Department of Biochemistry, University of Connecticut Health Center, Farmington, CT 06032, USA1
Author for correspondence: Peter Setlow. Tel: +1 860 679 2607. Fax: +1 860 679 340. e-mail: setlow{at}sun.uchc.edu
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ABSTRACT |
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Keywords: spore resistance, spore killing, membrane damage
Abbreviations: DPA, dipicolinic acid; PON, peroxynitrite; SASP, small acid-soluble protein
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INTRODUCTION |
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METHODS |
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PON synthesis and treatment of cells or spores.
PON was synthesized by rapid mixing of 10 ml ice-cold 250 mM sodium nitrite and 270 µl 22% hydrogen peroxide followed by rapid addition of 5 ml ice-cold 1 mol HCl l-1 and 5 ml 1·5 mol NaOH l-1. Unreacted hydrogen peroxide was removed and the PON was stored as described (Beckman et al., 1994 ). PON concentrations were determined immediately prior to use by measurement of the OD302 of appropriate dilutions in 1·2 M NaOH (Beckman et al., 1994
).
Spores and growing cells were incubated with PON at an OD600 of 1 in 200 mmol potassium phosphate l-1 (pH 7·4) for 15 min at 24 °C, with PON added last. For control incubations, PON was diluted into the buffer prior to addition of spores or growing cells, the mix incubated for 15 min at 24 °C to allow PON breakdown, spores or growing cells added, and the mix incubated a further 15 min at 24 °C. Aliquots of PON-treated or control mixtures were diluted into phosphate-buffered saline (0·2 g KCl l-1, 0·24 g KH2PO4 l-1, 8 g NaCl l-1, 1·44 g Na2HPO4 l-1, pH 7·4), appropriate dilutions plated on LB agar plates (Setlow & Setlow, 1996 ) and colonies counted after 2436 h incubation at 3037 °C; incubation for longer times gave no increase in colonies. All killing experiments were repeated at least twice with two independent spore preparations, always with essentially identical results, as the slopes of killing curves for the same strain never varied by more than 25%. However, in all experiments reported here, killing of spores of different strains was always carried out at the same time. In some experiments, catalase or pyruvate was added to the plates used for analysis of PON killing as described (Flowers et al., 1977
; Hood et al., 1990
).
The disinfectant Sterilox was prepared and analysed as described previously (Loshon et al., 2001 ); Sterilox preparations had pH values of
6·3 and available free chlorine levels of
240 mg l-1. Spores were treated with Sterilox without prior decoating, the Sterilox inactivated and spores recovered and killing measured as described (Loshon et al., 2001
).
Spore decoating, spore germination and analytical procedures.
Spores were decoated by incubation for 30 min at 65 °C in 100 mmol NaOH l-1, 100 mM NaCl, 5 g SDS l-1 and 100 mmol DTT l-1, and the spores washed as described (Bagyan et al., 1998 ). Note that this decoating procedure also removes much, if not all, of the spores outer membrane (Buchanan & Neyman, 1986
). Spores with or without PON treatment were tested for mutagenesis to asporogeny or auxotrophy as described by Fairhead et al. (1993)
. The pyridine-2,6-dicarboxylic acid [dipicolinic acid (DPA)] content of spores with or without PON treatment was assayed after DPA extraction by boiling as described (Rotman & Fields, 1967
; Nicholson & Setlow, 1990
). Spores with or without prior PON or Sterilox treatment were incubated in water at an OD600 of 59 for 30 min at various temperatures, cooled on ice for 10 min, centrifuged in a microcentrifuge and DPA in the supernatant fluid assayed directly.
After a heat shock for 30 min at 70 °C in water, spores were germinated at an OD600 of 1 in either 2x YT medium (Setlow & Setlow, 1996 ) plus 4 mmol L-alanine l-1 or 10 mmol Tris/HCl l-1 (pH 8·3) plus 8 mmol L-alanine l-1. DPA release, hexosamine release from spore cortex peptidoglycan and the percentage of spores that had turned phase dark or swelled were determined as described (Popham et al., 1996
; Loshon et al., 2001
). For analysis of light production during germination of spores carrying the V. harveyi luxAB genes, spores were germinated in 2x YT medium plus L-alanine as described above; at various times aliquots of 500 µl were mixed with 500 µl fresh medium and dodecanal added to 0·1 g l-1 as described (Hill et al., 1994
; Loshon et al., 2001
). Light production was measured in a Turner TD-20/20 Luminometer over three consecutive intervals of 10 s and values extrapolated to the time of dodecanal addition, since light production from the V. harveyi LuxA and B gene products in B. subtilis decays, as seen previously (Karp, 1989
; Ciarciaglini et al., 2000
; Loshon et al., 2001
). Recovery of decoated spores by treatment with lysozyme in a hypertonic medium was as described by Popham et al. (1996)
. Spores were stained with acridine orange and examined by fluorescence microscopy as described by Setlow et al. (2001)
.
