and Germination under a Combination of Pressure and Heat Treatment

To find the range of pressure required for effective high-pressure inactivation of bacterial spores and to investigate the role of α/β-type small, acid-soluble proteins (SASP) in spores under pressure treatment, mild heat was combined with pressure (room temperature to 65°C and 100 to 500 MPa) and applied to wild-type and SASP-α−/β−Bacillus subtilis spores. On the one hand, more than 4 log units of wild-type spores were reduced after pressurization at 100 to 500 MPa and 65°C. On the other hand, the number of surviving mutant spores decreased by 2 log units at 100 MPa and by more than 5 log units at 500 MPa. At 500 MPa and 65°C, both wild-type and mutant spore survivor counts were reduced by 5 log units. Interestingly, pressures of 100, 200, and 300 MPa at 65°C inactivated wild-type SASP-α+/β+ spores more than mutant SASP-α−/β− spores, and this was attributed to less pressure-induced germination in SASP-α−/β− spores than in wild-type SASP-α+/β+ spores. However, there was no difference in the pressure resistance between SASP-α+/β+ and SASP-α−/β− spores at 100 MPa and ambient temperature (approximately 22°C) for 30 min. A combination of high pressure and high temperature is very effective for inducing spore germination, and then inactivation of the germinated spore occurs because of the heat treatment. This study showed that α/β-type SASP play a role in spore inactivation by increasing spore germination under 100 to 300 MPa at high temperature.

Crystallization and preliminary X-ray analysis of the complex between aBacillus subtilisα/β-type small acid-soluble spore protein and DNA

An engineered variant of an alpha/beta-type small acid-soluble spore protein (SASP) from Bacillus subtilis was crystallized in a complex with a ten-base-pair double-stranded DNA by the hanging-drop vapor-diffusion method using ammonium sulfate as a precipitating agent. Crystals grew at 281 K using sodium cacodylate buffer pH 5.5 and these crystals diffracted X-rays to beyond 2.4 A resolution using synchrotron radiation. The crystallized complex contains two or three SASP molecules bound to one DNA molecule. The crystals belong to the hexagonal space group P6(1)22 or P6(5)22, with unit-cell parameters a = b = 87.0, c = 145.4 A, alpha = beta = 90.0, gamma = 120.0 degrees. Diffraction data were 96.6% complete to 2.4 A resolution, with an R(sym) of 8.5%. Structure solution by the multiwavelength/single-wavelength anomalous dispersion method using isomorphous crystals of selenomethionine-labeled protein is in progress.

UV-radiation-induced formation of DNA bipyrimidine photoproducts in Bacillus subtilis endospores and their repair during germination.

The spore photoproduct (SP) is the main DNA lesion after UV-C irradiation, and its repair is crucial for the resistance of spores to UV. The aims of the present study were to assess the formation and repair of bipyrimidine photoproducts in spore DNA of various Bacillus subtilis strains using a sensitive HPLC tandem mass spectrometry assay. Strains deficient in nucleotide excision repair, spore photoproduct lyase, homologous recombination (recA), and with wild-type repair capability were investigated. Additionally, one strain deficient in the formation of major small, acid-soluble spore proteins (SASPs) was tested. In all SASP wild-type strains, UV-C irradiation generated almost exclusively SP (>95 %) but also a few by-photoproducts. In the major SASP-deficient strain, SP and by-photoproducts were generated in equal quantities. The status time of 60 min, >75% of the SP was repaired in wild-type strains and in the SASP-deficient strain, while half of the photoinduced SP was removed in the recA-deficient strain. SP-lyase-deficient spores repaired 20% of the SP produced. Thus, SP lyase, with respect to nucleotide excision repair, has a remarkable impact on the removal of SP upon spore germination.

Bacillus cereus strains fall into two clusters (one closely and one more distantly related) to Bacillus anthracis according to amino acid substitutions in small acid-soluble proteins as determined by tandem mass spectrometry

