Department of Molecular Biology and Biotechnology, University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, UK1
Author for correspondence: Simon J. Foster. Tel: +44 114 2224411. Fax: +44 114 2728697. e-mail: s.foster{at}sheffield.ac.uk
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ABSTRACT |
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Keywords: spores, peptidoglycan, cortex, resistance, lytic transglycosylase
Abbreviations: AGFK, a mixture of L-asparagine, glucose, fructose and KCl; A2pm, diaminopimelic acid; GSLE, germination-specific lytic enzyme
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INTRODUCTION |
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Recent findings have shown that cortex hydrolysis during germination is a complex process with at least three GSLEs involved: a glucosaminidase, a lytic transglycosylase and a possible amidase (Atrih et al., 1998 , 1999
; Atrih & Foster, 1999
; Boland et al., 2000
). Another non-hydrolytic activity, suggested to be an epimerase, has also been noted (Atrih et al., 1998
). Two components involved in cortex hydrolysis during B. subtilis germination have been identified. Spores of a sleB mutant germinate more slowly than the wild-type and analysis of peptidoglycan dynamics during germination has revealed that the gene is likely to encode a lytic transglycosylase (Boland et al., 2000
). The cwlJ gene product is homologous to SleB (Ishikawa et al., 1998
). A cwlJ mutation results in slower germination and a sleB/cwlJ double mutant cannot hydrolyse the cortex during germination (Ishikawa et al., 1998
).
A number of GSLEs have been purified from germinated or broken spores of Bacillus megaterium, Bacillus cereus and Clostridium perfringens (Chen et al., 1997 , 2000
; Foster & Johnstone, 1987
; Makino et al., 1994
; Miyata et al., 1995
; Moriyama et al., 1996
). The common property shared by these GSLEs is the requirement for the presence of the muramic
-lactam moiety on the spore cortex for activity. Purified GSLEs extracted from spores are not unusually heat resistant (Chen et al., 1997
, 2000
; Foster & Johnstone, 1987
; Moriyama et al., 1996
; Warth, 1978
); therefore they must be protected against heat within the internal environment of spores. Unfortunately in B. subtilis, attempts to isolate active GSLEs from germinated or physically broken spores have been unsuccessful (T. J. Smith & S. J. Foster, unpublished). In C. perfringens heat and NaOH inactivate GSLEs involved in cortex hydrolysis, since treated spores can be partially rescued by lysozyme (Duncan et al., 1972
; Labbe & Chang, 1995
). However, the role of the multiple GSLEs of B. subtilis and their potential as sporicidal targets is still unknown. In this study we have analysed the combined function of the GSLEs of B. subtilis in germination under different environmental conditions and in response to sporicidal treatments.
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METHODS |
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Germination of spores at different pH values.
Washed spores of strain HR and strain FB101 were suspended at a final concentration of 3 mg dry wt ml-1 in distilled water, and heat-shocked at 70 °C for 30 min. Spores were then cooled on ice and used within 1 h (Atrih et al., 1998 ). The following germination buffer solutions were used at a final concentration of 20 mM in the presence of 20 mM KCl: sodium acetate/acetic acid (pH 3·55), potassium phosphate buffer (pH 57), and Tris/HCl buffer (pH 810). Germination was triggered by L-alanine (1 mM; a saturating concentration for germination) and monitored by recording the decrease of OD600 as previously described (Atrih et al., 1998
). After 2 h, germination exudates were collected by centrifugation (14000 g, 8 min, 4 °C), boiled for 3 min and the pH was adjusted to 5 before lyophilization. Germination was carried out for 2 h, a time at which >90% of total OD600 loss had occurred. All germination experiments were repeated at least twice and showed less than 10% variation.
Spore heat treatment.
Spores were suspended at a final concentration of 3 mg dry wt ml-1 in distilled water and heat-treated at 90 °C for different times. The heat-treated spores were immediately cooled on ice and used for germination experiments within 1 h, without further heat shock.
Spore alkali treatment.
Spores (4 mg dry weight ml-1) were suspended in distilled water (on ice), and NaOH was added to give a final concentration of 0·25 M. After treatment the spores were recovered by centrifugation (12000 g, 8 min, 4 °C) and washed three times by centrifugation and resuspension with 20 mM phosphate buffer at pH 6, then twice with distilled water at 4 °C. Spores were heat shocked at 65 °C for 25 min and germination was then performed as indicated above.
Lysozyme recovery of heat- or alkali-treated spores.
Lysozyme was added to molten nutrient agar (48 °C) to give a final concentration of 0·15, 0·3, 0·6 and 1·0 µg ml-1 prior to pouring the plates. The experiments were performed in duplicate and plate counts were determined after 48 h. The lysozyme concentrations used in this study do not affect the total viable count of HR untreated spores.
Muropeptide purification and amino acid analysis.
