Role of DNA Repair and Protective Components in Bacillus subtilis Spore Resistance to Inactivation by 400-nm-Wavelength Blue Light.

The high intrinsic decontamination resistance of Firmicutes spores is important medically (disease) and commercially (food spoilage). Effective methods of spore eradication would be of considerable interest in the health care and medical product industries, particularly if the decontamination method effectively killed spores while remaining benign to both humans and sensitive equipment. Intense blue light at a ∼400 nm wavelength is one such treatment that has drawn significant interest. This work has determined the resistance of spores to blue light in an extensive panel of Bacillus subtilis strains, including wild-type strains and mutants that (i) lack protective components such as the spore coat and its pigment(s) or the DNA protective α/ß-type small, acid-soluble spore proteins (SASP); (ii) have an elevated spore core water content; or (iii) lack enzymes involved in DNA repair, including those for homologous recombination and nonhomologous end joining (HR and NHEJ), apurinic/apyrimidinic endonucleases, nucleotide and base excision repair (NER and BER), translesion synthesis (TLS) by Y-family DNA polymerases, and spore photoproduct (SP) removal by SP lyase (SPL). The most important factors in spore blue light resistance were determined to be spore coats/pigmentation, α/ß-type SASP, NER, BER, TLS, and SP repair. A major conclusion from this work is that blue light kills spores by DNA damage, and the results in this work indicate at least some of the specific DNA damage. It appears that high-intensity blue light could be a significant addition to the agents used to kill bacterial spores in applied settings.IMPORTANCE Effective methods of spore inactivation would be of considerable interest in the health care and medical products industries, particularly if the decontamination method effectively killed spores while remaining benign to both humans and sensitive equipment. Intense blue light radiation is one such treatment that has drawn significant interest. In this work, all known spore-protective features, as well as universal and spore-specific DNA repair mechanisms, were tested in a systematic fashion for their contribution to the resistance of spores to blue light radiation.

Changes in Bacillus Spore Small Molecules, rRNA, Germination, and Outgrowth after Extended Sublethal Exposure to Various Temperatures: Evidence that Protein Synthesis Is Not Essential for Spore Germination.

rRNAs of dormant spores of Bacillus subtilis were >95% degraded during extended incubation at 50°C, as reported previously (E. Segev, Y. Smith, and S. Ben-Yehuda, Cell 148:139-114, 2012, doi:http://dx.doi.org/10.1016/j.cell.2011.11.059), and this was also true of spores of Bacillus megaterium Incubation of spores of these two species for ∼20 h at 75 to 80°C also resulted in the degradation of all or the great majority of the 23S and 16S rRNAs, although this rRNA degradation was slower than nonenzymatic hydrolysis of purified rRNAs at these temperatures. This rRNA degradation at high temperature generated almost exclusively oligonucleotides with minimal levels of mononucleotides. RNase Y, suggested to be involved in rRNA hydrolysis during B. subtilis spore incubation at 50°C, did not play a role in B. subtilis spore rRNA breakdown at 80°C. Twenty hours of incubation of Bacillus spores at 70°C also decreased the already minimal levels of ATP in dormant spores 10- to 30-fold, to ≤0.01% of the total free adenine nucleotide levels. Spores depleted of rRNA were viable and germinated relatively normally, often even faster than starting spores. Their return to vegetative growth was also similar to that of untreated spores for B. megaterium spores and slower for heat-treated B. subtilis spores; accumulation of rRNA took place only after completion of spore germination. These findings thus strongly suggest that protein synthesis is not essential for Bacillus spore germination.IMPORTANCE A recent report (L. Sinai, A. Rosenberg, Y. Smith, E. Segev, and S. Ben-Yehuda, Mol Cell 57:3486-3495, 2015, doi:http://dx.doi.org/10.1016/j.molcel.2014.12.019) suggested that protein synthesis is essential for early steps in the germination of dormant spores of Bacillus subtilis If true, this would be a paradigm shift in our understanding of spore germination. We now show that essentially all of the rRNA can be eliminated from spores of Bacillus megaterium or B. subtilis, and these rRNA-depleted spores are viable and germinate as well as or better than spores with normal rRNA levels. Thus, protein synthesis is not required in the process of spore germination.

