Mineralization and responses of bacterial spores to heat and oxidative agents

Transcription

1 FEMS Microbiology Reviews 14 (1994) Federation of European Microbiological Societies /94/$26.00 Published by Elsevier 375 FEMSRE Mineralization and responses of bacterial spores to heat and oxidative agents Robert E. Marquis * and Soon Young Shin Department of Microbiology and Immunology, The Unieersity of Rochester Medical Center, Box 672 Rochester, NY , USA Abstract: Mineralization of bacterial spores with Ca 2+ and a variety of other mineral cations enhances resistance to heat damage. Part of the enhancement is associated with increased dehydration of the mineralized protoplast or spore core, while part is independent of dehydration and effective for resistance even to dry heat. Spore mineralization was found also to enhance resistance to oxidative damage caused by agents such as tertiary butyl hydroperoxide or H20 2. In contrast, mineral cations in the environment increased oxidative damage, presumably by catalyzing radical formation. Metal ion chelators such as o-phenanthroline protected spores against such damage. Key" words: Mineralization; Spores; Heat resistance; Resistance to oxidative chemicals; Bacillus General background Bacterial endospores are of major importance in human affairs, in large part because of their phenomenal resistance to heat, radiation and germicides. A large component of industry is focused on their destruction. For example, in the food industry, the canning process is designed specifically to inactivate spores of organisms such as Clostridium botulinum, which produces one of the most potent exotoxins known. The need to reduce spore populations in foods is not just to avoid disease but also to avoid spoilage by vegetative organisms formed from spores that germinate during storage. The most reliable means to destroy spores is with moist heat. However, spores have high levels of resistance to moist heat, and * Corresponding author. Tel.: (716) ; Fax: (716) also to dry heat. Ionizing radiation has had more limited application in the food industry because it can create off flavors in many foods. However, its use is growing for limited applications such as reducing microbial and spore numbers in spices or on the surfaces of fruits and vegetables. In addition, oxidative agents are used widely in the food industry, for example, in sterilizing packaging materials for use with food sterilized by hightemperature, short-time processes. There is also a very large industry supplying sterile materials for use in medicine, dentistry and science. Radiation sterilization is common in this industry, as are processes based on moist or dry heat and on sporicidal chemicals. The major function of bacterial spores is to preserve the organisms during difficult periods in the environment, particularly in soil. Bacterial endospores are the most dormant cells known, and their extremely low level of metabolism can allow for survival of individuals for centuries. SSDI (94)00016-R

2 376 Thus spores are resistant to aging as well as to deleterious factors such as heat, radiation and chemicals. The longevity of spores is useful for preserving sporogenous bacteria in the laboratory but can be of concern in environmental release and persistence of the organisms. Fortunately, many types of asporogenous mutants are available for genetic engineering projects. The impetus to understand and control spore resistance is clear. The outstanding resistance of the organisms is to heat, and in the extreme, super resistant spores can survive autoclaving at 121 C for 20 min. The upper temperature for growth of the extreme thermophiles from places such as the hydrothermal vents of the ocean floor appears to be some 120 C to 130 C [1]. The most resistant spores can remain viable at temperatures of some C higher than this limit, but of course, the spores are dormant. However, spores still are the most thermotolerant forms of life and set the upper temperature limit for life, albeit life in the dormant state. The resistance of spores to moist heat involves multiple known components, as reviewed in detail by Gerhardt and Marquis [2]. One is so-called inherent or molecular resistance related to the heat stabilities of biopolymers produced by the organisms. In general, thermophilic spore-formers produce spores that are more