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 $~$ 10% of the dry weight of sporesof Bacillus species. Although DPA has long been implicated inspore resistance to wet heat and spore stability, definitiveevidence on the role of this abundant molecule in spore propertieshas generally been lacking. Bacillus subtilis strain FB122 (sleBspoVF) produced very stable spores that lacked DPA, and sporulationof this strain with DPA yielded spores with nearly normal DPAlevels. DPA-replete and DPA-less FB122 spores had similar levelsof the DNA protective ${alpha}$ /ß-type small acid-soluble sporeproteins (SASP), but the DPA-less spores lacked SASP- ${gamma}$ . The DPA-lessFB122 spores exhibited similar UV resistance to the DPA-repletespores but had lower resistance to wet heat, dry heat, hydrogenperoxide, and desiccation. Neither wet heat nor hydrogen peroxidekilled the DPA-less spores by DNA damage, but desiccation did.The inability to synthesize both DPA and most ${alpha}$ /ß-typeSASP in strain PS3664 (sspA sspB sleB spoVF) resulted in sporesthat lost viability during sporulation, at least in part dueto DNA damage. DPA-less PS3664 spores were more sensitive towet heat than either DPA-less FB122 spores or DPA-replete PS3664spores, and the latter also retained viability during sporulation.These and previous results indicate that, in addition to ${alpha}$ /ß-typeSASP, DPA also is extremely important in spore resistance andstability and, further, that DPA has some specific role(s) inprotecting spore DNA from damage. Specific roles for DPA inprotecting spore DNA against damage may well have been a majordriving force for the spore's accumulation of the high levelsof this small molecule.

INTRODUCTION

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Introduction

Spores of Bacillus species are dormant and extremely resistantto many environmental stresses including, heat, desiccation,radiation, and a variety of toxic chemicals (9, 17, 33). Asa consequence, spores can survive for extremely long periods,certainly hundreds of years and perhaps much longer (5, 15,37). Spore resistance is due to a variety of factors, includingthe outer spore coats and the relative impermeability of thespore's inner membrane (6, 9, 17). There are also novel featuresof the spore's central region or core, the site of spore DNA,which play major roles in spore resistance. These include therelatively low content of core water and the saturation of sporeDNA with a group of small acid-soluble proteins (SASP) of the ${alpha}$ /ß type (9, 11, 23, 31). The low core water protectsspores against wet heat, while the ${alpha}$ /ß-type SASP protectDNA against damage due to wet and dry heat, desiccation, andgenotoxic chemicals (6, 17, 31, 33). The binding of the ${alpha}$ /ß-typeSASP also drastically alters the DNA's UV photochemistry, andthis change is a major factor in the elevated resistance ofdormant spores to UV light (17, 18).

Another novel feature of the spore core is the presence of highlevels ( $~$ 20% of core dry weight) of pyridine-2,6-dicarboxylicacid (dipicolinic acid [DPA]), that likely exists in the coreas a 1:1 chelate with divalent cations, predominantly Ca²⁺ (11).One role of DPA is to lower the core water content, since DPAloss early in germination is paralleled by its replacement withwater, and genetically DPA-less dormant spores have more corewater than wild-type dormant spores (10, 21, 32). The role ofDPA in spore properties has long been unclear. There are dataindicating that increasing spore DPA levels are associated withincreased spore wet heat resistance (11, 21). However, mutantshave been isolated that produce heat-resistant but DPA-lessspores (4, 12). Unfortunately, there has never been a thoroughgenetic analysis of these strains, and they may well have hadmultiple mutations.

DPA-less spores of B. subtilis can also be generated by mutationof the spoVF operon that encodes DPA synthetase (7, 11, 21).However, the DPA-less spoVF spores are unstable and lyse duringsporulation (21, 36). In contrast, when sporulated with exogenousDPA, spoVF spores accumulate near wild-type DPA levels and arestable (21, 36). DPA-less spoVF spores can also be stabilizedsomewhat, albeit not completely, by deleting the genes encodingthe three receptors that trigger spore germination in responseto nutrient germinants (termed the ger3 mutations) (21). DPA-lessand DPA-replete ger3 spoVF spores have relatively similar resistanceto UV radiation, but the DPA-less spores are more sensitiveto wet heat and hydrogen peroxide (21).

