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Department of Biochemistry, University of Connecticut Health Center, Farmington, Connecticut 06032
Received 29 August 2003/ Accepted 21 December 2003
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ABSTRACT |
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INTRODUCTION |
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One group of agents that have been tested as microbial biocides are Fenton reagents (34) that use various metal ions plus hydrogen peroxide or ascorbic acid to generate highly reactive hydroxyl radicals (2, 21, 22, 23). The reactivity of hydroxyl radicals means that they are most effective when they are generated in the immediate vicinity of their target, which is often DNA (8, 9, 19). Fenton reagent formulations with Cu2+ and hydrogen peroxide or ascorbic acid kill spores of Bacillus species (2, 21, 22, 23). Bacillus globigii spore killing by CuCl2-ascorbic acid requires oxygen, presumably for the generation of hydroxyl radicals, and killing is increased by the addition of NaCl or a surfactant (2). Since spores killed by Fenton reagent with Cu2+ and ascorbic acid were not visibly damaged, it was suggested that the Cu2+ and ascorbic acid had penetrated into the spore core and with dissolved oxygen had generated hydroxyl radicals that caused spore death by damage to DNA or enzymes (2). While this is a logical possibility, penetration of ascorbic acid into the spore core seems unlikely, since hydrophilic molecules such as monosaccharides do not normally penetrate the core of Bacillus cereus spores (6). Indeed, even a molecule as small as methylamine penetrates the spore core slowly (32). Since Cu2+-ascorbic acid may be used to kill spores in applied settings, it seemed appropriate to determine how this reagent acts on spores. Of particular interest are the following issues: (i) whether this reagent kills spores by DNA damage; (ii) whether the reagent acts in the spore core or on some more external spore layer; and (iii) what spore features are important in protection against this reagent.
Spore DNA is well protected against damage from toxic chemicals by the proteinaceous spore coats, the low permeability of the spore's inner membrane, the spore core's low water content, and the saturation of spore DNA with 
/ß-type small, acid-soluble spore proteins (SASP) (13, 31). The DNA protection afforded by these mechanisms is so great that many potentially genotoxic agents that kill spores, including many oxidizing agents, do not do so by causing DNA damage (5, 10, 13, 31, 37).
To test whether Cu2+-ascorbic acid kills spores by causing DNA damage, the killing of spores of B. subtilis strains lacking various DNA protective mechanisms was examined. The B. subtilis strains were isogenic derivatives of strain 168. PS533 is the wild-type strain and carries plasmid pUB110, which contains a kanamycin resistance marker (30). Two genes (sspA and sspB) that encode the two major /ß-type SASP were deleted from strain 168 to create strain PS578, which carries pUB110 (30). The recA gene was deleted from strain 168 and replaced with a gene providing resistance to macrolide antibiotics (MLSr) to create strain PS2318 (30). The sspA and sspB genes were deleted from strain 168 and the recA gene was mutated to create strain PS2319 (30). Strain PS3394 carries pUB110 and contains a tetracycline resistance cassette inactivating the cotE gene essential for proper spore coat assembly (3, 37). Strain PS3379 contains the luxAB genes from Vibrio harveyi under the control of the forespore-specific promoter of the sspB gene (10).
Spores were prepared at 37°C on 2xSG medium plates without antibiotics and washed and cleaned as described previously (14, 15). Spore preparations used were free (>98%) from growing cells, cell debris, or spores that had germinated, as determined by examination with a phase-contrast microscope. Spores were treated at an optical density at 600 nm (OD600) of 
1.0 (108 CFU/ml) at room temperature (24°C) with 54 mM CuCl2 and 90 mM ascorbic acid unless noted otherwise; the pH of this solution was 1.5. At various times, aliquots were diluted 100-fold in a solution containing 2.5 g of Na2S2O3 per liter to neutralize the Cu2+-ascorbic acid, and further diluted in water, and aliquots were spotted in duplicate on Luria-Bertani (LB) medium (15, 33, 37) plates. The plates were incubated for 24 h at 30 to 37°C, and colonies were counted to assess spore viability. Spores of all strains tested were not killed by being incubated at pH 1.5 for 2 h, as found previously (27).
Spores of the wild-type B. subtilis strain (PS533) were killed by CuCl2-ascorbic acid (Fig. 1A), although more slowly than spores of B. globigii (2). Spores of strain PS578 that lack most of the DNA-protective /ß-type SASP (termed –ß– spores) exhibited slightly greater sensitivity to CuCl2-ascorbic acid, but in either a wild-type or –ß– background, loss of the recA gene responsible for much DNA repair in B. subtilis (36) did not reduce spore resistance (Fig. 1A). Previous work has shown that –ß– or recA spores are 3- to 10-fold more sensitive to agents that cause DNA damage than are wild-type spores (13, 33). Thus, the slight (1.5-fold) decrease in spore sensitivity to CuCl2-ascorbic acid upon loss of SASP- and SASP-ß may not be due to killing of the –ß– spores through DNA damage. Instead, this small difference is likely due to global changes in gene expression during sporulation of the –ß– strain, because of the absence of accumulation of most /ß-type SASP in the developing –ß– forespore (28). This alteration in gene expression causes slight changes in the properties of the –ß– spores, as seen previously in the decreased resistance of –ß– spores to other agents that do not cause DNA damage in spores (5, 28, 33, 37). In particular, the lack of synthesis of SASP- and -ß during sporulation has major effects on expression of genes coding for spore coat proteins (28), and as shown below, the spore coats are a major factor in spore resistance to CuCl2-ascorbic acid.