Small acid-soluble proteins (SASPs) are located in the core region of Bacillus spores and have been previously demonstrated as reliable biomarkers for differentiating Bacillus anthracis and Bacillus cereus. Using MS and MS-MS analysis of SASPs further phylogenetic correlations among B. anthracis and B. cereus strains are described here. ESI was demonstrated to be a more comprehensive method, allowing for the analysis of intact proteins in both MS and MS-MS mode, thus providing molecular weight (MW) and sequence information in a single analysis, and requiring almost no sample preparation. MALDI MS was used for determination of MW of intact proteins; however, MS-MS analysis can only be achieved after enzymatic digestion of these proteins. It was demonstrated that the combination of the two different approaches provides confirmatory and complementary information, allowing for unambiguous protein characterization and sequencing. This study established that B. cereus strains fall into two clusters (one closely and one more distantly related) to B. anthracis as exhibited by amino acid substitutions. The closely related cluster was characterized by a beta-SASP with a single amino acid substitution, localized either close to the C terminus (phenylalanine-->tyrosine, 16 masses change) or close to the N terminus (serine-->alanine serine, also 16 masses change). The more distantly related cluster displayed both amino acid substitutions (32 masses change). One strain of B. cereus isolated from a patient with severe pneumonia (an anthrax-like disease) fell into the more distantly related cluster implying that pathogenicity and phylogenicity are not necessarily correlated features. Unlike PCR and DNA sequencing, protein sequence variation assessed by ESI MS-MS, essentially occurs in real-time, and involves simply extracting the protein and injecting into the instrument for analysis.

The impact of functional genomics on microbiological food quality and safety

In the food processing industry, unwanted occurrence and growth of spoilage and pathogenic microorganisms is a key concern. A prime example is the extremely heat resistant bacterial endospores, microbial survival structures, that create problems due to their ability to survive classical thermal treatments and their ability to subsequently germinate and form actively growing vegetative cells. Research on food spoilage Bacillus subtilis isolates using the Amplified Fragment Length Polymorphism (AFLP) technology and micro-array technology has identified a number of genomic factors correlated to the level of spore heat resistance. Strains could be classified according to these genomic markers. In addition, it was shown with the sequenced B. subtilis laboratory strain that sporulation in the presence of in particular calcium ions in a cocktail of calcium, magnesium, iron, manganese and potassium promotes thermal resistance of developing spores. This physiological observation correlated with an increased expression during sporulation of genes encoding small acid soluble spore proteins. Screening of ingredients using DNA-chip based techniques identifying the above indicated molecular markers, should allow in the future the identification of the occurrence of spoilage and pathogenic bacteria and prediction of their thermal preservation stress resistance. Currently various projects aiming at the integration of genomics data and micro(nano)-technology, a prerequisite if the alluded to ingredient Quality Control is going to succeed, are running or are being set-up. Information from these projects will be used together with the requirements of product organoleptic quality to derive robust integrated food safety and food quality processing parameters. Such parameters should form the basis of future food Quality Assurance systems.

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.

Role of Dipicolinic Acid in Resistance and Stability of Spores of Bacillus subtilis with or without DNA-Protective α/β-Type Small Acid-Soluble Proteins

Dipicolinic acid (DPA) comprises approximately 10% of the dry weight of spores of Bacillus species. Although DPA has long been implicated in spore resistance to wet heat and spore stability, definitive evidence on the role of this abundant molecule in spore properties has generally been lacking. Bacillus subtilis strain FB122 (sleB spoVF) produced very stable spores that lacked DPA, and sporulation of this strain with DPA yielded spores with nearly normal DPA levels. DPA-replete and DPA-less FB122 spores had similar levels of the DNA protective alpha/beta-type small acid-soluble spore proteins (SASP), but the DPA-less spores lacked SASP-gamma. The DPA-less FB122 spores exhibited similar UV resistance to the DPA-replete spores but had lower resistance to wet heat, dry heat, hydrogen peroxide, and desiccation. Neither wet heat nor hydrogen peroxide killed the DPA-less spores by DNA damage, but desiccation did. The inability to synthesize both DPA and most alpha/beta-type SASP in strain PS3664 (sspA sspB sleB spoVF) resulted in spores that lost viability during sporulation, at least in part due to DNA damage. DPA-less PS3664 spores were more sensitive to wet heat than either DPA-less FB122 spores or DPA-replete PS3664 spores, and the latter also retained viability during sporulation. These and previous results indicate that, in addition to alpha/beta-type SASP, DPA also is extremely important in spore resistance and stability and, further, that DPA has some specific role(s) in protecting spore DNA from damage. Specific roles for DPA in protecting spore DNA against damage may well have been a major driving force for the spore's accumulation of the high levels of this small molecule.

History of science – spores.