Muropeptide purification and quantification in the germination exudate were performed as previously described (Atrih et al., 1996 , 1998
). Diaminopimelic acid (A2pm), a peptidoglycan-specific amino acid, was used to measure the amount of peptidoglycan released in the exudate by the Pico-Tag method (Atrih et al., 1996
, 1998
). All experiments were carried out in duplicate. The results shown are representative; there was <5% variability between experiments.
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RESULTS |
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Germination of alkali-treated spores
Treatment of dormant B. subtilis HR spores with 0·25 M NaOH for 130 min did not affect the peptidoglycan structure significantly, although a minor change of a 1·5% increase of muropeptides with an open lactam was noted (results not shown) (Atrih et al., 1996 ). Alkali-treated spores were evaluated for their germination ability and cortex hydrolysis. The loss of OD600 during germination actually increased after 1 h in 0·25 M NaOH treatment, from 41% (untreated) to 47%, a slight but reproducible effect (Fig. 4
). The loss of epimerase and glucosaminidase products in alkali-treated spores followed a similar pattern to heat-treated spores: 88% and 89% of epimerase and glucosaminidase products, respectively, were lost within 15 min (Fig. 4
). Lytic transglycosylase activity was not only alkali resistant, but actually increased after treatment (Fig. 4
). The amount of A2pm released from 1 h alkali-treated spores was 40% lower than that released from untreated spores. Heat- and alkali-treated spores were also germinated in the presence of AGFK and similar results to those obtained with L-alanine were noted (results not shown).
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Effect of heat or NaOH on spore survival
As GSLEs are crucial enzymes in cortex hydrolysis their inactivation, by heat or NaOH, may affect spore survival. Heat treatment (90 °C) of B. subtilis HR spores led to a decline in viable cell numbers (as measured by c.f.u.) resulting in only 0·2% recovery after 130 min treatment. Spores of FB101 (sleB) were considerably more heat sensitive, with only 0·01% recovery after 130 min heat treatment (Fig. 5). Wild-type spores of B. subtilis were not affected by alkali treatment (0·25 M) for up to 130 min (Fig. 5
). However, the lack of SleB in strain FB101 resulted in sensitivity to alkali. Indeed, 97% of spores had lost viability within the 130 min treatment (Fig. 5
).
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DISCUSSION |
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Glucosaminidase activity has been previously reported to have a minor role in cortex hydrolysis at pH 7 (Atrih et al., 1998 ). Here we have shown that glucosaminidase activity is optimum at pH 5. Germination at pH 5 also resulted in a maximal release of peptidoglycan fragments as shown by the amount of A2pm in the exudate. This is in contrast with the lower loss of OD600 compared to spores germinated at pH 710. The loss of OD600 has already been shown to occur in spores without any cortex hydrolysis (Atrih et al., 1998
; Popham et al., 1996
; Sekiguchi et al., 1995
). Therefore, it is likely to be due to the loss of other spore components such as dipicolinic acid, and the uptake of water (Atrih & Foster, 1999
; Sekiguchi et al., 1995
). The effect of pH on germination of B. megaterium and B. subtilis has been previously investigated (Ciarciaglini et al., 2000
; Stewart et al., 1981
). In both organisms optimum absorbance loss occurs at slightly basic pH 79, which correlates with our results.
Another important feature of B. subtilis GSLEs is their activities over a pH range of 410. These observations concur with previous reports on various purified GSLEs from other spore-forming bacteria (Chen et al., 1997 , 2000
; Makino et al., 1994
). The differential activities of the GSLEs at different pH values may be important in underlining their potential role in spore germination in diverse environments.
GSLEs isolated from spore-forming bacteria are not unusually heat resistant and are generally denatured at temperatures lower than those required in inactivating whole spores (Chen et al., 2000 ; Makino et al., 1994
). The extensive studies of Warth (1980)
also indicate that enzymes of central metabolism are inactivated at temperatures 2446 °C lower than those required to inactivate the same enzymes within intact spores.
Heat or alkali treatment of B. subtilis spores resulted in rapid loss of epimerase and glucosaminidase activities. These results suggest that the enzymes themselves, or their activation mechanism(s), are sensitive to heat and alkali. The fact that the two distinct treatments affect the same enzymes suggests that the localization or the mechanisms of activation of these enzymes are different to that of the lytic transglycosylase. The NaOH effect implies a close association or location of some germination components with the alkali-soluble proteins, which are removed by such treatment (Duncan et al., 1972 ; Gould et al., 1970
). The lytic transglycosylase is not affected by heat or NaOH and thus this enzyme is protected to a much greater degree than the other enzymes. Alternatively, the glucosaminidase and epimerase may be protected, but their activation mechanism(s) is not.