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–ß– spores surviving CuCl2-ascorbic acid were tested for the presence of auxotrophic or asporogenous mutations by examining growth of survivors on plates containing rich, minimal, and sporulation media as described previously (4, 5, 10, 33, 37). In contrast to spores that survive treatments that kill by damaging DNA, 15% of which have obvious mutations (4, 33), wild-type spores surviving CuCl2-ascorbic acid (8% survival) had no more mutations (0 in 416 survivors) than the untreated spores (0 in 416 spores tested). The same finding was made for –ß– spores (83% killed) treated with CuCl2-ascorbic acid (6 mutants in 416 survivors; 6 mutants in 416 untreated –ß– spores examined). The significant level of mutations in untreated –ß– spores has been seen previously and ascribed to mutagenesis during spore preparation and storage (4, 5, 10, 33, 37). In contrast to the absence of DNA damage detected in spores killed by CuCl2-ascorbic acid, growing bacteria treated with a Fenton reagent do exhibit significant mutagenesis, and cell killing is accelerated by a recA mutation (35). The spore coat helps protect spores against many chemical agents (3, 5, 10, 31, 37). Indeed, B. subtilis spores with defective coats are 10- to 100-fold times more sensitive to many oxidizing agents than intact spores are (5, 10, 37). To assess the role of the coat in spore resistance to CuCl2-ascorbic acid, we examined wild-type spores that were chemically decoated (1) as well as cotE spores (3). Not surprisingly, both the decoated and cotE spores were much more sensitive to this reagent than were untreated wild-type spores (Fig. 1B). The protection against CuCl2-ascorbic acid by the spore coat may explain the observation that a surfactant sensitized B. globigii spores to this reagent (2). Perhaps the surfactant used in that study (2) removes some coat protein, allowing easier access to targets located inside the spore.
It was reported that CuCl2-ascorbic acid killing of spores is increased significantly by the addition of NaCl (2), and we also found this to be the case (Fig. 1C). Why NaCl would increase spore killing by CuCl2-ascorbic acid if these chemicals were to react in the spore core to generate hydroxyl radicals is not clear. However, perhaps the agent killing the spores is not the hydroxyl radical but an oxidizing agent formed by reaction of hydroxyl radicals with chloride ions in free solution. A number of chlorine-containing species can be generated by the Fenton reaction in the presence of chloride ions, including chlorine atoms and hypochlorite (24, 25), and hypochlorite kills spores efficiently, especially as the pH is decreased (37). If the reaction of hydroxyl radicals with chloride ions does generate the killing species, then spores incubated with CuSO4-ascorbic acid should not be killed. This was indeed the case for both intact and decoated wild-type spores, and spore killing by CuSO4-ascorbic acid was restored by the addition of NaCl (Fig. 1C). The lack of spore killing by CuSO4-ascorbic acid further indicates that enhancement by NaCl of spore killing by Cu2+-ascorbic acid was not an ionic strength effect but is more likely a specific effect of the Cl– ion. Indeed, 0.5 M Na2SO4 did not enhance spore killing by CuCl2-ascorbic acid (data not shown).
The data noted above suggest that spore killing by CuCl2-ascorbic acid is through generation of oxidizing chlorine-containing species. In addition, since there is no evident DNA damage in spores killed by CuCl2-ascorbic acid, even in spores lacking the DNA protective /ß-type SASP, it seems likely that the killing agent does not act on the spore core but on some more exterior spore layer. Recent studies examining spore killing by hypochlorite and chlorine dioxide, as well as the new disinfectant Sterilox (which also contains a variety of oxidizing chlorine-containing compounds), have shown that these agents do not damage the DNA in the spore core but appear to kill spores by damaging the spore's inner membrane in some fashion (5, 10, 37). This damage, in particular by hypochlorite, can also greatly diminish the spore's ability to germinate (37).
To assess the effect of CuCl2-ascorbic acid on spore germination, wild-type spores (94 to 99% of wild-type spores killed by CuCl2-ascorbic acid) were tested for spore germination with nutrients, with a 1:1 mixture of Ca2+ and dipicolinic acid (DPA), and with dodecylamine. Ca2+-DPA and dodecylamine initiate germination of spores of many Bacillus species, including Bacillus subtilis, but are not nutrients (16, 26). The CuCl2-ascorbic acid-killed spores initiated germination normally with nutrients, judging from the conversion of the spores from the bright to dark phase using a phase-contrast microscope (Table 1). Spores killed 99% by CuCl2-ascorbic acid also germinated in LB medium plus L-alanine as rapidly as untreated spores, as assessed by the decrease in OD600 of spore suspensions (data not shown), and after 90 min, these germinated spores appeared viable in an assay based on the inability of bacteria with a severely damaged plasma membrane to exclude the dye propidium iodide (Table 1). As judged by their ability to give rise to colonies, 
95% of these spores were actually dead, indicating that the spores have indeed been damaged in some fashion. A similar discrepancy between these two viability assays was seen when chlorine dioxide or a superoxidized water, Sterilox, was used to kill spores, as a large fraction of spores killed by these reagents as assessed by colony formation appeared alive as measured by the exclusion of propidium iodide (10, 37). Presumably, the CuCl2-ascorbic acid-treated spores had accumulated sufficient damage such that they could not give rise to colonies but not so much damage that spores germinated 90 min in LB medium do not exclude propidium iodide.