Bacterial endospores were first studied 130 years ago by Cohn in 1876 and independently by Koch in the same year. Although spore dormancy and resistance have been much studied since then, questions still remain concerning the basic mechanisms and the kinetics of heat inactivation in particular. Likewise, the extreme dormancy and longevity of spores was recognized early on and later greatly extended but still evade complete understanding. Evidence has accumulated for the involvement of specific spore components such as calcium, dipicolinic acid, small acid soluble proteins in the core and peptidoglycan in the cortex. Involvement of physical factors too, such as the relative dehydration of the core, maybe in a high-viscosity state or even in a glassy state, has added to appreciation of the multicomponent nature of dormancy and resistance. Spore-former morphology formed the basis for early classification systems of sporeformers from about 1880 and consolidated in the mid-1900s, well prior to the use of modern genetic procedures. With respect to sporulation, groundbreaking sequence studies in the 1950s provided the basis for later elucidation of the genetic control widely relevant to many cell differentiation mechanisms. With respect to the breaking of dormancy (activation and germination), the elucidation of mechanisms began in the 1940s following the observations of Hills at Porton who identified specific amino acid and riboside 'germinants', and laid the basis for the later genetic analyses, the identification of germinant receptor genes and the elucidation of key germination reactions. The nonexponential nature of germination kinetics has thwarted the development of practical Tyndallization-like processing. So inactivation by heat remains the premier method of spore control, the basis of a huge worldwide industry, and still relying on the basic kinetics of inactivation of Clostridium botulinum spores, and the reasoning regarding safety first evolved by Bigelow et al. in 1920 and Esty and Meyer in 1922. 'Newer' processes such as treatment with ionizing radiation (first proposed in 1905) and high hydrostatic pressure (first proposed in 1899) may be introduced if consumer resistance and some remaining technical barriers could be overcome.

Proteins and dipicolinic acid released during heat shock activation of Bacillus subtilis spores probed by optical spectroscopy

UV fluorescence and absorption spectroscopy from Bacillus subtilis spores detected proteins and dipicolinic acid (DPA) released into the supernatant after heat treatments ranging from 20 degrees to 90 degrees C. The protein and DPA concentration in the supernatant was greater with higher heat treatment temperatures, undergoing a substantial increase for T > or = 60 degrees C, and supporting the theory that spores undergo a phase transition from a glassylike to a rubberylike state at 56 degrees C. Gel electrophoresis detected several small proteins with molecular weights between 6 and 11 kDa. These proteins may be small acid-soluble spore proteins that are present in spores but break down during germination. A 30 kDa protein extracted above 60 degrees C is related to the rubber-glass phase transition.

Identification of marker proteins for Bacillus anthracis using MALDI‐TOF MS and ion trap MS/MS after direct extraction or electrophoretic separation

Direct extraction of bacterial vegetative cells or spores followed by matrix-assisted laser desorption ionization/time of flight mass spectrometry (MALDI TOF MS) has become popular for bacterial identification, since it is simple to perform and mass spectra are readily interpreted. However, only high-abundance proteins that are of low mass and ionize readily are observed. In the case of B. anthracis spores, small acid-soluble spore proteins (SASPs) have been the most widely studied. Additional information can be obtained using tandem mass spectrometry (MS-MS) to confirm the identity of proteins by sequencing. This is most readily accomplished using ion trap (IT) MS-MS. However, enzymatic digestion of these proteins is needed to generate peptides that are within the mass range of the ion trap. The use of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), or other forms of electrophoresis, allows one to focus on specific proteins of interest (e.g. the high mass exosporium glycoproteins BcIA and BcIB) that provide additional species- and strain-specific discrimination.

Rapid characterization of Bacillus spores targeting species‐unique peptides produced with an atmospheric pressure matrix‐assisted laser desorption/ionization source

New and improved strategies are eagerly sought for the rapid identification of microorganisms, particularly in mixtures. Mass spectrometry remains a powerful tool for this purpose. Small acid-soluble proteins (SASPs), which are relatively abundant in Bacillus spores, represent potential biomarkers for species characterization. Despite sharing extensive sequence homology, these proteins differ sufficiently in sequence for discrimination between species. This work focuses on the differences in sequence between SASPs from various Bacillus species. Compilation of SASP sequences from protein database searches, followed by in silico trypsin digestion and analysis of the resulting fragments, identified several species-specific peptides that could be targeted for analysis using mass spectrometry. This strategy was tested and found to be successful in the characterization of Bacillus spores both from individual species and in mixtures. Analysis was performed using an ion trap mass spectrometer with an atmospheric pressure MALDI source. This instrumentation offers the advantage of increased speed of analysis and accurate precursor ion selection for tandem mass spectrometric analysis compared with vacuum matrix-assisted laser desorption/ionization and time-of-flight instruments. The identification and targeting of species-specific peptides using this type of instrumentation offers a rapid, efficient strategy for the identification of Bacillus spores and can potentially be applied to different microorganisms.