Although the latter differences in resistance between sporeswith or without DPA are striking, it is not clear whether thesedifferences are due only to the higher core water content ofthe DPA-less spores or to specific effects of DPA on spore resistance.Indeed, DPA can have striking effects on spore properties, inparticular spore DNA properties (8, 26). In particular, UV irradiationof spores lacking DPA is much less efficient in generating thethyminyl-thymine adduct termed the spore photoproduct in DNAthan is UV irradiation of spores containing normal DPA levels(8). This difference is even larger in spores with or withoutDPA that also lack ${alpha}$ /ß-type SASP (8). These latterstudies were carried out with spores of a strain lacking bothspoVF and sleB. The latter gene encodes one of the two redundantenzymes responsible for the hydrolysis of the spore's peptidoglycancortex in the first min of germination (32). The second cortex-lyticenzyme, CwlJ, is present in sleB spoVF spores. However, CwlJrequires DPA for its activation (20). Consequently, DPA-lesssleB spoVF spores do not germinate either spontaneously or withnutrients and are much more stable than ger3 spoVF spores (20,21). However, DPA-less sleB spoVF spores can be germinated eitherby exogenous Ca²⁺-DPA or by lysozyme treatment of decoated sporesin a hypertonic medium (20, 21).

Given the extreme stability of the sleB spoVF spores that lackDPA, we have reexamined the role of DPA in the resistance ofthese spores. We have also examined the survival and resistanceof spores that lack both DPA and ${alpha}$ /ß-type SASP. Wereport here the results of these studies.

MATERIALS AND METHODS

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Materials and Methods

Strains used. All strains used are isogenic with PS832, a prototrophic laboratorystrain of B. subtilis 168 and are listed in Table 1. StrainPS3664 (8) lacks the sspA and sspB genes which encode SASP- ${alpha}$ and -ß that together constitute $~$ 80% of the spore'spool of ${alpha}$ /ß-type SASP; consequently, PS3664 sporesare termed ${alpha}$ ^–ß^–. Strains PS3664 and FB122also lack the spoVF operon that encodes DPA synthetase, andthe DPA-less spores produced by these strains are stabilizedby the loss of the sleB gene that encodes one of the spore'scortex lytic enzymes (8, 20). Strains made in current studywere constructed by transformation to appropriate drug resistancewith chromosomal DNA (1). Transformants were screened to ensurethat they retained all antibiotic markers and, in the case ofstrains PS3747 and PS3755, that the spores of these strainslacked SASP- ${alpha}$ and -ß.

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TABLE 1. B. subtilis strains used

Spore formation. Spores of FB122 were routinely prepared by growth at 37°Con 2xSG medium agar plates (19) without antibiotics for 72 h.The plates were then incubated at 23°C for 3 to 4 days,and spores were harvested, cleaned over 10 to 14 days at 4°C,and stored as described previously (19). In some experimentsstrain FB122 was sporulated on 2xSG medium plates containing200 µg of DPA/ml. FB122 spores made without added DPAhad <2% of the DPA of wild-type spore DPA levels, whereasspores made with DPA had $~$ 85% of wild-type spore DNA levels,as found previously (20, 21). Spores of strain FB112 used inthis study were free (>98%) of growing or sporulating cells,germinated spores, or cell debris as observed in a phase-contrastmicroscope. In one experiment FB122 spores were prepared at37°C in liquid 2xSG medium (19) without DPA.

Spores of strain PS3664 were initially prepared on plates withor without DPA as described above. However, while spores ofthis strain made with DPA were fully viable after cleaning,the DPA-less spores were not (see Results). Consequently, infurther work this strain was grown at 37°C in liquid 2xSGmedium (19) with or without DPA. At various times in sporulation1-ml aliquots were harvested by centrifugation in a microcentrifugeand treated with decoating solution at an optical density at600 nm (OD₆₀₀) of $~$ 5 (see below). The decoated preparation waswashed, suspended in hypertonic medium at an OD₆₀₀ of 1, andgerminated with lysozyme, and viable counts were determinedas described below. In other experiments, washed decoated preparationsof sporulating cells of various strains were subjected to wetheat treatment at an OD₆₀₀ of 2, and viable counts were determinedas described below.