heat resistant than those of mesophiles, which in turn produce spores more heat resistant than psychrophilic spores [3]. The other major components of spore heat resistance are cellular and peculiar to the spore state. In general, molecules such as enzymes are stable within spores at temperatures some 40 C higher than within vegetative cells or extracts of disrupted spores [4]. This stabilization depends on dehydration of the spore core or protoplast and on specific mineralization, primarily with calcium but also with other minerals [5]. Mineralization begins usually in stage 4 of sporulation, somewhat before synthesis of dipicolinate, and is associated with development of specific mineral transport systems in the sporangeal cells. Spore heat resistance can generally be increased by raising the temperature for growth and sporulation within the limits permitted by the temperature range of the spore former. However, the increase appears to be due to enhanced dehydration at higher temperatures and not to changes in the inherent resistances of biological structures to heat damage [6]. Minerals and heat resistance Procedures for altering mineral contents of spores Mineral contents of spores can be manipulated by altering the mineral content of the medium used for growth and sporulation [7]. However, this approach is limited because of sensitivities of growth to mineral levels, especially those of toxic minerals such as Mn. Once cells in batch culture have committed to sporulation after the end of exponential growth, they can be removed to a simpler medium for so-called endotrophic sporulation. Minerals added to the endotrophic sporulation medium are then incorporated into the developing spores [8]. The best means to manipulate spore mineral contents is through ion-exchange, as first described by Alderton and Snell [9], In subsequent studies, Bender and Marquis [10] developed procedures for controlled total mineral exchange of a variety of spore types. The procedures allowed for total removal of minerals in acidified media and total replacement with a range of desired minerals, all without any reduction in the viability of the populations used. Hierarchy for protection Bender and Marquis [10] concluded from an extensive series of experiments that the hierarchy for protection against moist heat damage for Bacillus megaterium ATCC 19213, Bacillus subtilis niger and Bacillus stearothermophilus ATCC 7953 was Ca > Mn > Mg > K. H and Na appeared to be ineffective for protection. The minerals tested were those normally found in quantity in native spores. When trace minerals such as Li, Sr, Cu and La were used for remineralization, the resultant spores had low levels of heat resistance, part of which may have been associated with toxicity. However, totally demineralized spores, or socalled H-spores, still were more heat resistant than germinated spores or vegetative cells of the

3 377 same strain. Therefore, protection against heat damage due to mineralization is imposed on a base of protection associated with the spore state, probably mainly due to dehydration of the spore core. Moreover, Beaman and Gerhardt [6] found that protoplasts of mineralized spores were more highly dehydrated than those of H-spores. Minerals and dipicolinic acid A long standing view in sporology is that divalent cations and dipicolinate (DPA) are associated in the spore, probably in the form of a chelate complex. However, it now seems that the two can go separate ways [2]. Generally, DPA uptake occurs slightly later in sporulation than the major uptake of minerals. Also, during ionexchange, minerals can be removed and added back independently of DPA. There is substantial evidence against any major role for DPA in heat resistance, including findings that fully resistant, DPA-less spores can be produced genetically or by ion-exchange. Clearly views of the importance of DPA in heat resistance are in flux, and there is now need for more work on the role of DPA in spore biology. States of minerals in spores Multiple approaches have been used to determine the locals of minerals in spores and their physicochemical states. The integuments as well as the protoplasts of spores appear on the basis of X-ray microanalyses, for example, those of Stewart et al. [11], to be mineralized. However, the mineral levels in the protoplast appear to be higher