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 which lack dipicolinic acid

Spores of Bacillus subtilis with a mutation in spoVF cannot synthesize dipicolinic acid (DPA) and are too unstable to be purified and studied in detail. However, the spores of a strain lacking the three major germinant receptors (termed Deltager3), as well as spoVF, can be isolated, although they spontaneously germinate much more readily than Deltager3 spores. The Deltager3 spoVF spores lack DPA and have higher levels of core water than Deltager3 spores, although sporulation with DPA restores close to normal levels of DPA and core water to Deltager3 spoVF spores. The DPA-less spores have normal cortical and coat layers, as observed with an electron microscope, but their core region appears to be more hydrated than that of spores with DPA. The Deltager3 spoVF spores also contain minimal levels of the processed active form (termed P(41)) of the germination protease, GPR, a finding consistent with the known requirement for DPA and dehydration for GPR autoprocessing. However, any P(41) formed in Deltager3 spoVF spores may be at least transiently active on one of this protease's small acid-soluble spore protein (SASP) substrates, SASP-gamma. Analysis of the resistance of wild-type, Deltager3, and Deltager3 spoVF spores to various agents led to the following conclusions: (i) DPA and core water content play no role in spore resistance to dry heat, dessication, or glutaraldehyde; (ii) an elevated core water content is associated with decreased spore resistance to wet heat, hydrogen peroxide, formaldehyde, and the iodine-based disinfectant Betadine; (iii) the absence of DPA increases spore resistance to UV radiation; and (iv) wild-type spores are more resistant than Deltager3 spores to Betadine and glutaraldehyde. These results are discussed in view of current models of spore resistance and spore germination.     

Role of dipicolinic acid in resistance and stability of spores of Bacillus subtilis with or without DNA-protective alpha/beta-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.     

Role of dipicolinic acid in the germination, stability, and viability of spores of Bacillus subtilis

Spores of Bacillus subtilis spoVF strains that cannot synthesize dipicolinic acid (DPA) but take it up during sporulation were prepared in medium with various DPA concentrations, and the germination and viability of these spores as well as the DPA content in individual spores were measured. Levels of some other small molecules in DPA-less spores were also measured. These studies have allowed the following conclusions. (i) Spores with no DPA or low DPA levels that lack either the cortex-lytic enzyme (CLE) SleB or the receptors that respond to nutrient germinants could be isolated but were unstable and spontaneously initiated early steps in spore germination. (ii) Spores that lacked SleB and nutrient germinant receptors and also had low DPA levels were more stable. (iii) Spontaneous germination of spores with no DPA or low DPA levels was at least in part via activation of SleB. (iv) The other redundant CLE, CwlJ, was activated only by the release of high levels of DPA from spores. (v) Low levels of DPA were sufficient for the viability of spores that lacked most alpha/beta-type small, acid-soluble spore proteins. (vi) DPA levels accumulated in spores prepared in low-DPA-containing media varied greatly between individual spores, in contrast to the presence of more homogeneous DPA levels in individual spores made in media with high DPA concentrations. (vii) At least the great majority of spores of several spoVF strains that contained no DPA also lacked other major spore small molecules and had gone through some of the early reactions in spore germination.     

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.     

Studies of the release of small molecules during pressure germination of spores of Bacillus subtilis

AIMS: To measure rates of release of small molecules during pressure germination of Bacillus subtilis spores, and the role of SpoVA proteins in dipicolinic acid (DPA) release. METHODS AND RESULTS: Rates of DPA release during B. subtilis spore germination with pressures of 150 or 500 megaPascals were much higher in spores with elevated levels of SpoVA proteins, and spores with a temperature-sensitive mutation in the spoVA operon were temperature-sensitive in DPA release during pressure germination. Spores also released arginine and glutamic acid, but not AMP, during pressure germination. CONCLUSIONS: Pressure germination of B. subtilis spores causes release of many small molecules including DPA. SpoVA proteins are involved in the release of DPA, perhaps because SpoVA proteins are a component of a DPA channel in the spore's inner membrane. SIGNIFICANCE AND IMPACT OF THE STUDY: This work provides new insight into the mechanism of pressure germination of spores of Bacillus species, a process that has significant potential for usage in the food industry.     