Complete sequences of small acid-soluble proteins from Bacillus globigii.

Three abundant small acid-soluble proteins (SASPs) from spores of Bacillus globigii were sequenced using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry with post-source decay and nanoelectrospray collision-induced dissociation tandem mass spectrometry. The proteins were extracted from spores with 1 M HCl. Scanning electron micrographs of spores before and after acid extraction show that the spores retain their overall structure but have a shriveled texture following the acid treatment. Extracted SASPs were purified by high-performance liquid chromatography and molecular masses of the SASPs were identified at 7068 (SASP-1), 7332 (SASP-2), and 8889 (gamma-SASP). De novo peptide sequencing was used to determine the protein sequences. The correct ordering of peptide sequences was aided by mapping overlapping enzymatic digests and by comparison with homologous SASPs from Bacillus stearothermophilus. B. globigii is used in many field tests as a surrogate for B. anthracis. Thus complete SASP sequences from B. globigii will facilitate the development of methods for rapid identification of bacteria based on mass spectrometry and the examination of taxonomic relationships between Bacillus species.

Analysis of Bacillus anthracis Spores in Milk Using Mass Spectrometry

New approaches for identifying biological threat agents in raw milk using spectroscopy were tested using Bacillus anthracis (BA) Sterne strain spores seeded into unpasteurized bulk tank milk. Direct filtration onto Tyvek membranes provided the optimal filtration approach from raw milk, but detection limits were not ideal. When beads coated with anti-BA antibodies were mixed with spores in raw milk, the beads were capable of concentrating the spores that could be later detected and characterized by MALDI spectroscopy based on presence of previously characterized small acid-soluble proteins (SASP's). This approach could provide a very rapid assessment of whether milk or milk products have been purposefully contaminated with BA spores. This work was fundamentally a proof-of-concept study demonstrating feasibility of the approach in milk. Other parameters could be changed to potentially lower detection limits, and additional studies are currently underway to improve the approach.

Mechanisms of Bacterial Spore Deactivation Using Ambient Pressure Nonthermal Discharges

Atmospheric pressure nonthermal plasmas have demonstrated rapid deactivation of bacterial spores on surfaces. For example, the lethal Ames strain of Bacillus anthracis, known as anthrax, has been deactivated after 1 min of ionized gas exposure. The hypothesized plasma-produced spore deactivation processes, such as ultraviolet light, hot gases, and intense electric fields, were isolated. Exposure to each of these assumed deactivation mechanisms has demonstrated little to no effect on the sterilization of bacterial spores at plasma discharge exposure levels. The focus of this study has therefore, been on the determination of the mechanism of spore lysis and resultant deactivation from the interaction of the ionized gas with the biological substances within the spore. The rapid lysis of bacterial spores and other cells by nonthermal plasma has led to the integration of this approach with pathogen detection instruments, such as mass spectrometers. The identification by mass spectrometry of the extracted proteins from plasmalyzed spores has facilitated the discovery and subsequent sequencing of small acid soluble proteins (SASP) within the spore responsible for protecting bacterial spores. The identified SASP surrounds the nucleic acids providing some measure of protection from insult in the environment. The plasma discharge rapidly interacts with this SASP causing deactivation of the spore.

Identification of salt-sensitive genes in the kidneys of Dahl rats.

Inherited differences in renal function underlie the effect of high salt diets on blood pressure in Dahl rats. We probed the kidneys of inbred Dahl SS/Jr and SR/Jr for anonymous and candidate genes whose expression was regulated by dietary sodium. mRNA quantitation of both candidate genes implicated in sodium excretion and anonymous gene products found by differential hybridization in the kidneys of salt-resistant (SR) and salt sensitive (SS) inbred Dahl rats on high and low salt diets for 21 days. Differential screening revealed a cDNA clone (H1) that showed increased dietary salt-dependent expression only in SS rats. Sequencing of the H1 cDNA showed it was the Dahl rat homologue to a perchloric acid soluble protein expressed in liver and kidney. Among candidate genes, transcript levels of arginosuccinate synthetase (AS) and arginosuccinate lyase (AL) were higher in SS on low salt diets, and AS mRNA increased in response to a high salt diet in SR. Renal mRNA for the ANP-A and the vasopressin type II receptors did not differ by strain or dietary conditions. Three new salt-sensitive genes were detected in the kidneys of inbred Dahl rats. Two genes encode enzymes in the biosynthesis of L-arginine. The upregulation of these genes by dietary salt indicates increased demand and biosynthesis of L-arginine in Dahl SS rats. A third gene encodes a small acid-soluble protein thought to influence the transcription/translation of numerous genes. Further studies will be needed to determine the nature of the association of these genes with salt-sensitivity and blood pressure.