than those in the integuments. The results of dielectric studies of fully viable spores [12] led to the conclusion that the mineral ions within the cells are nearly completely immobilized, even for high-frequency electric current of some 50 MHz. In fact, spores represent a biological extreme in terms of low electrical conductivity. The idea of a nonconducting spore protoplast is in line with knowledge of dehydration and of extreme dormancy and resistance of spores. However, the finding of low conductivity even for the peptidoglycan cortex is very much contrary to the usual views of a highly charged, loosely cross-linked structure [13]. The minerals in spores do not appear to form crystalline phases but presumably form complexes with spore polymers and lowmolecular-mass anions. Mechanisms of protection Knowledge of the molecular mechanisms by which minerals enhance heat resistance is very sketchy. Enhanced protection due to dehydration associated with mineralization can be related to known stabilization of proteins against heat damage in the dry state [14]. However, just how dehydration of the protoplast confers protection is not entirely clear because protoplasts of spores in aqueous media contain more than 27 g water per 100 g wet protoplast weight, or more than sufficient to hydrate spore biopolymers. Thus, the protoplast has reduced water content but is certainly not dry. Water in the protoplast appears to be fully mobile on the basis of its nuclear magnetic resonance and dielectric behavior [15,16] and not in a glassy state. The stabilization of spores by minerals that is indepcndent of dehydration could involve reduced thermal mobilities of molecules at high temperatures because of electrostatic bonding. At this time, there just is not sufficient information to be other than speculative about stabilization mechanisms. One interesting case of stabilization is that described by Ando and Tsuzuki [17] for Clostridiltm perfringens NCTC 8238 in which minerals appear specifically to protect enzymes required for cortex lysis during germination. Thus, heated, demineralized spores become superdormant because of damage to the enzymes. Treatment of the superdormant spores with alkali and plating on media with added lysozyme returned the ceils to life. However, this situation is not common, and heat-killed spores generally are not simply in a superdormant state. Minerals and oxidative damage Protective role of minerals Oxidative chemicals such as H202 or organic peroxides such as tertiary butyl hydroperoxide (t-booh) are sporicidal. It is generally considered that these agents are not themselves lethal

4 378 but must be converted to toxic radicals such as hydroxyl radical. Radical scavengers have been found to be protective [18]. Radical formation in biological systems commonly involves metabolism. However, since spores are extremely dormant, spore killing presumably depends on non-metabolic chemical formation of radicals. Spore killing by these agents has a very marked dependence on temperature, and for example, we have found that the dose of t-booh required to kill spores of B. megaterium ATCC decreased by a factor of ten for each 15 C increase in temperature in the range from 0 to 70 C, a range below the threshold for heat killing. As shown by the data presented in Fig. 1, H spores of this organism demineralized by means of standard acid extraction procedures [10] were more sensitive to killing by t-booh than were fully mineralized spores, here at a ph value of 6.0 at 50 C. Remineralization of the H spores with Ca + resulted in a regain of native resistance. Int,oh,ement of enttironmental minerals" in damage Cu 2+ ions have been found to be sensitizers for damage to spores caused by H20 2 [19,20]. We were not able to demonstrate this type of sensitization for t-booh killing of spores of B. megaterium ATCC 19213, nor for H20 2 killing, presumably because these spores have sufficient o 6 o i i - i Hourm Fig, 1. Enhanced killing of demineralized spores of B. rnegaterium ATCC by 72 mm t-booh at 50 C and ph 6./). Spores were demineralized by the procedure of Bender and Marquis [10]. Data are shown for native spores (0) and for demineralized H spores ( ). g g i1 o ~ 8 0 _J 7 6 i i! i Hours Fig. 2. Protection by o-phenanthroline against killing of spores of B. rnegaterium ATCC by 72 mm t-booh at 50 C and