EtfA catalyses the formation of dipicolinic acid in Clostridium perfringens

Dipicolinic acid (DPA) is a major component of bacterial endospores, comprising 5–15% of the spore dry weight, and is important for spore stability and resistance properties. The biosynthetic precursor to DPA, dihydro-dipicolinic acid (DHDPA), is produced by DHDPA synthase within the lysine biosynthesis pathway. In Bacillus subtilis , and most other bacilli and clostridia, DHDPA is oxidized to DPA by the products of the spoVF operon. Analysis of the genomes of the clostridia in Cluster I, including the pathogens Clostridium perfringens , Clostridium botulinum and Clostridium tetani , has shown that no spoVF orthologues exist in these organisms. DPA synthase was purified from extracts of sporulating C. perfringens cells. Peptide sequencing identified an electron transfer flavoprotein, EtfA, in this purified protein fraction. A C. perfringens strain with etfA inactivated is blocked in late stage sporulation and produces ≤ 11% of wild-type DPA levels. C. perfringens EtfA was expressed in and purified from Escherichia coli , and this protein catalysed DPA formation in vitro . The sequential production of DHDPA and DPA in C. perfringens appears to be catalysed by DHDPA synthase followed by EtfA. Genome sequence data and the taxonomy of spore-forming species suggest that this may be the ancestral mechanism for DPA synthesis.     

Germination of individual Bacillus subtilis spores with alterations in the GerD and SpoVA proteins, which are important in spore germination

Release of Ca(2+) with dipicolinic acid (CaDPA) was monitored by Raman spectroscopy and differential interference contrast microscopy during germination of individual spores of Bacillus subtilis strains with alterations in GerD and SpoVA proteins. Notable conclusions about germination after the addition of nutrient were as follows. (i) Following L-alanine addition, wild-type and gerD spores and spores with elevated SpoVA protein levels (↑SpoVA spores) slowly released ～10% of their CaDPA during a variable (6- to 55-min) period ending at T(lag), the time when faster CaDPA release began. (ii) T(lag) times were lower for ↑SpoVA spores than for wild-type spores and were higher for gerD spores. (iii) The long T(lag) times of gerD spores were partially due to slow commitment to germinate. (iv) The intervals between the commitment to germinate and CaDPA release were similar for wild-type and ↑SpoVA spores but longer for gerD spores. (v) The times for rapid CaDPA release, ΔT(release) = T(release) - T(lag) (with T(release) being the time at which CaDPA release was complete), were similar for wild-type, gerD, and ↑SpoVA spores. (vi) Spores with either one of two point mutations in the spoVA operon (spoVA(1) and spoVA(2) spores) exhibited a more rapid rate of CaDPA release beginning immediately after L-alanine addition leading to ～65% CaDPA release prior to T(lag). (vii) T(lag) times for spoVA(1) and spoVA(2) spores were longer than for wild-type spores. (viii) The intervals between spoVA(1) and spoVA(2) spores' commitment and CaDPA release were similar to those for wild-type spores, but commitment occurred later. In contrast to germination after the addition of nutrient, T(lag) and ΔT(release) times were relatively similar during dodecylamine germination of spores of the five strains. These findings suggest the following. (i) GerD plays no role in CaDPA release during spore germination. (ii) SpoVA proteins are involved in CaDPA release during germination with nutrients, and probably with dodecylamine. (iii) Spores release significant CaDPA before commitment. (iv) CaDPA release during T(lag) and ΔT(release) may signal subsequent germination events.     

Role of SpoVA proteins in release of dipicolinic acid during germination of Bacillus subtilis spores triggered by dodecylamine or lysozyme

The release of dipicolinic acid (DPA) during the germination of Bacillus subtilis spores by the cationic surfactant dodecylamine exhibited a pH optimum of approximately 9 and a temperature optimum of 60 degrees C. DPA release during dodecylamine germination of B. subtilis spores with fourfold-elevated levels of the SpoVA proteins that have been suggested to be involved in the release of DPA during nutrient germination was about fourfold faster than DPA release during dodecylamine germination of wild-type spores and was inhibited by HgCl(2). Spores carrying temperature-sensitive mutants in the spoVA operon were also temperature sensitive in DPA release during dodecylamine germination as well as in lysozyme germination of decoated spores. In addition to DPA, dodecylamine triggered the release of amounts of Ca(2+) almost equivalent to those of DPA, and at least one other abundant spore small molecule, glutamic acid, was released in parallel with Ca(2+) and DPA. These data indicate that (i) dodecylamine triggers spore germination by opening a channel in the inner membrane for Ca(2+)-DPA and other small molecules, (ii) this channel is composed at least in part of proteins, and (iii) SpoVA proteins are involved in the release of Ca(2+)-DPA and other small molecules during spore germination, perhaps by being a part of a channel in the spore's inner membrane.     

Use of a lacZ gene fusion to determine the dependence pattern and the spore compartment expression of sporulation operon spoVA in spo mutants of Bacillus subtilis

A spoVAA::lacZ gene fusion has been used to study expression of the spoVA operon during sporulation in Bacillus subtilis. beta-Galactosidase activity, encoded by the fusion gene, begins to be produced about 2.5 h after the induction of sporulation, well before the phenotypic consequences of spoVA mutations are manifested. spoVA expression is dependent on all of the known spo0 and spoII loci and on some of the 'early' spoIII loci, but not on 'later' loci. Several lines of evidence suggest that spoVA expression occurs only in the spore compartment. The implications of this observation for models of the overall regulation of gene expression during sporulation are discussed.     