Water and Small-Molecule Permeation of Dormant Bacillus subtilis Spores.

UNLABELLED: We use a suspended microchannel resonator to characterize the water and small-molecule permeability of Bacillus subtilis spores based on spores' buoyant mass in different solutions. Consistent with previous results, we found that the spore coat is not a significant barrier to small molecules, and the extent to which small molecules may enter the spore is size dependent. We have developed a method to directly observe the exchange kinetics of intraspore water with deuterium oxide, and we applied this method to wild-type spores and a panel of congenic mutants with deficiencies in the assembly or structure of the coat. Compared to wild-type spores, which exchange in approximately 1 s, several coat mutant spores were found to have relatively high water permeability with exchange times below the ∼200-ms temporal resolution of our assay. In addition, we found that the water permeability of the spore correlates with the ability of spores to germinate with dodecylamine and with the ability of TbCl3 to inhibit germination with l-valine. These results suggest that the structure of the coat may be necessary for maintaining low water permeability. IMPORTANCE: Spores of Bacillus species cause food spoilage and disease and are extremely resistant to standard decontamination methods. This hardiness is partly due to spores' extremely low permeability to chemicals, including water. We present a method to directly monitor the uptake of molecules into B. subtilis spores by weighing spores in fluid. The results demonstrate the exchange of core water with subsecond resolution and show a correlation between water permeability and the rate at which small molecules can initiate or inhibit germination in coat-damaged spores. The ability to directly measure the uptake of molecules in the context of spores with known structural or genetic deficiencies is expected to provide insight into the determinants of spores' extreme resistance.

Uptake and levels of the antibiotic berberine in individual dormant and germinating Clostridium difficile and Bacillus cereus spores as measured by laser tweezers Raman spectroscopy.

OBJECTIVES: Spores of Clostridium difficile and Bacillus cereus are major causes of nosocomial diarrhoea and foodborne disease. Our aim was to measure the dynamics of the uptake of the antibiotic berberine by individual germinating spores and the levels of berberine accumulated in germinated spores. METHODS: Laser tweezers Raman spectroscopy (LTRS) and differential interference contrast microscopy were used to measure levels of berberine accumulated in single germinating spores and to monitor berberine uptake and germination of individual C. difficile and B. cereus spores. RESULTS: MIC values of berberine for C. difficile and B. cereus spores were 640 and 256 mg/L, respectively. Levels of berberine accumulated at the berberine MICs in individual germinated spores were heterogeneous, with values of 17.1 ±â€Š5.4 and 12.7 ±â€Š5.5 g/L for C. difficile and B. cereus spores, respectively. These values were 25-50-fold higher than the MIC values. However, berberine did not affect the germination of C. difficile and B. cereus spores, but did block germinated spores' outgrowth. Berberine uptake kinetics were similar for these two kinds of spores. After the addition of germinants, berberine began to enter germinating spores at the time (Tlag) when rapid release of the spore core's large depot of the 1:1 chelate of Ca(2+) with dipicolinic acid began, and the level of berberine taken up was maximal shortly after spore cortex lysis was completed (Tlysis). CONCLUSIONS: LTRS can be used to measure uptake and levels of berberine in single cells. High levels of berberine can enter spores of C. difficile and B. cereus soon after germination is initiated, thus inhibiting spore outgrowth and minimizing hazards posed by germinated spores.

Effects of High-Pressure Treatment on Spores of Clostridium Species.

