J. appl. Bact. 1976,40,229-244

Fifth Stenhouse-WiII iams Memorial Lecture Oxygen and the Obligate Anaerobe

J.

G. MORRIS

Department of Botany & Microbiology, School of Biological Sciences, University College of Wales, Aberystwyth SY23 3DA, Wales, U.K.

Contents Introduction . . . . . . . . . . .. . . Measuring differences in oxygen sensitivities . . . . . . Oxygen sensitivity and culture E h values .. .. .. Possible causes of oxygen toxicity .. . . . . .. Chemistry and biochemistry of oxygen and its derivatives . . (a) Oxygen . . .. . . . . . . . . .. (b) Peroxide anion . . . . .. . . . . .. .. (c) Superoxide anion and hydroxyl radical . . . . (d) Superoxide dismutase . . .. .. . . . . 6. The superoxide dismutase theory of obligate anaerobiosis . . 7. Which derivative of oxygen is potentially most hazardous? . . 8. Summary . . .. .. . . .. .. . . .. 9. What of the future? . . . . . . . . . . .. 10. References . . . . . . .. . . .. .. . . 1. 2. 3. 4. 5.

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Introduction SOMEOF THE terms currently used to describe the oxygen relations of living organisms are so unhelpful that I feel the need at the outset to define what I mean by an obligate anaerobe. I would describe an obligate anaerobe as an anoxybiontic, aero-intolerant organism, that is, an organism which (1) is capable of generating energy and synthesizing its substance without recourse to molecular oxygen, and (2) demonstrates a singular degree of adverse sensitivity to oxygen which renders it unable to grow in air at 1 atmosphere. It is with the second of these properties that I shall be concerned here i.e. the nature of aero-intolerance. But first let us examine the trawl of organisms that are caught in the twin-stranded net of my definition of an obligate anaerobe. It includes very few eukaryotes and it has been suggested that in these, obligate anaerobiosis may be a secondarily acquired characteristic selected for by retrogressive evolution in specialized oxygen-free habitats. In fact, amongst eukaryotes, the only strict anaerobes are some protozoa isolated from highly anaerobic locations e.g. from the digestive tracts of termites or herbivores, or from deep bottom muds. Thus, it is amongst the prokaryotes that we find the vast majority of obligate anaerobes, and I am certain that to a gathering of Applied Bacteriologists I have no need to spell out the very great diversity of bacterial genera represented, or the biochemical diversity that these

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display in the variety of organic and inorganic compounds that between them they can exploit as electron donors and acceptors in anaerobic energy generation-in the processes of fermentation, non-oxygen-generating photosynthesis and anaerobic respiration. With the improved techniques now available to us for the isolation and examination of aero-intolerant bacteria, we are daily being made aware not only of the existence of new species with novel biochemical attributes, but more particularly of the widespread distribution of so many of these organisms. Whereas it has been usual to associate an obligately anaerobic microflora with oxygen-free habitats, we now recognize that obligate anaerobes may flourish in what at first sight would appear to be wholly inappropriate aerobic locations. Whereas it does not astound us to learn that over 97 % of the human and faecal microflora is composed of anaerobic bacteria (Moore & Holdeman, 1972) it continues to surprise students at least, that on the surface of the human skin anaerobes outnumber aero-tolerant organisms by 10 : 1, whilst on the mucous membrane of the mouth the ratio (30 : 1) is even more in favour of the strict anaerobes (Rosebury, 1962). A second misconception that must be dispelled is that the obligate anaerobes are a homogenous group of organisms, for this is not true even of their oxygen relations. Whilst inability to grow in 0.2 atm. of oxygen is of enormous practical significance in that it dictates the manner in which the organism must be isolated and handled in the laboratory, it is important to realize that oxygen is potentially toxic to all living cells (aero-intolerant and aero-tolerant species alike). However, organisms diEer in their sensitivity to oxygen poisoning, such that one discerns a continuous spectrum of oxygen tolerance from the most sensitive, strict anaerobe to the least sensitive hyperaerobe. Tolerance of air at 1 atm. containing 20% 0 2 , represents but one line drawn on this spectrum of oxygen sensitivity, which extends below, as well as above, this particular concentration of oxygen. It is helpful when considering the problem of oxygen toxicity to recall the evolutionary history of our contemporary biosphere. Geologists, paleobiologists and phylogeneticists tell us that the first four-fifths of the total span of life on this earth was accomplished under anaerobic to semi-anaerobic conditions. When the first prokaryotic life forms made their appearance some 3.5 x lo9 years ago, they emerged on a planet whose surface was highly reduced and whose atmosphere was totally devoid of oxygen. Even when the first oxygen-evolving photosynthetic organisms made their appearance (first the cyanobacteria and later the green algae), the oxygen content of the atmosphere increased only slowly, possibly due to the considerable quantities of auto-oxidizable inorganic compounds (e.g. Fez ions) then present in solution in the oceans. Geological evidence suggests that the most abrupt increase in the oxygen content of the atmosphere occurred only some 640 million years ago when these reservoirs of reductants were exhausted. It requires little imagination to see why the availability of molecular oxygen opened up startling new vistas of evolutionary advance. Its ready diffusibility and reactivity meant that it supplied the requirement for an almost universally accessible electron acceptor of pleasingly high potential, the major product of whose reduction (Hz0) was innocuous and disposed of easily. Yet that very reactivity which was the basis of its effectivenessas an electron acceptor in aerobic respiration and as a substrate in new oxygenase-mediated biosynthetic and catabolic processes, also posed a threat to +