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RESULTS |
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DISCUSSION |
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While removal of much coat protein by either mutation or decoating greatly reduced spore PON resistance, decoated or cotE spores are much more PON resistant than are growing cells. Although the coat protein remaining in decoated or cotE spores could provide significant protection against PON, it seems more likely that there are factors in addition to the spore coat which are involved in spore PON resistance. Other factors that have been identified as involved in spore resistance to one or more chemicals include the relative impermeability of the spores inner membrane, the dehydration and mineralization of the spore core, the protection of spore DNA from damage by its saturation with /ß-type SASP, and the repair of damage, in particular to DNA, in the early minutes of spore germination (Setlow, 2000
). Neither DNA repair nor the
/ß-type SASP appears to play any role in spore PON resistance, as PON treatment of neither wild-type nor
-ß- spores caused mutagenesis and a recA mutation did not decrease spore PON resistance. Since growing bacteria are mutagenized by PON and DNA repair is a crucial factor in their PON resistance (Routledge, 2000
; Spek et al., 2001
), these findings imply that PON causes no significant DNA damage in spores and thus that PON levels in the spore core, the site of spore DNA, must never become very high. Consequently, the dehydration and mineralization of the spore core, which probably reduce the rates of reactions of toxic chemicals with spore core targets (Setlow, 2000
), may not be particularly important in spore PON resistance. This analysis thus suggests that a major factor in spore PON resistance may be the relative impermeability of the spores inner membrane to PON. This latter compound is largely an anion at neutral pH and previous work has shown that charged molecules penetrate the spore core extremely poorly (Gerhardt et al., 1972
; Khairutdinov et al., 2000
). In contrast, the protonated form of PON, peroxynitrous acid, is a neutral species which is thought to readily cross biological membranes (Khairutdinov et al., 2000
), and the spore core is permeable to small uncharged molecules <150 Da in size (Gerhardt et al., 1972
). However, the rate of permeation of small uncharged molecules, for example unprotonated methylamine, into the spore core is extremely slow, possibly because of the compressed state of the spores inner membrane (Setlow & Setlow, 1980
; Driks & Setlow, 1999
). Consequently the slow permeation of peroxynitrous acid across the inner spore membrane may restrict most PON action to targets on the outer surface of or exterior to the spores inner membrane. It might be expected that PON would be more effective in killing bacteria at lower pH values because the higher levels of the more permeable peroxynitrous acid at lower pH values would result in more PON inside cells. However, this has not been observed by others, perhaps due to the greater lability of peroxynitrous acid relative to PON (Hurst & Lymar, 1997
; Khairutdinov et al., 2000
). It is also worth noting that enzymic detoxification of PON by reduction, a significant factor in the PON resistance of growing bacteria (Chen et al., 1998
; Bryk et al., 2000
), is almost certainly not important in spore PON resistance. Whilst at least one enzyme that is reported to detoxify PON, alkyl hydroperoxide reductase subunit C, is present in the spore core, this enzyme as well as catalase has been shown to play no role in dormant spore resistance to peroxides, most likely because of the inactivity of enzymes in the dormant spore core (Casillas-Martinez & Setlow, 1997
; Chen et al., 1998
).
A tentative conclusion from the material presented above is that spores are most likely killed by PON action on the outer surface of, or exterior to, the spores inner membrane. This region of the spore contains the receptors for germinants as well as the enzymes that are involved in the depolymerization of the spore cortex (Paidhungat & Setlow, 2001 ). However, spore germination is not abolished in PON-killed spores, as these spores release DPA and initiate metabolism upon mixing of spores with germinants. It is true that cortex fragments are not released into the medium during this latter process with PON-killed spores. However, the cortex of PON-killed germinated spores must be significantly depolymerized, since initiation of metabolism and complete staining of the germinated spore by acridine orange are observed, and both these processes have been shown to require cortex depolymerization (Setlow et al., 2001
). However, whilst PON-killed spores do initiate germination, these germinated spores have severe permeability defects as shown by their uptake of propidium iodide, a hallmark of bacteria with a damaged cytoplasmic membrane; in germinated spores this latter membrane is derived from the spores inner membrane. The precise nature of the inner membrane damage caused by PON is not clear, but PON is a strong oxidant and at concentrations well below those used in the current work can damage both membrane lipids and proteins (Soszynski & Bartosz, 1996
; Gadhela et al., 1997
; Mallozzi et al., 1997
). That PON-killed dormant spores have suffered some type of membrane damage is also suggested by their poor retention of DPA upon subsequent heat treatment. The precise barrier to DPA loss from dormant spores is not known, but a major role for the inner spore membrane in the process seems most likely. Therefore, a likely scenario for PON killing of B. subtilis spores is that this agent causes some type of damage to the spores inner membrane that does not breach the permeability barrier of this membrane, perhaps because the inner membrane is extremely compressed in the dormant spore. However, the permeability barrier of this membrane is weakened by the PON damage, resulting in more rapid loss of DPA on spore heating. Upon spore germination and the inner membrane expansion to accommodate the rapid twofold expansion of the cores volume in the absence of membrane synthesis (Paidhungat & Setlow, 2001
), the effects of the inner-membrane damage caused by PON become much more severe, resulting in spore death even though early events in spore germination are relatively normal. Interestingly, essentially all the findings reported here on PON killing of B. subtilis spores are extremely similar to those made both previously and in the current work examining spore killing by the superoxidized water, Sterilox (Loshon et al., 2001
). This suggests that both these agents kill B. subtilis spores by similar mechanisms, possibly through damage to the inner spore membrane as we suggest here. The challenge now is to identify and quantitate this membrane damage.
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ACKNOWLEDGEMENTS |
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Received 18 June 2001;
revised 7 September 2001;
accepted 24 September 2001.