UNLABELLED: This work analyzes the high-pressure (HP) germination of spores of the food-borne pathogen Clostridium perfringens (with inner membrane [IM] germinant receptors [GRs]) and the opportunistic pathogen Clostridium difficile (with no IM GRs), which has growing implications as an emerging food safety threat. In contrast to those of spores of Bacillus species, mechanisms of HP germination of clostridial spores have not been well studied. HP treatments trigger Bacillus spore germination through spores' IM GRs at ∼150 MPa or through SpoVA channels for release of spores' dipicolinic acid (DPA) at ≥400 MPa, and DPA-less spores have lower wet heat resistance than dormant spores. We found that C. difficile spores exhibited no germination events upon 150-MPa treatment and were not heat sensitized. In contrast, 150-MPa-treated unactivated C. perfringens spores released DPA and became heat sensitive, although most spores did not complete germination by fully rehydrating the spore core, but this treatment of heat-activated spores led to almost complete germination and greater heat sensitization. Spores of both clostridial organisms released DPA during 550-MPa treatment, but C. difficile spores did not complete germination and remained heat resistant. Heat-activated 550-MPa-HP-treated C. perfringens spores germinated almost completely and became heat sensitive. However, unactivated 550-MPa-treated C. perfringens spores did not germinate completely and were less heat sensitive than spores that completed germination. Since C. difficile and C. perfringens spores use different mechanisms for sensing germinants, our results may allow refinement of HP methods for their inactivation in foods and other applications and may guide the development of commercially sterile low-acid foods. IMPORTANCE: Spores of various clostridial organisms cause human disease, sometimes due to food contamination by spores. Because of these spores' resistance to normal decontamination regimens, there is continued interest in ways to kill spores without compromising food quality. High hydrostatic pressure (HP) under appropriate conditions can inactivate bacterial spores. With growing use of HP for food pasteurization, advancement of HP for commercial production of sterile low-acid foods requires understanding of mechanisms of spores' interactions with HP. While much is known about HP germination and inactivation of spores of Bacillus species, how HP germinates and inactivates clostridial spores is less well understood. In this work we have tried to remedy this information deficit by examining germination of spores of Clostridium difficile and Clostridium perfringens by several HP and temperature levels. The results may give insight that could facilitate more efficient methods for spore eradication in food sterilization or pasteurization, biodecontamination, and health care.

The effects of heat activation on Bacillus spore germination, with nutrients or under high pressure, with or without various germination proteins.

Nutrient germination of spores of Bacillus species occurs through germinant receptors (GRs) in spores' inner membrane (IM) in a process stimulated by sublethal heat activation. Bacillus subtilis spores maximum germination rates via different GRs required different 75 °C heat activation times: 15 min for l-valine germination via the GerA GR and 4 h for germination with the L-asparagine-glucose-fructose-K(+) mixture via the GerB and GerK GRs, with GerK requiring the most heat activation. In some cases, optimal heat activation decreased nutrient concentrations for half-maximal germination rates. Germination of spores via various GRs by high pressure (HP) of 150 MPa exhibited heat activation requirements similar to those of nutrient germination, and the loss of the GerD protein, required for optimal GR function, did not eliminate heat activation requirements for maximal germination rates. These results are consistent with heat activation acting primarily on GRs. However, (i) heat activation had no effects on GR or GerD protein conformation, as probed by biotinylation by an external reagent; (ii) spores prepared at low and high temperatures that affect spores' IM properties exhibited large differences in heat activation requirements for nutrient germination; and (iii) spore germination by 550 MPa of HP was also affected by heat activation, but the effects were relatively GR independent. The last results are consistent with heat activation affecting spores' IM and only indirectly affecting GRs. The 150- and 550-MPa HP germinations of Bacillus amyloliquefaciens spores, a potential surrogate for Clostridium botulinum spores in HP treatments of foods, were also stimulated by heat activation.

Function of the SpoVAEa and SpoVAF proteins of Bacillus subtilis spores.

The Bacillus subtilis spoVAEa and spoVAF genes are expressed in developing spores as members of the spoVA operon, which encodes proteins essential for the uptake and release of dipicolinic acid (DPA) during spore formation and germination. SpoVAF is likely an integral inner spore membrane protein and exhibits sequence identity to A subunits of the spore's nutrient germinant receptors (GRs), while SpoVAEa is a soluble protein with no obvious signals to allow its passage across a membrane. However, like SpoVAD, SpoVAEa is present on the outer surface of the spore's inner membrane, as SpoVAEa was accessible to an external biotinylation agent in spores and SpoVAEa disappeared in parallel with SpoVAD during proteinase K treatment of germinated spores. SpoVAEa and SpoVAD were also distributed similarly in fractions of disrupted dormant spores. Unlike spoVAD, spoVAEa is absent from the genomes of some spore-forming members of the Bacillales and Clostridiales orders, although SpoVAEa's amino acid sequence is conserved in species containing spoVAEa. B. subtilis strains lacking SpoVAF or SpoVAEa and SpoVAF sporulated normally, and the spores had normal DPA levels. Spores lacking SpoVAF or SpoVAEa and SpoVAF also germinated normally with non-GR-dependent germinants but more slowly than wild-type spores with GR-dependent germinants, and this germination defect was complemented by ectopic expression of the missing proteins.