OXYGEN AND THE OBLIGATE ANAEROBE

23 1

organisms which did not simultaneously evolve means to deal with undesirable by-products of its consumption, or to protect or modify key ‘autoxidizable’ cellular components. It is by examining (1) the possible nature of toxic products of oxygen consumption and of the cellular ‘targets’ most prone to attack by these and/or oxygen itself, and (2) the various defence mechanisms against oxygen toxicity that contemporary aero-tolerant organisms now possess, that we can hope to obtain rational explanations for the apparent super oxygen-sensitivity of present day aerointolerant species. Taking this evolutionary stance, we are led to expect no singular conformity in the reasons why contemporary obligate anaerobes are aero-intolerant. Furthermore we are encouraged to presume that any information we can glean concerning the cause of oxygen toxicity in aero-tolerant organisms (even though this will be displayed only at unusually high concentrations of oxygen), could prove to be relevant to the plight of the obligate anaerobe.

Measuring Differences in Oxygen Sensitivity When atmospheric oxygen is rigidly excluded during the primary isolation of anaerobic bacteria from gingival debris, some 50 to 60% of the total microscopic count can be recovered as single colonies. When the same specimen is manipulated in room atmosphere and then incubated anaerobically, only 15 to 30% of the total count will grow (Loesche, 1969). Similar findings are made whenever we attempt to isolate the constituents of any mixed anaerobic bacterial community, and we conclude that even obligate anaerobes differ significantly in their sensitivity to oxygen. It is by no means easy, however, to quantify these differences. Methods so far employed include : (a) exposure of spread plates to air for increasing time intervals prior to anaerobic incubation (Loesche, 1969); (b) incubation of spread plates under artificial atmospheres containing different concentrations of 02 (Loesche, 1969); (c) exposure to test atmospheres of organisms deposited on the surface of a membrane filter (Brown & Huggett, 1968), and (d) estimation of the depth of oxygen inhibition zones in serially diluted deep agar cultures incubated under test atmospheres (Schwartz, 1973, 1975). From his findings with a selection of anaerobes, Loesche (1969) concluded that at least three subgroups were identifiable viz: (1) strict anaerobes which would grow on spread plates only when the atmosphere contained less than 0.5 % 0 2 e.g. Treponema denticola, Selenomonas ruminatium, Clostridium haemolyticum ; (2) moderate anaerobes which would generally grow on plates when the atmosphere contained less than 10 % 0 2 e.g. Bacteroides fragilis, Clostridium novyi Type A, Peptostreptococcus elsdenii; ( 3 ) microaerophiles which require a low concentration of 0 2 for optimal growth but are still unable to grow in air e.g. Vibriofetus. In all such surveys the main basis for concern is that to satisfy the growth requirements of all organisms a common, nutrient-rich medium must be employed, even though this will sustain different growth rates of the test species. In his deep agar procedure, Schwartz (1973) has attempted to minimize some of the potential sources of error by making corrections for different media, growth rates and differences in cell concentration in test cultures, obtaining, thereby, comparable values which he terms the ‘constant of oxygen sensitivity’ and the ‘modification factor’ for each organism. Thus, when he compared the oxygen sensitivity of Clostridium sticklandii with that of Propionibacterium shermanii PZ3, although