ph 6.0. Data are shown for suspensions with ([]) or without ( ) 10 mm o-phenanthroline. transition metals so that supplementation does not enhance radical formation. However, as shown by the data in Fig. 2, a metal chelator, o-phenanthroline, at a concentration of 10 mm was highly protective against oxidative killing involving t-booh. The net conclusion is that environmental ions, specifically transition metal ions, are detrimental to the spores because they catalyze non-metabolic formation of radicals, which can then penetrate into the spore and cause damage. However, internal minerals appear to be protective against damage, possibly because the highly oxidized state of the spore interior [21] allows for non-damaging electrons transfer involving radicals. Acknowledgement The work of the authors was supported by award DAAL03-90-G-0146 from the US Army Research Office. References 1 Pool, R. (1990) Pushing the envelope of life. Science 247, Gerhardt, P. and Marquis, R.E. (1989) Spore thermoresistance mechanisms. In: Regulation of Procaryotic Develop-

5 379 ment (Smith, I, Slepecky, R.A. and Setlow, P., Eds.), pp American Society for Microbiology, Washington, D.C. 3 Warth, A.D. (1978) Relationship between the heat resistance of spores and the optimum and maximum growth temperatures of Bacillus species, J. Bacteriol. 134, 699 7(15. 4 Warth, A.D. (1981) Stabilization of spore enzymes to heat by reduction in water activity. In: Sporulation and Germination (Levinson, H.S., Sonenshein, A.L. and Tipper, D.J., Eds.), pp American Society for Microbiology, Washington, D.C. 5 Murrel[, W.G. and Warth, A.D. (1965) Composition and heat resistance of bacterial spores. In: Spores III (Campbell, L.L. and Halvorson, H.O., Eds.), pp American Society for Microbiology, Ann Arbor, MI. 6 Beaman, T.C. and Gerhardt, P. (1986) Heat resistance of bacterial spores correlated with protoplast dehydration, mineralization, and thermal adaptation. Appl. Environ. Microbiol. 52, Crosby, W.H., Greene, R.A. and Slepecky, R.A. (19711 The relationship of metal content to dormancy, germination and sporulation in Bacillus megaterium. In: Spore Research 1971 (Barker, A.N., Gould, G.W. and Wolf, J., Eds.) pp Academic Press, I~ndon. 8 Foerster, H.F. and Foster, J.W. (19661 Endotrophic calcium, strontium, and barium spores of Bacillus rnegateriurn and Bacillus cereus. J. Bacteriol, 91, Alderton, G. and Snell, N. (1963) Base exchange and heat resistance in bacterial spores. Biochem. Biophys. Res. Commun. 10, Bender, G.R. and Marquis, R.E. (1985) Spore heat resistance and specific mineralization. Appl. Environ. Microbiol. 50, Stewart, M., Somylo, A.P., Somylo, A.V., Shuman, H., Lindsay, J,A. and Murrell, W.G. (1980) Distribution of calcium and other elements in cryo-sectioned Bacillus cereus T spores determined by high-resolution scanning electron probe X-ray microanalysis. J. Bacteriol. 143, Cartensen, E.L., Marquis, R.E., Child, S.Z. and Bender, G.R. (19791 Dielectric properties of native and decoated spores of Bacillus megaterium. J. Bacteriol. 14(I Warth, A.D. (1978) Molecular structure of the bacterial spore, Adv. Microb. Physiol. 17, Klibanov, A.M. and Ahern, T.J. (1987)Thermal stability of proteins. In: Protein Engineering (Oxender, D.L. and Fox, C.F., Eds.) pp Alan R. Liss, New York, NY. 15 Carstensen, E.L., Marquis, R.E. and Gerhardt, P. (1971) Dielectric study of the physical state of electrolytes and water within Bacillus cereus spores. J. Bacteriol. 107, Bradbury, J.H., Foster, J.R., Hammer, B., Lindsay, J. and Murrell, W.G. (1981) The source of the heat resistance of bacterial spores; study of water in spores by NMR. Biochim. Biophys. Acta 678, Ando, Y. and Tsuzuki, T. (19831 Mechanism of chemical manipulation of heat resistance of Clostridium perfrmgens spores. J. Appl. Bacteriol. 54, Ando, Y. and Tsuzuki, T. (1986) The effect of hydrogen peroxide on spores of Clostridium perfringens. Lett. Appl. Microbiol. 2, King, W.L. and Gould, G.W. (1969) Lysis of bacterial spores with hydrogen peroxide. J. Appl. Bacteriol. 32, Bayliss, C.E. and Waites, W.M. (19761 The effect of hydrogen peroxide on spores of Clostridium bifermentans. J. Gen. Microbiol. 96, Singh, R.P., Setlow, B. and Setlow, P. (19771 Levels of small molecules and enzymes in the mother cell compartment and the forespore of sporulating Bacillus megaterium. J. Bacteriol. 130,