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.

Mobility of core water in Bacillus subtilis spores by 2H NMR.

Bacterial spores in a metabolically dormant state can survive long periods without nutrients under extreme environmental conditions. The molecular basis of spore dormancy is not well understood, but the distribution and physical state of water within the spore is thought to play an important role. Two scenarios have been proposed for the spore's core region, containing the DNA and most enzymes. In the gel scenario, the core is a structured macromolecular framework permeated by mobile water. In the glass scenario, the entire core, including the water, is an amorphous solid and the quenched molecular diffusion accounts for the spore's dormancy and thermal stability. Here, we use (2)H magnetic relaxation dispersion to selectively monitor water mobility in the core of Bacillus subtilis spores in the presence and absence of core Mn(2+) ions. We also report and analyze the solid-state (2)H NMR spectrum from these spores. Our NMR data clearly support the gel scenario with highly mobile core water (~25 ps average rotational correlation time). Furthermore, we find that the large depot of manganese in the core is nearly anhydrous, with merely 1.7% on average of the maximum sixfold water coordination.

Activity and regulation of various forms of CwlJ, SleB, and YpeB proteins in degrading cortex peptidoglycan of spores of Bacillus species in vitro and during spore germination.

Germination of Bacillus spores requires degradation of a modified layer of peptidoglycan (PG) termed the spore cortex by two redundant cortex-lytic enzymes (CLEs), CwlJ and SleB, plus SleB's partner protein, YpeB. In this study, in vitro and in vivo analyses have been used to clarify the roles of individual SleB and YpeB domains in PG degradation. Purified mature Bacillus cereus SleB without its signal sequence (SleB(M)) and the SleB C-terminal catalytic domain (SleB(C)) efficiently triggered germination of decoated Bacillus megaterium and Bacillus subtilis spores lacking endogenous CLEs; previously, SleB's N-terminal domain (SleB(N)) was shown to bind PG but have no enzymatic activity. YpeB lacking its putative membrane anchoring sequence (YpeB(M)) or its N- and C-terminal domains (YpeB(N) and YpeB(C)) alone did not exhibit degradative activity, but YpeB(N) inhibited SleB(M) and SleB(C) activity in vitro. The severe germination defect of B. subtilis cwlJ sleB or cwlJ sleB ypeB spores was complemented by ectopic expression of full-length sleB [sleB(FL)] and ypeB [ypeB(FL)], but normal levels of SleB(FL) in spores required normal spore levels of YpeB(FL) and vice versa. sleB(FL) or ypeB(FL) alone, sleB(FL) plus ypeB(C) or ypeB(N), and sleB(C) or sleB(N) plus ypeB(FL) did not complement the cortex degradation defect in cwlJ sleB ypeB spores. In addition, ectopic expression of sleB(FL) or cwlJ(FL) with a Glu-to-Gln mutation in a predicted active-site residue failed to restore the germination of cwlJ sleB spores, supporting the role of this invariant glutamate as the key catalytic residue in SleB and CwlJ.

Identification of new proteins that modulate the germination of spores of bacillus species.