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under uncontrolled conditions it would appear that CI. sticklandii is decidedly the more fastidious anaerobe, by his criteria, the two species actually differ very little in their sensitivity to oxygen (Schwartz, 1975). It is plain that there is scope for considerable experimentation in devising novel means of assessing the oxygen sensitivities of different bacterial species. However, it is doubtful whether any determined ‘constant’ of oxygen sensitivity will prove to be as invariable as one would wish, even for a single strain. It is a not uncommon finding that some organisms which when first isolated grow only under strictly anaerobic conditions, may become more oxygen tolerant after two or three subcultures (Willis, 1969), whilst in batch growth, cells harvested from exponential phase cultures may be more oxygen sensitive than those taken from the succeeding stationary phase (Smith, 1973). Again, as Louis Smith (1973) has pointed out, individual cells in a pure culture can vary tremendously in their oxygen sensitivities. For example, if viable counts are performed on a culture of Propionibacterium acnes using strictly anaerobic (Hungate roll-tube) procedures, a thousand times greater recovery will be obtained than if the count is performed by surface inoculating blood agar plates and then incubating these anaerobically. Nor are all such individual differences phenotypic in character. Recently, both aero-tolerant and aero-sensitive mutant strains of Clostridium perfringens Type A, strain 19P were isolated, which also differed in their toxicity, ability to sporulate and capacity to produce butyric acid from glucose (Zhvadovh, Mikulik & Sebald, 1974).

Oxygen Sensitivity and Culture Eh Values It is universally recognized that even total exclusion of molecular oxygen is not sufficient to ensure growth of most strict anaerobes. These organisms develop and maintain a low E h in their cultures, and reducing agents which facilitate the establishment of an agreeably low redox potential are generally useful in aiding initiation of growth of all anaerobes, especially from small inocula. The standard growth media for anaerobic bacteria are therefore supplemented with SH-compounds such as thioglycollate, or cysteine plus or minus dithiothreitol, or with other reductants such as ascorbate. Though it is virtually impossible to define what we measure as the E h of a bacterial culture (Morris, 1975), we all accept that shifts in this value reflect changes in the balance between the contending oxidizing and reducing agencies that are its prime determinants. Considerable effort has gone into delineating the maximum E h values compatible with growth of various species of anaerobes. Thus upper limits between + 30 mV and +250 mV have been reported for various Clostridium spp., but these values were determined in media of disparate composition and widely differing pH values, with further complications being introduced by vagaries in the size and constitution of the inocula employed. Since oxygen can be one important determinant of the culture E h , input of oxygen tending to increase its value, it has frequently been proposed that oxygen exerts its toxic action through its elevation of the culture Eh. This argument is not easily disposed of, especially when examples can be cited of moderate anaerobes which are seemingly more tolerant of oxygen when their culture E h is kept at a relatively low value by electrical means (Smith, 1973). Of course, contrary evidence can also be cited, for example of the behaviour of Clostridium acetobutylicum which grew at an E h of + 370 mV when this was artificially maintained by ferricyanide, though not at + 100 mV when this E h was produced by aeration.

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Furthermore aeration of the ferricyanide-poised culture then halted growth even though there was no further increase in culture Eh (O’Brien & Morris, 1971). From the wealth of anecdotal evidence of this kind that is recorded in the literature, it seems reasonable to conclude that anaerobes generally grow best under so-called ‘reducing conditions’ wherein there is minimal drainage from the organisms of reducing power which otherwise they could more productively employ for energy-yielding or biosynthetic purposes. The establishment and maintenance of these desirable equilibrium conditions calls for a reducing contribution from the organisms themselves, but anaerobes differ in their capacity to generate the necessary excess (disposable) reducing power. Those with the least capacity possibly have a minimally flexible, tightly coupled cycle of endogenous electron generation and acceptance which leaves little scope for diversion of electron flow for reduction of exogenous oxidants. These, therefore, are the species that are most threatened by addition to the medium of oxidants and whose growth is most benefited by exogenous reductants. These will be the organisms unable to grow in media that have not been pre-poised at a low Eh. If this were all, then oxygen should pose no greater threat to the anaerobes than any other equally potent exogenous oxidant (though as we well know, oxygen as a biological electron acceptor is unique in its effectiveness and catholicity of action). However, there is evidently more to oxygen’s action than this; having conceded its ability to serve as a general oxidant, we must still conclude that specific toxic effects flow from oxygen consumption. It is in the search for the causes of this specifically injurious action of oxygen that progress has been relatively slow. Possibly this is not surprising, for it is not a simple matter to separate the twin effects of oxygen as oxidant and oxygen as toxic agent. For example, the possibility that some of the toxic effects of oxygen are mediated via oxygen-free radicals, leads us to reassess the role of those thiols known to be particularly beneficial to the growth of anaerobes and which we had previously viewed merely as convenient reducing agents. These are now invested with the additional virtue of being potential scavengers of free radicals. Other components of the growth medium might similarly not serve merely as nutrients or reductants/oxidants, they might themselves generate or neutralize toxic by-products of oxygenation. Even the hydrogen present in most standard anaerobic gas mixtures acquires a new significance. Not only is it a prime reductant capable of aiding the process of lowering culture Eh by reducing certain constituents of the medium and ‘priming’ any inoculum which possesses hydrogenase activity, it must also be considered a possible protective against oxygen-free radicals. [Indeed, hyperbaric hydrogen therapy has been proposed as a cancer remedy on the basis that H2 destroys oxygenfree radicals (Dole, Wilson & Fife, 1975).] No wonder it is impossible for us to make simple correlations between culture Eh limits and the effects of oxygen on all anaerobes in all growth media. Following each thread of evidence we soon find ourselves enmeshed in a tangled skein of multi-stranded components, which is thereafter very difficult to unravel.