In B. subtilis, sleB and the adjacent downstream ypeB gene form a bicistronic operon (Moriyama et al., 1999 ), and both genes are necessary for cortex hydrolysis (Boland et al., 2000
). SleB is most likely a lytic transglycosylase (Boland et al., 2000
) and is located in a mature form on the outside of the cortex in the dormant spore (Moriyama et al., 1999
). This localization of the enzyme does not correlate with its exceptional resistance to heat and alkali, suggesting that it may have a unique protection mechanism. Studies using FB101 (sleB) highlight the pH dependence of germination enzyme activities. In FB101 (sleB) the maximal release of peptidoglycan material during germination occurred at pH 5, the pH at which glucosaminidase activity is greatest. The role of the likely epimerase in cortex hydrolysis is not clear, as it is not a hydrolytic enzyme. However, it has been previously suggested to cause an alteration in peptidoglycan conformation which may affect the activity of the GSLEs (Atrih et al., 1998
). The present study suggests that the epimerase is not necessary for cortex hydrolysis since the lytic transglycosylase is active in the absence of significant epimerase activity.
CwlJ is another apparent GSLE involved in cortex hydrolysis (Ishikawa et al., 1998 ). The hydrolytic bond-specificity of this enzyme is still unknown and the double mutant sleB/cwlJ is unable to hydrolyse the cortex as evidenced by the absence of peptidoglycan fragments in the germination exudate (Ishikawa et al., 1998
). Thus, the epimerase and glucosaminidase activities are unable to initiate cortex hydrolysis. Also, CwlJ activity or its activation mechanism must be destroyed by heating, as FB101 (sleB) spores show reduced heat resistance compared to the parent. The lytic transglycosylase is probably the only significant enzyme which survives heat treatment and is able to hydrolyse the cortical peptidoglycan to allow outgrowth. The heat sensitivity of FB101 (sleB) spores is not due to the core dehydration level, as the core wet density of the mutant is comparable to that of wild-type spores (results not shown).
From previous work it was not apparent whether the products of glucosaminidase or lytic transglycosylase activities are acted on by the epimerase (Atrih et al., 1998 ). In this study, the lytic transglycosylase products are not altered by epimerase because their retention time is identical in the presence or absence of epimerase. Similarly, partially purified glucosaminidase from B. megaterium generated products with identical retention time to those obtained in the presence of epimerase (Atrih et al., 1999
). It is possible that epimerase products are resistant to the action of other enzymes, and that the subtle modification of the peptidoglycan may direct hydrolysis to optimize the hydrolytic effect of the other GSLEs. Thus, the increase in lytic transglycosylase activity in both the heat- and alkali-treated spores could be explained by the availability of substrate in the absence of the other enzymes.
The loss of spore viability during heat treatment cannot be correlated directly with GSLE activities. Indeed, after 1 h treatment of spores at 90 °C only 8% of spores remained viable but the lytic transglycosylase was still fully active. Thus, even though cortex hydrolysis is taking place in this case, it is likely that outgrowth or vegetative growth components have been damaged, thus preventing colony formation. This is confirmed by the inability of lysozyme to recover heat-treated spores of C. perfringens (Duncan et al., 1972 ; Labbe & Chang, 1995
). Enzymes and other proteins are the main targets for heat-killing of spores (Belliveau et al., 1992
; Marquis et al., 1994
; Nicholson et al., 2000
); however, it is still unclear which proteins are the critical targets for such treatment (Nicholson et al., 2000
).
Alkali treatment of spores, although it affects glucosaminidase and epimerase activities, does not affect spore viability. Also, CwlJ activity must be inactivated by alkali as sleB spores are much more sensitive than the wild-type to this treatment. Thus, the glucosaminidase, epimerase and CwlJ activities are not crucial for germination in alkali-treated spores; however SleB certainly is. In C. perfringens both heat and alkali treatment reduced the apparent viability of spores to a comparable level (Duncan et al., 1972 ). Furthermore, the loss of viability in alkali-treated spores was linked to inactivation of GSLEs. This implies that although the bacterial spores share a conserved peptidoglycan structure, the GSLEs involved in its hydrolysis show some diversity (Atrih & Foster, 1999
, 2001
; Foster & Johnstone, 1988
; Moriyama et al., 1999
).
Previous studies have shown that heat combined with additional controlling factors (pH, organic acids, preservatives) affects Bacillus species spore viability (Oloyede & Scholefield, 1994 ), outgrowth and germination (Ciarciaglini et al., 2000
). However, none of these studies identified the molecular target of the treatments. Here we have shown that the major GSLE, SleB, is a critical resistance determinant for B. subtilis spores in response to heat, and in particular NaOH. Thus, development of inhibitors of GSLEs coupled with sporicidal treatments may provide novel combinatorial approaches to prevent outgrowth of potentially deleterious spore-formers. It is by a further understanding of the molecular mechanisms of spore germination and resistance that rational spore control measures can be developed.
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ACKNOWLEDGEMENTS |
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Received 6 June 2001;
revised 13 July 2001;
accepted 20 August 2001.