A number of operons encoding the nutrient germinant receptors (GRs) in dormant spores of Bacillus megaterium and Bacillus subtilis species have small open reading frames (ORFs) of unknown function within or immediately adjacent to the operons. Inactivation of the genes in these ORFs, encoding proteins now termed D proteins, either significantly increased or decreased spore germination via the associated GR but had no effects on germination via non-GR-dependent germinants. These effects on GR-dependent germination were complemented by ectopic expression of the appropriate D gene (gene encoding D protein). However, substitution of noncognate D genes in two GR operons resulted in inhibition of germination via the GR manipulated, although ectopic overexpression of a D gene had no effect on overall GR-dependent germination. The various D genes studied were expressed in the forespore during sporulation in parallel with the associated GR operon, and transcription of a B. subtilis D gene was controlled by RNA polymerase sigma factor σ(G). These results indicate that proteins encoded by small ORFs within or adjacent to operons encoding GRs play major roles in modulating GR function in spores of Bacillus species. In B. subtilis, deletion of a D gene (B. subtilis gerKD [gerKDbs]) adjacent to the gerK operon encoding the GerK GR or ectopic expression or overexpression of gerKDbs had no major effect on the levels of GR subunits or of two other germination proteins.

Crystal structure of the catalytic domain of the Bacillus cereus SleB protein, important in cortex peptidoglycan degradation during spore germination.

The SleB protein is one of two redundant cortex-lytic enzymes (CLEs) that initiate the degradation of cortex peptidoglycan (PG), a process essential for germination of spores of Bacillus species, including Bacillus anthracis. SleB has been characterized as a soluble lytic transglycosylase that specifically recognizes spore cortex PG and catalyzes the cleavage of glycosidic bonds between N-acetylmuramic acid (NAM) and N-acetylglucosamine residues with concomitant formation of a 1,6-anhydro bond in the NAM residue. We found that like the full-length Bacillus cereus SleB, the catalytic C-terminal domain (SleB(C)) exhibited high degradative activity on cortex PG in vitro, although SleB's N-terminal domain, thought to bind PG, was inactive. The 1.85-Å crystal structure of SleB(C) reveals an ellipsoid molecule with two distinct domains dominated by either α helices or ß strands. The overall fold of SleB closely resembles that of the catalytic domain of the family 1 lytic transglycosylases but with a completely different topological arrangement. Structural analysis shows that an invariant Glu157 of SleB is in a position equivalent to that of the catalytic glutamate in other lytic transglycosylases. Indeed, SleB bearing a Glu157-to-Gln mutation lost its cortex degradative activity completely. In addition, the other redundant CLE (called CwlJ) in Bacillus species likely has a three-dimensional structure similar to that of SleB, including the invariant putative catalytic Glu residue. SleB and CwlJ may offer novel targets for the development of anti-spore agents.

Role of a SpoVA protein in dipicolinic acid uptake into developing spores of Bacillus subtilis.

The proteins encoded by the spoVA operon, including SpoVAD, are essential for the uptake of the 1:1 chelate of pyridine-2,6-dicarboxylic acid (DPA(2,6)) and Ca(2+) into developing spores of the bacterium Bacillus subtilis. The crystal structure of B. subtilis SpoVAD has been determined recently, and a structural homology search revealed that SpoVAD shares significant structural similarity but not sequence homology to a group of enzymes that bind to and/or act on small aromatic molecules. We find that molecular docking placed DPA(2,6) exclusively in a highly conserved potential substrate-binding pocket in SpoVAD that is similar to that in the structurally homologous enzymes. We further demonstrate that SpoVAD binds both DPA(2,6) and Ca(2+)-DPA(2,6) with a similar affinity, while exhibiting markedly weaker binding to other DPA isomers. Importantly, mutations of conserved amino acid residues in the putative DPA(2,6)-binding pocket in SpoVAD essentially abolish its DPA(2,6)-binding capacity. Moreover, replacement of the wild-type spoVAD gene in B. subtilis with any of these spoVAD gene variants effectively eliminated DPA(2,6) uptake into developing spores in sporulation, although the variant proteins were still located in the spore inner membrane. Our results provide direct evidence that SpoVA proteins, in particular SpoVAD, are directly involved in DPA(2,6) movement into developing B. subtilis spores.

Effects of the SpoVT regulatory protein on the germination and germination protein levels of spores of Bacillus subtilis.