The effect of metal ions commonly present in food on gene expression of sporulating Bacillus subtilis cells in relation to spore wet heat resistance

Bacillus subtilis is a food spoilage spore-forming bacterium. The spores can be very heat-resistant and may cause problems in the production of foods. Varying the metal concentration in the sporulation media is known to influence the heat resistance of the spores. The effect of changing the metal composition of the sporulation media was monitored by means of transcriptome analysis. Heat resistance was measured based on the method described by Kooiman and the transcriptome analysis was performed with micro-arrays of B. subtilis wt168 DNA. The micro-array analysis showed that genes coding for the two small acid soluble proteins (SASPs a and b) were induced earlier during sporulation in the media that contained higher metal concentrations and produced thermal-resistant spores. The results also showed that the spores of a product isolate (strain B. subtilis A163) were much more heat-resistant than the spores produced under the same environmental conditions by strain B. subtilis PS832. In addition to other genes, SASP a was also significantly induced in cells of strain B. subtilis A163 at early to middle stages of sporulation.

The Role of Calcium in Bacterial Spore Germination

The germination of bacterial endospores involves the following structural and physiological events: binding of nutrients to specific receptors located in the spore, the release of cations and calcium dipicolinate (CaDPA) from the core, degradation of the spore cortex, and the breakdown of small acid soluble proteins (SASP) protecting the DNA in the core. This minireview focuses specifically on the action of germination-specific lytic enzymes (GSLE) and the role of calcium ions in the degradation and collapse of the spore cortex. Several key questions for future research are discussed.

Effect of glutamate synthase (GOGAT) activity on Bacillus subtilis spore properties.

The role of glutamate as osmoprotector was investigated through the study of a mutation in its biosynthetic pathway. A glt::Tn917-lacZ-cat insertion mutant (N1) conferring glutamate auxotrophy and enhanced beta-galactosidase expression on high-salt media was selected. Co-transformation experiments and PCR analysis allowed locating the insertion into the gltB gene corresponding to the small unit of the glutamate synthase (GOGAT). The N1 mutant strain presented a glutamate requirement for growth and a tenfold decrease in GOGAT activity. Transcriptional activity of GOGAT, measured as beta-galactosidase from the transposon fusion, correlated with enzymatic activity; expression was enhanced at the stationary phase and in high-ionic-strength media. However, osmotolerance of cultures of N1 mutant were as wild-type (wt), at least in semi-rich medium. In contrast, sporulation was slightly reduced (75% of wt), and spores were less resistant to UV, heat, and osmolarity, properties linked to the content of small, acid-soluble proteins (SASP). The content of these proteins was, in fact, reduced, in particular the SASP-gamma type. The peptidoglycan-cortex, however, was not impaired since spores maintained lysozyme resistance. Addition of glutamate during sporulation partially rescued spore resistance, but germination and outgrowth remained impaired. Deficiencies in germination and outgrowth were also observed with spores from a gltA mutant strain. Taken together, these results pointed to the importance of GOGAT activity during sporulation, in particular for the synthesis SASPs.

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.

The p32K structural protein of the atadenovirus might have bacterial relatives.

The primary structure of a novel adenoviral protein referred to as p32K and found exclusively in members of the proposed new genus Atadenovirus was analyzed. The p32K gene sequence was determined from two bovine and one snake adenovirus types. Altogether five different p32K sequences were examined, two of them were obtained from the Gene Bank. The C-terminal part of the protein is conserved and shares similarity with certain bacterial small acid soluble proteins (SASPs). The sequence similarity seems coupled with functional relatedness, i.e. both protein groups are found in structures where the genome of the "dormant" organism is packaged in tight nucleoprotein complexes. In these complexes the DNA is protected against harmful environmental effects until the new reproductive cycle is started with specific protease cleavage of the packaging proteins. Although there is no experimental clue about the role of the p32K proteins, we hypothesize phylogenetic relationship between the two protein groups based on the sequence similarity and the supposed functional similarity. The alignments of these protein groups shows that the conserved part of the p32Ks probably is the result of the duplication of a shorter sequence similar to the SASPs of the Bacilli.