Measurement of spore resistance. Spores of strain FB122 or PS3664 with or without DPA were decoatedand washed (3) prior to measurement of resistance to heat andoxidizing agents to allow spore germination by lysozyme treatmentin a hypertonic medium. This method was chosen for spore germinationrather than germination with Ca²⁺-DPA, since CwlJ, the enzymeactivated by Ca²⁺-DPA, is relatively sensitive to treatmentssuch as wet heat and oxidizing agents, more sensitive than isspore viability (2, 39, 40). The decoating procedure was asdescribed previously (3) but at 45°C for 60 min. The sporesat an OD₆₀₀ of 1 to 20 in hypertonic medium were germinatedwith lysozyme (22), diluted serially in hypertonic medium, andaliquots were spotted onto LB medium plates (21) containingan appropriate antibiotic, generally spectinomycin at 100 µg/ml.The plates were incubated for $~$ 36 h at 37°C, and colonieswere counted, although generally >90% of colonies appearedin 24 h. Decoating has no significant effect on B. subtilisspore resistance to wet or dry heat or UV radiation but doesdecrease spore resistance to hydrogen peroxide slightly (17,18, 24).

For wet heat resistance, decoated spores at an OD₆₀₀ of 2 wereincubated in water at various temperatures. At various timessamples (0.5 ml) were diluted with 0.5 ml of cold water, centrifuged,and suspended in 1 ml of hypertonic medium. For UV resistance,decoated spores at an OD₆₀₀ of 1 in water were irradiated insmall petri dishes rotating beneath a UVS-11 lamp (UVP, Inc.,San Gabriel, CA) with a maximum output at 254 nm. At varioustimes aliquots (1 ml) were centrifuged and suspended in 1 mlof hypertonic medium. For hydrogen peroxide resistance, sporesat an OD₆₀₀ of 1 were incubated at 23°C in 25 mM KPO₄ buffer(pH 7.4) with 1.5% H₂O₂. At various times samples (1 ml) werecentrifuged (1 min) and rapidly suspended in 1 ml of hypertonicmedium containing catalase (40 µg/ml) as described previously(21, 27). For dry heat resistance, decoated spores (200 µlof a suspension with an OD₆₀₀ of 10) were lyophilized in glasstubes and exposed to dry heat in an oil bath. After heating,the tubes were cooled on ice, 1 ml of hypertonic medium added,and the suspension sonicated in a bath sonicator to dispersethe spores, followed by lysozyme treatment.

For desiccation resistance, 2 ml of intact spores at an OD₆₀₀of 10 were pelleted in a 2-ml plastic centrifuge tube and freeze-driedovernight. The dried spores were hydrated in 2 ml of water ina cold room at 4°C overnight, and the OD₆₀₀ of the suspensionmeasured to assess spore recovery. An aliquot of the sporeswas diluted 1/10 in 50 mM Ca²⁺-DPA at pH 8.3 and incubated for2 h at 23°C (20), and viability was assessed by platingas described above. The centrifugation, freeze-drying, and hydrationsteps were then repeated.

Analytical procedures. Extraction of SASP from spores and their analysis by polyacrylamidegel electrophoresis at low pH was as described previously (21).Spore core water content was determined on decoated spores byisopycnic density gradient centrifugation as described previously(11). The level of mutations in spores of different strainswith or without various treatments was determined by transferringcolonies of either untreated or treated spore preparations obtainedfrom LB medium plates to either 2xSG medium plates or Spizizenminimal medium plates without Casamino Acids as described previously(10, 34). A lack of growth on the minimal medium plates indicatesan auxotrophic mutation, while the colonies on 2xSG medium plateswere examined for sporulation.

RESULTS

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Results

Characterization of DPA-less sleB spoVF spores. Previous work on DPA-less spores (i.e., spoVF spores) of Bacillusspecies has shown that such spores lyse readily and are thusextremely difficult if not impossible to work with (21, 36).Deletion of the genes encoding the three functional nutrientgerminant receptors (termed the ger3 mutations) stabilizes spoVFspores to some degree, such that they can be isolated and characterized(21). However, even these ger3 spoVF spores germinate spontaneouslymuch more readily than wild-type spores (21). In contrast tothe ger3 mutations, a sleB mutation stabilizes spoVF sporescompletely, since sleB spoVF spores exhibit no spontaneous germination(20; data not shown). Indeed, these spores even germinate extremelypoorly with nutrients, because the remaining spore cortex-lyticenzyme, CwlJ, requires DPA for its activation (20). However,DPA-less sleB spoVF spores do germinate with exogenous Ca²⁺-DPA,and decoated sleB spoVF spores can also be germinated by lysozymetreatment in a hypertonic medium (20).