Bacillus subtilis isolates lacking the SpoVT protein, which regulates gene expression in developing forespores, gave spores that released their dipicolinic acid (DPA) via germinant receptor (GR)-dependent germination more rapidly than wild-type spores. Non-GR-dependent germination via dodecylamine was more rapid with spoVT spores, but germination via Ca-DPA was slower. The effects of a spoVT mutation on spore germination were seen with spores made in rich and poor media, and levels of SpoVT-LacZ were elevated 2-fold in poor-medium spores; however, elevated SpoVT levels were not the only cause of the slower GR-dependent germination of poor-medium spores. The spoVT spores had ≥5-fold higher GerA GR levels, ∼2-fold elevated GerB GR levels, wild-type levels of a GerK GR subunit and the GerD protein required for normal GR-dependent germination, ∼2.5-fold lower levels of the SpoVAD protein involved in DPA release in spore germination, and 30% lower levels of DNA protective α/ß-type small, acid-soluble spore proteins. With one exception, the effects on protein levels in spoVT spores are consistent with the effects of SpoVT on forespore transcription. The spoVT spores were also more sensitive to UV radiation and outgrew slowly. While spoVT spores' elevated GR levels were consistent with their more rapid GR-dependent germination, detailed analysis of the results suggested that there is another gene product crucial for GR-dependent spore germination that is upregulated in the absence of SpoVT. Overall, these results indicate that SpoVT levels during spore formation have a major impact on the germination and the resistance of the resultant spores.

Extremely variable conservation of γ-type small, acid-soluble proteins from spores of some species in the bacterial order Bacillales.

γ-Type small, acid-soluble spore proteins (SASP) are the most abundant proteins in spores of at least some members of the bacterial order Bacillales, yet they remain an enigma from both functional and phylogenetic perspectives. Current work has shown that the γ-type SASP or their coding genes (sspE genes) are present in most spore-forming members of Bacillales, including at least some members of the Paenibacillus genus, although they are apparently absent from Clostridiales species. We have applied a new method of searching for sspE genes, which now appear to also be absent from a clade of Bacillales species that includes Alicyclobacillus acidocaldarius and Bacillus tusciae. In addition, no γ-type SASP were found in A. acidocaldarius spores, although several of the DNA-binding α/ß-type SASP were present. These findings have elucidated the phylogenetic origin of the sspE gene, and this may help in determining the precise function of γ-type SASP.

Maturation of released spores is necessary for acquisition of full spore heat resistance during Bacillus subtilis sporulation.

The first ∼10% of spores released from sporangia (early spores) during Bacillus subtilis sporulation were isolated, and their properties were compared to those of the total spores produced from the same culture. The early spores had significantly lower resistance to wet heat and hypochlorite than the total spores but identical resistance to dry heat and UV radiation. Early and total spores also had the same levels of core water, dipicolinic acid, and Ca and germinated similarly with several nutrient germinants. The wet heat resistance of the early spores could be increased to that of total spores if early spores were incubated in conditioned sporulation medium for ∼24 h at 37°C (maturation), and some hypochlorite resistance was also restored. The maturation of early spores took place in pH 8 buffer with Ca(2+) but was blocked by EDTA; maturation was also seen with early spores of strains lacking the CotE protein or the coat-associated transglutaminase, both of which are needed for normal coat structure. Nonetheless, it appears to be most likely that it is changes in coat structure that are responsible for the increased resistance to wet heat and hypochlorite upon early spore maturation.

Levels and localization of mechanosensitive channel proteins in Bacillus subtilis.

Log phase Bacillus subtilis cells lacking the mscL gene encoding the mechanosensitive (MS) channel of large conductance are sensitive to an osmotic downshock > or =0.5 M. However, B. subtilis mscL cells develop osmotic downshock resistance in late log and early stationary phase growth that is partially dependent on three likely MS channel proteins of small conductance (MscS), YfkC, YhdY, and YkuT. Bacillus subtilis MS proteins were fused with green fluorescent protein (GFP) at their C termini; at least the MscL-, YfkC-, and YkuT-GFP fusions were functional and overexpression of YkuT-GFP, or YkuT alone abolished log phase mscL cells' osmotic downshock sensitivity. Western blot analysis found high levels of MscL-GFP in early exponential phase cells with levels subsequently decreasing greatly. MscS-GFP proteins were present in exponential phase cells, but again disappeared almost completely in stationary phase cells and these proteins were not detected in spores. Western blot analyses further showed that MS-GFP proteins were associated with the plasma membrane, as expected. Fluorescence microscopy confirmed the localization of MscL-GFP and YhdY-GFP to the plasma membrane, with non-uniform distribution of these proteins along this membrane consistent with but by no means proving that these proteins are present in a helical array.