Nucleotide sequence and complementation analysis of a polycistronic sporulation operon, spoVA, in Bacillus subtilis

We have determined the nucleotide sequence of a 3706 bp stretch of Bacillus subtilis chromosomal DNA that complements all known spoVA mutations. The sequence contains five consecutive large open reading frames capable of encoding proteins of molecular weights ranging from approximately 15000 to 36000. Analysis using integrational plasmids suggests that the region is likely to be transcribed as a single mRNA. A novel form of complementation analysis, based on derivatives of bacteriophage phi 105 carrying the cloned spoVA locus, has been used to define four distinct complementation groups among the eight previously characterized spoVA mutations. The spoVA locus is the largest polycistronic sporulation operon yet characterized.     

Localization of SpoVAD to the inner membrane of spores of Bacillus subtilis

The products of the hexacistronic spoVA operon of Bacillus subtilis may be involved in the transport of dipicolinic acid into the forespore during sporulation and its release during spore germination. The major hydrophilic coding region of B. subtilis spoVAD was cloned, the protein was expressed in Escherichia coli as a His tag fusion protein, and a rabbit antiserum was raised against the purified protein. Western blot analyses of fractions from B. subtilis spores showed that SpoVAD is an integral inner membrane protein present at levels >50-fold higher than those of the spore's nutrient germinant receptors that are also present in the inner membrane. SpoVAD also persisted in outgrowing spores.     

Synthesis of spoIIA and spoVA mRNA in Bacillus subtilis

The expression of the spoIIA and spoVA sporulation loci of Bacillus subtilis was examined by using DNA-RNA hybridization to detect the time of appearance of their corresponding mRNA molecules in wild-type and asporogenous mutants of B. subtilis. From the size of the mRNA molecules it is clear that both the spoIIA and spoVA loci are polycistronic operons. Neither of the mRNA molecules is polyadenylated. The results also indicate the spoIIA operon is regulated by two promoters which become functional at different times.     

Properties of Bacillus anthracis spores prepared under various environmental conditions

Bacillus anthracis makes highly stable, heat-resistant spores which remain viable for decades. Effect of various stress conditions on sporulation in B. anthracis was studied in nutrient-deprived and sporulation medium adjusted to various pH and temperatures. The results revealed that sporulation efficiency was dependent on conditions prevailing during sporulation. Sporulation occurred earlier in culture sporulating at alkaline pH or in PBS than control. Spores formed in PBS were highly sensitive towards spore denaturants whereas, those formed at 45°C were highly resistant. The decimal reduction time (D-10 time) of the spores formed at 45°C by wet heat, 2 M HCl, 2 M NaOH and 2 M H₂O₂ was higher than the respective D-10 time for the spores formed in PBS. The dipicolinic acid (DPA) content and germination efficiency was highest in spores formed at 45°C. Since DPA is related to spore sensitivity towards heat and chemicals, the increased DPA content of spores prepared at 45°C may be responsible for increased resistance to wet heat and other denaturants. The size of spores formed at 45°C was smallest amongst all. The study reveals that temperature, pH and nutrient availability during sporulation affect properties of B. anthracis spores.     

Characterization of the germination of Bacillus megaterium spores lacking enzymes that degrade the spore cortex

Aims:  To determine roles of cortex lytic enzymes (CLEs) in Bacillus megaterium spore germination.
Methods and Results:  Genes for B. megaterium CLEs CwlJ and SleB were inactivated and effects of loss of one or both on germination were assessed. Loss of CwlJ or SleB did not prevent completion of germination with agents that activate the spore's germinant receptors, but loss of CwlJ slowed the release of dipicolinic acid (DPA). Loss of both CLEs also did not prevent release of DPA and glutamate during germination with KBr. However, cwlJ sleB spores had decreased viability, and could not complete germination. Loss of CwlJ eliminated spore germination with Ca²⁺ chelated to DPA (Ca-DPA), but loss of CwlJ and SleB did not affect DPA release in dodecylamine germination.
Conclusions:  CwlJ and SleB play redundant roles in cortex degradation during B. megaterium spore germination, and CwlJ accelerates DPA release and is essential for Ca-DPA germination. The roles of these CLEs are similar in germination of B. megaterium and Bacillus subtilis spores.
Significance and Impact of the Study:  These results indicate that redundant roles of CwlJ and SleB in cortex degradation during germination are similar in spores of Bacillus species; consequently, inhibition of these enzymes will prevent germination of Bacillus spores.