Previous work found that DPA-less ger3 spoVF spores had verylow levels of SASP- ${gamma}$ compared to SASP- ${gamma}$ levels in wild-type orDPA-replete ger3 spoVF spores (21). SASP- ${gamma}$ was also absent fromDPA-less sleB spoVF spores (Fig. 1). In contrast, the levelsof the DNA binding SASP- ${alpha}$ and -ß were similar in DPA-lessand DPA-replete sleB spoVF spores (Fig. 1), as found previouslywith ger3 spoVF spores (21). The DPA-less sleB spoVF sporesalso had $~$ 20% higher levels of core water than did DPA-repletespores (data not shown), similar to results with ger3 spoVFspores (21).

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FIG. 1. Levels of SASP in sleB spoVF spores prepared with or without DPA. SASP were extracted from spores of strain FB122 (sleB spoVF) prepared at 37°C on plates with or without DPA. Extracts were prepared and subjected to polyacrylamide gel electrophoresis at low pH, and the gel was stained with Coomassie blue as described in Materials and Methods. The samples run in the lanes are from 0.5 mg of dry spores and are presented as spores with DPA (lane 1) and spores without DPA (lane 2). The symbols ${alpha}$ , ß, and ${gamma}$ denote the migration positions of SASP- ${alpha}$ , -ß, and - ${gamma}$ , respectively.

Resistance of sleB spoVF spores. The higher level of core water in DPA-less sleB spoVF sporesmade it likely that these spores would be more sensitive towet heat than their DPA-replete counterparts (11, 16), and thiswas the case (Fig. 2A). The DPA-less spores were also more sensitiveto hydrogen peroxide and dry heat than were the DPA-repletespores (Fig. 2C and D). Since the levels of SASP- ${alpha}$ and -ßwere similar in spores with or without DPA, it seemed likelythat these spores would have similar UV resistance (17, 18,31), and this was again the case (Fig. 2B). While DPA-less sleBspoVF spores were more sensitive to wet heat and hydrogen peroxidethat their DPA-replete brethren, the DPA-less spores were notkilled by DNA damage. Analysis of auxotrophic and asporogenousmutations in survivors of DPA-less sleB spoVF spores killed $~$ 95% by wet heat or hydrogen peroxide revealed no significantincrease in mutation frequency (Table 2). In contrast, 15 to20% of the survivors of ${alpha}$ ^–ß^– spores treatedwith hydrogen peroxide or wet heat, treatments that kill thesespores via DNA damage, have auxotrophic or asporogenous mutations(10, 27). Dry heat is also known to kill even wild-type sporesby DNA damage (28).

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FIG. 2. Resistance of sleB spoVF spores prepared with or without DPA. Spores of strain FB122 (sleB spoVF) prepared on plates at 37°C with or without DPA were decoated (A, C, and D) or not (B) and treated with wet heat (70°C) at an OD₆₀₀ of 2 (A), UV radiation at an OD₆₀₀ of 1 (B), hydrogen peroxide at an OD₆₀₀ of 1 (C), or dry heat (200 µl at an OD₆₀₀ of 10) at 105°C (D). Spores were germinated with lysozyme in hypertonic medium (A, C, and D) or by Ca²⁺-DPA treatment (B), and viability was determined as described in Materials and Methods. Symbols: ${circ}$ , spores with DPA; •, spores without DPA.