Release of small molecules during germination of spores of Bacillus Species.

Free amino acids, dipicolinic acid, and unidentified small molecules were released early in Bacillus spore germination before hydrolysis of the peptidoglycan cortex, but adenine nucleotides and 3-phosphoglycerate were not. These results indicate that early in germination there is a major selective change in the permeability of the spore's inner membrane.

Characterization of Clostridium perfringens spores that lack SpoVA proteins and dipicolinic acid.

Spores of Clostridium perfringens possess high heat resistance, and when these spores germinate and return to active growth, they can cause gastrointestinal disease. Work with Bacillus subtilis has shown that the spore's dipicolinic acid (DPA) level can markedly influence both spore germination and resistance and that the proteins encoded by the spoVA operon are essential for DPA uptake by the developing spore during sporulation. We now find that proteins encoded by the spoVA operon are also essential for the uptake of Ca(2+) and DPA into the developing spore during C. perfringens sporulation. Spores of a spoVA mutant had little, if any, Ca(2+) and DPA, and their core water content was approximately twofold higher than that of wild-type spores. These DPA-less spores did not germinate spontaneously, as DPA-less B. subtilis spores do. Indeed, wild-type and spoVA C. perfringens spores germinated similarly with a mixture of l-asparagine and KCl (AK), KCl alone, or a 1:1 chelate of Ca(2+) and DPA (Ca-DPA). However, the viability of C. perfringens spoVA spores was 20-fold lower than the viability of wild-type spores. Decoated wild-type and spoVA spores exhibited little, if any, germination with AK, KCl, or exogenous Ca-DPA, and their colony-forming efficiency was 10(3)- to 10(4)-fold lower than that of intact spores. However, lysozyme treatment rescued these decoated spores. Although the levels of DNA-protective alpha/beta-type, small, acid-soluble spore proteins in spoVA spores were similar to those in wild-type spores, spoVA spores exhibited markedly lower resistance to moist heat, formaldehyde, HCl, hydrogen peroxide, nitrous acid, and UV radiation than wild-type spores did. In sum, these results suggest the following. (i) SpoVA proteins are essential for Ca-DPA uptake by developing spores during C. perfringens sporulation. (ii) SpoVA proteins and Ca-DPA release are not required for C. perfringens spore germination. (iii) A low spore core water content is essential for full resistance of C. perfringens spores to moist heat, UV radiation, and chemicals.

Characterization of spores of Bacillus subtilis that lack most coat layers.

Spores of Bacillus subtilis have a thick outer layer of relatively insoluble protein called the coat, which protects spores against a number of treatments and may also play roles in spore germination. However, elucidation of precise roles of the coat in spore properties has been hampered by the inability to prepare spores lacking all or most coat material. In this work, we show that spores of a strain with mutations in both the cotE and gerE genes, which encode proteins involved in coat assembly and expression of genes encoding coat proteins, respectively, lack most extractable coat protein as seen by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, as well as the great majority of the coat as seen by atomic force microscopy. However, the cotE gerE spores did retain a thin layer of insoluble coat material that was most easily seen by microscopy following digestion of these spores with lysozyme. These severely coat-deficient spores germinated relatively normally with nutrients and even better with dodecylamine but not with a 1:1 chelate of Ca(2+) and dipicolinic acid. These spores were also quite resistant to wet heat, to mechanical disruption, and to treatment with detergents at an elevated temperature and pH but were exquisitely sensitive to killing by sodium hypochlorite. These results provide new insight into the role of the coat layer in spore properties.