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Further evidence for damage to CuCl2-ascorbic acid-killed spores came from analysis of metabolism upon germination using spores that contain high levels of the V. harveyi LuxA and LuxB proteins. Untreated B. subtilis spores initiate metabolism soon after the initiation of germination and generate FMNH2, and FMNH2, LuxA, LuxB, and dodecanal result in light production by germinating spores (5, 7, 10). This was also observed in the current work (Fig. 2), when light production was measured as described previously (5, 10, 37). However, spores killed 99% by CuCl2-ascorbic acid and then germinated exhibited 
1% of the light production of germinated untreated spores (Fig. 2). Perhaps slight damage by CuCl2-ascorbic acid to the inner spore membrane that becomes the germinated spore's plasma membrane has compromised energy metabolism by these germinated spores.
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95% with CuCl2-ascorbic acid and decoated and treated with lysozyme in a hypertonic medium to degrade the spore's peptidoglycan and induce germination, the killed spores became phase dark (data not shown), an indicator of cortex lysis. However, the CuCl2-ascorbic acid-treated spores were not revived by this treatment (data not shown). In contrast, spores not treated with CuCl2-ascorbic acid remained viable after the same treatment (data not shown), as expected (20).
The data presented in this communication allow three new conclusions. First, spore killing by Cu2+-ascorbic acid largely if not completely requires Cl–, suggesting that the killing agent is some oxidizing chlorine-containing species. Second, the spore coat provides major protection against spore killing by CuCl2-ascorbic acid, as they do against spore killing by a number of other oxidizing agents (5, 10, 37). Third, spore killing by CuCl2-ascorbic acid is not due to DNA damage, at least DNA damage that is either mutagenic, repaired in a RecA-dependent process, or prevented by /ß-type SASP. In addition, since wild-type and –ß– spores exhibit relatively similar sensitivities to CuCl2-ascorbic acid, it seems unlikely that this reagent generates hydroxyl radicals within the spore core. In contrast, hydrogen peroxide may generate hydroxyl radicals in the spore core, since –ß– spores are much more sensitive to hydrogen peroxide than wild-type spores and –ß– spores (but not wild-type spores) surviving treatment with hydrogen peroxide exhibit high levels of mutations (29).
A remaining area of uncertainty about spore killing by CuCl2-ascorbic acid concerns the mechanism of killing. Previous work on spore killing by oxidizing chlorine-containing agents has shown that these agents do not kill spores by DNA damage but likely cause some damage to the spore's inner membrane that either prevents spore germination or results in loss of membrane integrity in the first minutes of spore germination (10, 37). However, there is no significant defect in the initiation of germination by CuCl2-ascorbic acid-killed spores, and these germinated spores appear to have retained their plasma membrane integrity, at least after 90 min of germination, although their ability to carry out energy metabolism is severely compromised. Consequently, the precise nature of the damage caused by CuCl2-ascorbic acid treatment that kills spores is not clear. Indeed, this damage may be different from that caused by other oxidizing chlorine-containing agents (10, 37), since these latter agents were used at pH values ranging from 3.5 to 11 and the CuCl2-ascorbic acid reagent has a pH of 1.5. An additional possible mechanism for spore killing by the CuCl2-ascorbic acid reagent is the inactivation of one or more enzymes within the spore core, in particular enzymes that are essential for spore metabolism. Inactivation of spore core enzymes accompanying spore killing by hydroperoxides has been observed previously (17, 18), although it is not clear if this is the cause of spore killing by such agents. However, as noted above, the absence of DNA damage in –ß– spores treated with CuCl2-ascorbic acid suggests that this reagent does not exert its killing action in the spore core. Clearly, determining the precise mechanism of spore killing by CuCl2-ascorbic acid remains a subject for further work.
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ACKNOWLEDGMENTS |
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This work was supported in part by a grant from the Army Research Office.
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FOOTNOTES |
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REFERENCES |
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), PS2318 (recA) (
), and PS2319 (
) were incubated with CuCl2-ascorbic acid. In each experiment shown, the viability of spores of all strains was determined in parallel and at least in duplicate as described in the text. (B) Spores of B. subtilis strains PS533 (wild type) (• and 
, and 
) or treated with CuCl2-ascorbic acid giving 99% killing (•) were germinated at 37°C and an OD600 of 1 in 2x YT medium (