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TABLE 2. Level of mutations in DPA-less sleB spoVF spores exposed to various treatments

The greater sensitivity of the DPA-less sleB spoVF spores todry heat suggested that these spores might also be sensitiveto desiccation alone. Indeed, while DPA-replete sleB spoVF sporeswere resistant to at least 10 cycles of freezing, desiccation,and rehydration, the DPA-less spores lost $~$ 50% of their viabilityin each cycle (Fig. 3). However, these DPA-less spores werenot sensitive to freezing and thawing (data not shown). Examinationof the survivors of four freeze-dry cycles showed that the DPA-repletespores had acquired no mutations, whereas the survivors of theDPA-less spores had (Table 2 and data not shown). These resultssuggested that desiccation had killed DPA-less sleB spoVF sporesby DNA damage. This was suggested further by the greater sensitivityof DPA-less recA sleB spoVF spores to desiccation, while DPA-repleterecA sleB spoVF spores remained resistant (Fig. 3). Previouswork found that a recA mutation alone does not sensitize B.subtilis spores to desiccation (30).

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FIG. 3. Effect of freezing, desiccation, and rehydration on spores prepared with or without DPA. Spores of strains FB122 (sleB spoVF) (circles) or PS3754 (squares) (recA sleB spoVF) (2 ml at an OD₆₀₀ of 10) prepared at 37°C with (open symbols) or without (solid symbols) DPA were subjected to freeze drying cycles as described in Materials and Methods. Spores were germinated by Ca²⁺-DPA treatment, and viability was determined as described in Materials and Methods. This procedure gave 2 x 10⁷ to 8 x 10⁷ CFU of spores/ml at an OD₆₀₀ of 1 at time zero for the four types of spores.

Loss of viability of DPA-less sspA sspB sleB spoVF spores. Another factor that is important in spore resistance and long-termsurvival is the ${alpha}$ /ß-type SASP (9, 17, 18, 31, 33).Consequently, it was of interest to analyze spores that lackedboth DPA and ${alpha}$ /ß-type SASP. A strain, PS3664, thatproduces such spores has been generated and has been used toshow that both DPA and ${alpha}$ /ß-type SASP play major rolesin determining the UV photochemistry of spore DNA (8). However,initial attempts to measure the resistance properties of cleanedDPA-less PS3664 spores made on plates failed because these cleanedspores at an OD₆₀₀ of 1 routinely gave $≤$ 0.001% of the CFU/mlgiven by the DPA-replete spores at the same concentration (Table3). The low viability of the DPA-less PS3664 spores when thespores were plated directly on nutrient plates was not surprising,since these spores are expected to germinate very poorly withnutrients, as noted above (20). However, these DPA-less sporeswere also not viable when either germinated with Ca²⁺-DPA orfirst decoated and then germinated with lysozyme in a hypertonicmedium (Table 3). In contrast, DPA-replete PS3664 spores atan OD₆₀₀ of 1 gave $~$ 10⁷ CFU/ml when decoated and germinated withlysozyme (Table 3). This latter value is similar to that obtainedin the present study and previously with spores of several B.subtilis strains germinated by this procedure (Table 3) (22,25). Purified spores of strain PS3747 (cotE sspA sspB sleB spoVF)prepared without DPA were also not viable when germinated withlysozyme in a hypertonic medium (Table 3). Spores of cotE strainsare germinated by lysozyme without prior decoating since, dueto the lack of the morphogenic CotE protein, their defectivecoat is permeable to lysozyme (9). Thus, the lack of recoveryof viable spores of DPA-less PS3664 spores was not due to theirdestruction by the decoating regimen used. Despite the extremelylow viability of the DPA-less PS3664 spores, the spores werebright and looked normal in the phase-contrast microscope (datanot shown).

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TABLE 3. Viability of spores of various strains prepared with or without DPA^a

Although the cleaned DPA-less PS3664 spores were not viable,it was possible that these spores were simply extremely labileand died during the $~$ 72 h of sporulation on plates at 37°C,the several days of incubation of the plates at 23°C, andthe $~$ 10 days of incubation at 4°C after the spores were harvestedand were being cleaned. Spores of strain PS3664 harvested after36 h of sporulation at 37°C in liquid medium without DPAand then cleaned over a period of $~$ 10 days were also not viable(Table 3 [values in parentheses]). However, we did recover viablespores early in the sporulation of such cultures by decoating,followed by lysozyme treatment in hypertonic medium (Fig. 4).In contrast, no viable forms ( $≤$ 10³ CFU/ml at an OD₆₀₀ of 1; datanot shown) were recovered in this manner from cultures of strainPS683, an asporogenous B. subtilis strain blocked late in sporulation(data not shown). The maximum viable count recovered from aculture of strain PS3664 sporulated without DPA (1.4 x 10⁶ CFU/mlat an OD₆₀₀ of 1; Fig. 4) was only $~$ 4-fold lower than for thisstrain sporulated with DPA recovered similarly (5 x 10⁶ CFU/mlat an OD₆₀₀ of 1; data not shown). However, the DPA-repletePS3664 spores maintained their viability throughout sporulation,harvesting, and cleaning (Table 3), whereas the DPA-less sporeslost viability continually as sporulation proceeded (Fig. 4).In contrast to strain PS3664 sporulated without DPA, strainFB122 sporulated without DPA did give fully viable spores afterharvesting and cleaning (Table 3).

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FIG. 4. Viability of DPA-less PS3664 spores during sporulation. Strain PS3664 (sspA sspB sleB spoVF)) was sporulated at 37°C in liquid without DPA, at various times samples (2 ml) were harvested, decoated, and germinated with lysozyme at an OD₆₀₀ of 1 in hypertonic medium, a process that took $≤$ 3 h, and viability was determined as described in Materials and Methods. Cultures were also examined in the phase-contrast microscope. The culture was inoculated $~$ 10 h prior to time zero, and arrows a and b denote the times at which surviving spores were tested for mutations. The number of phase bright spores in the culture did not decrease ( $≤$ 20%) after 75 h as determined by phase-contrast microscopy.

Analysis of mutations in the PS3664 spores prepared in liquidmedium with or without DPA showed that <1% of the DPA-repletespores had auxotrophic or asporogenous mutations (Table 4).However, of the colonies from DPA-less PS3664 spores harvestedat several times after the point of maximum spore viability,12 to 34% had obvious mutations (Table 4). This finding suggestedthat DPA-less PS3664 spores were dying because of DNA damage.Consistent with this suggestion, when sporulated without DPA,the recA derivative of strain PS3664 (strain PS3755) lost viabilitymore rapidly than did strain PS3664 (Fig. 5).

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TABLE 4. Levels of mutations in spores of strain PS3664 prepared with or without DPA^a

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FIG. 5. Effect of a recA mutation on the viability of DPA-less ${alpha}$ ^–ß^– spores during sporulation. Strains PS3664 (sspA sspB sleB spoVF) and PS3755 (recA sspA sspB sleB spoVF) were sporulated at 37°C in liquid 2xSG medium without DPA. At various times samples were harvested and decoated, spores were germinated at an OD₆₀₀ of 1 with lysozyme in hypertonic medium, and the viability was determined as described in Materials and Methods. Cultures were inoculated $~$ 10 h prior to time zero, and the times of development in the two cultures were aligned by examination with a phase-contrast microscope. Symbols: ${circ}$ , strain PS3664; •, strain PS3755.

Wet heat resistance of DPA-less PS3664 spores. Given the relatively rapid loss in viability of PS3664 sporesduring sporulation without DPA, it was reasonable to assumethat these spores would have much lower resistance than DPA-repletePS3664 spores or DPA-less spores that contained ${alpha}$ /ß-typeSASP. Indeed, when sporulating cultures were harvested shortlyafter the achievement of maximum spore viability, DPA-less PS3664spores died rapidly upon incubation at 65°C, a temperatureto which the DPA-replete PS3664 spores were relatively resistant(Fig. 6). DPA-less FB122 spores were also much more resistantto this temperature than were the DPA-less PS3664 spores (Fig.6; see also Fig. 2). Given the significant DNA damage in DPA-lessPS3664 spores even just after their release from the sporangium,we carried out no further studies of the resistance of theseDPA-less spores.

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FIG. 6. Resistance of spores of various strains to wet heat. Strains FB122 (sleB spoVF) and PS3664 (sspA sspB sleB spoVF) were sporulated with or without DPA, and samples were harvested after $~$ 80% of spores had been released from the sporangium, treated with decoating solution, washed, treated with wet heat at 65°C, and germinated with lysozyme in hypertonic medium. The spore viability was determined as described in Materials and Methods. Symbols: ${blacksquare}$ , PS3664 sporulated with DPA; ${square}$ , PS3664 sporulated without DPA; ${circ}$ , FB122 sporulated without DPA.

DISCUSSION

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Discussion

Some of the results presented here with DPA-less spores of strainFB122 (sleB spoVF) confirm results obtained with DPA-less sporesof a ger3 spoVF strain (21). These confirmatory results withDPA-less FB122 spores include (i) the absence of SASP- ${gamma}$ , (ii)the presence of normal levels of SASP- ${alpha}$ and -ß, and(iii) their increased sensitivity to wet heat and hydrogen peroxidecompared to that of their DPA-replete brethren. It was suggestedpreviously (21) that the increased sensitivity to wet heat andhydrogen peroxide of DPA-less ger3 spoVF spores is due to theincreased water content of these DPA-less spores, since increasedcore water content increases spore sensitivity to both wet heatand hydrogen peroxide (11, 23). The increased core water contentof DPA-less FB122 spores is certainly consistent with this suggestion.The increased core water content is also the likely reason forthe absence of SASP- ${gamma}$ from DPA-less FB122 spores. Presumably,the SASP-specific germination protease (GPR) that becomes activatedlate in forespore development is able to digest SASP- ${gamma}$ in thedeveloping DPA-less forespore due to these forespore's higherlevels of hydration (14, 21, 28). However, the binding of SASP- ${alpha}$ and -ß to DNA protects these proteins against GPRdigestion (29, 31). In contrast, SASP- ${gamma}$ does not bind to sporeDNA and is thus not well protected against GPR digestion (31).

Previous work showed that DPA-less ger3 spoVF spores were slightlymore UV resistant than were DPA-replete spores, but DPA-lesssleB spoVF spores exhibited similar UV resistance to the comparableDPA-replete spores. The retention of spore UV resistance inthe DPA-less spores is not surprising, since these spores retaintheir ${alpha}$ /ß-type SASP, the major factor causing sporeUV resistance (17, 18, 31, 33). Previous work (21) also foundvery little difference in the dry heat resistance of DPA-lessand DPA-replete ger3 spoVF spores, while we now observed significantlylower dry heat resistance of DPA-less sleB spoVF spores. Wedo not know the reason for the differences between previousand current findings. Possible contributing factors may be that(i) our current measurements were more extensive than thosemade previously and (ii) the DPA-less sleB spoVF spores aremuch more stable than the DPA-less ger3 spoVF spores.

Another new finding was that DPA-less sleB spoVF spores weresensitive to repeated desiccation, a treatment that was notinvestigated thoroughly with DPA-less ger3 spoVF spores (21).The high level of mutations in survivors of DPA-less sleB spoVFspores subjected to multiple freeze-drying cycles suggests thatdesiccation is killing these spores by DNA damage. This is consistentwith the sensitization of these DPA-less spores to desiccationby a recA mutation that eliminates much DNA repair in B. subtilis(30, 38).

Certainly the most striking finding in the present study wasthat spores that lacked both DPA and ${alpha}$ /ß-type SASPrapidly lost viability during sporulation and storage. In contrast,spores that lacked only DPA or only ${alpha}$ /ß-type SASP arestable for weeks to years during sporulation and subsequentstorage, as found previously and in current study (10, 20, 21;data not shown). ${alpha}$ ^–ß^– spores stored inwater at 12°C do lose viability on extended storage, since90% viability is lost in $~$ 2 years (10). However, DPA-less ${alpha}$ ^–ß^–spores lost $~$ 90% viability in 10 to 15 h at 37°C. The highlevel of mutations in DPA-less ${alpha}$ ^–ß^– sporesrecovered early in sporulation suggests that these spores weredying largely because of DNA damage, and again this was consistentwith the more rapid decline in viability of DPA-less recA ${alpha}$ ^–ß^–spores. ${alpha}$ ^–ß^– spores do accumulate DNA damageduring sporulation, harvesting, and cleaning (10, 39, 40). However,the levels of obvious mutations in ${alpha}$ ^–ß^–spores just after the spores are cleaned, a process that cantake up to 2 weeks, are only $~$ 1% of the population (39, 40),a value more than 10-fold lower than for DPA-less ${alpha}$ ^–ß^–spores immediately after their release from the sporangium.The heightened sensitivity of DPA-less ${alpha}$ ^–ß^–spores to wet heat is a further indication of the potentiallability of DNA in DPA-less spores, since wet heat treatmentkills ${alpha}$ ^–ß^– spores by DNA damage (10, 28,30).

Although DPA was identified as a major component of spores ofBacillus species over 50 years ago, the role of this compoundin spores has been difficult to clarify. This has been in largepart because of the instability of spores from strains whoseonly lesion is that they cannot make DPA. The results in thiscommunication as well as previous work indicate that stableDPA-less spores can be prepared from B. subtilis strains thathave defined mutations in genes of known function (20, 21).In current and previous work the analysis of these spores hasshown that DPA plays a number of roles in spores including thefollowing. (i) DPA in the spore core displaces water such thatDPA-replete spores have less core water than isogenic DPA-lessspores (21). This is a major reason for the role of DPA in sporewet heat resistance, since there is an inverse correlation betweencore water content and spore wet heat resistance (11). (ii)DPA plays a major role in the UV photochemistry of spore DNA,affecting both the photoreactivity of spore DNA and the natureof the photoproducts produced (8, 26). Since at least some ofthe effects of DPA on spore DNA are most likely via energy transferfrom an excited state of DPA to DNA (8), DPA must be in closeproximity to spore DNA. (iii) The likely close proximity ofDPA and DNA noted above is consistent with spore protectionagainst dry heat damage by DPA, since dry heat kills sporesby DNA damage (28). (iv) The loss in viability of DPA-less sporesupon desiccation, with killing of these DPA-less spores duein large part to DNA damage, is further evidence that DPA playsa major role in stabilizing spore DNA. (v) Finally, the extremelability of DPA-less ${alpha}$ ^–ß^– spores indicatesagain that DPA is an important factor in protecting spore DNAfrom damage, since in ${alpha}$ ^–ß^– spores the strongprotective effect of ${alpha}$ /ß-type SASP on DNA is lost.

DPA in spores thus has two main general functions. One is tolower the content of core water by replacing some core waterwith DPA. This elevates spore wet heat resistance by protectingcore proteins from inactivation or denaturation by wet heat.The second function of DPA in spores is to contribute to theprotection of spore DNA against many types of damage. In wild-typespores, the protection of DNA against wet heat by DPA may notbe especially important, since this is provided by ${alpha}$ /ß-typeSASP (10). However, when ${alpha}$ /ß-type SASP are absent,DNA protection by DPA becomes important even in hydrated spores.In dry spores, DNA is not completely protected against heatdamage by the ${alpha}$ /ß-type SASP (28). Consequently, DPAprotects the DNA in dry spores from heat damage and also againstdesiccation. The mechanism whereby DPA protects DNA againstdry heat and desiccation is not known. However, the DNA damagecaused by dry heat and desiccation treatment of spores is differentthan that caused by wet heat or spontaneous mutations (13).

Since core water content is significantly higher in DPA-lessspores, this raises the possibility of there being a low levelof aerobic metabolic activity in such spores, and aerobic metabolismcan generate compounds such as superoxide and hydrogen peroxidethat directly or indirectly can cause DNA damage. In otherwisewild-type spores, the ${alpha}$ /ß-type SASP would protect DNAagainst such oxidative damage (27), but in the absence of theseprotective proteins such DNA damage could lead to spore death.Currently available evidence suggests that metabolism in dormantspores, even DPA-less spores, is minimal, if it takes placeat all (25). However, a very low level of metabolism in DPA-lessspores would be extremely difficult to detect.

Spores of most Bacillus species are soil organisms and thusare likely to spend a large amount of time in the dry state.Consequently, the protection of spore DNA against damage dueto dry heat and desiccation may be extremely important in sporesurvival in the environment. The ability of DPA to contributesignificantly to spore DNA protection may thus have been a majordriving force in the evolution of the capacity for DPA synthesisduring sporulation and the accumulation of high DPA levels inspores.

ACKNOWLEDGMENTS

This study was supported by a grant from the Army Research Office.

FOOTNOTES

* Corresponding author. Mailing address: Department of Molecular, Microbial and Structural Biology, University of Connecticut Health Center, Farmington, CT 06030-3305. Phone: (860) 679-2607. Fax: (860) 679-3408. E-mail: setlow{at}nso2.uchc.edu.

REFERENCES

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