Possible Causes of Oxygen Toxicity It might be as well at this point to summarize the main hypotheses proposed over the years to explain the oxygen intolerance of obligate anaerobes.

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(a) Oxygen is toxic Oxygen is toxic by virtue of its excellence as an oxidant. (i) Presence of free oxygen in the culture medium is incompatible with the attainment and maintenance of the low culture Eh value required for growth. (ii) Preferential reduction of exogenous oxygen drains the organism of reducing power which it requires for biosynthetic purposes. (iii) The anaerobe is particularly vulnerable to oxygen because its metabolism and growth depend on key components liable to ‘autoxidation’ (SH compounds, iron sulphur proteins, tetrahydropteridines, flavoproteins, have been singled out for special mention). (iv) Growth is inhibited due to oxidation of a key metabolic regulator, presumed either to react directly with oxygen or to be in equilibrium with another auto-oxidizable redox couple. (This is a somewhat more sophisticated variant of hypotheses (ii) and (iii).) (b) Oxygen is not itself toxic Oxygen toxicity is attributable to products of the interaction of oxygen with the organisms and/or components of their culture media. Unlike aerobes, obligate anaerobes lack the means of detoxifying these substances. (i) Toxic products accumulate in nutrient media exposed to oxygen (organic peroxides, aldehydes, free radicals etc.). (ii) Toxic products (or incidental by-products) are invariably formed by the interaction of oxygen with reduced cell constituents such as iron sulphur proteins, tetrahydropteridines, flavoproteins, as well as by the action of certain oxidases and flavin dehydrogenases (hydrogen peroxide, superoxide anion, hydroxyl radical and singlet oxygen are the chief contenders).

Those hypotheses (a) based on oxygen acting as a preferred oxidant, have been with us for many years and they have been fully discussed in several reviews (Smith, 1967; Morris & O’Brien, 1971; Morris, 1975). I need therefore do no more than remind you that there is evidence that several moderate anaerobes, though they may not grow in air, can withstand exposure to aeration for considerable periods, recovering rapidly and totally when anaerobic conditions are restored. For these organisms, at least in the short term, oxygenation is bacteriostatic rather than bactericidal and the ill-effects of oxygenation are readily reversible-which would tend to argue against gross cellular damage and in favour of diversion of reducing power and displacement of endogenous redox equilibria of the type envisaged in these hypotheses. The cessation of butyrate production in favour of acetate formation in aerated cultures of saccharolytic Clostridium spp. (Aubel & Perdigon, 1945; O’Brien & Morris, 1971) provides evidence of diversion of reducing power, and the abrupt decrease in intracellular ATP content in aerated cultures of CI. acetobutylicum with the equally abrupt rise in ATP level when anaerobic conditions are re-established, illustrates the rapidity of these events (O’Brien & Morris, 1971). Some anaerobes are, therefore, able to sustain their viability for a period when preferential reduction of oxygen causes elevation of intra-cellular E h and consequent, but reversible, metabolic disfunction. This might be thought of as the bacteriostatic Phase 1 of oxygen action, which has its counterpart in those aerobic bacteria which divert reducing power to scavenge molecular oxygen so

OXYGEN AND THE OBLIGATE ANAEROBE

235

as to shield key oxygen-labile components from direct contact with oxygen (Jones, Brice, Wright & Ackrell, 1973). Presumably this defensive mechanism is not available to those strict anaerobes which have no 'spare' reducing power which they may divert to these ends, or which cannot accomplish this reduction of oxygen in an innocuous manner. These would immediately, and at low concentrations of oxygen, be prey to the sort of irreversible damage suffered by moderate anaerobes in the bactericidal Phase 2 of oxygen toxicity (when the reductive Phase 1 defences of the organism have been overwhelmed by continued, excessive oxygenation). It is this potentially lethal damage that is contemplated by those who emphasize the oxygen lability of key cellular components or the devastation that may be wrought by oxygen-derived free radicals (hypotheses b).

Chemistry and Biochemistry of Oxygen and its Derivatives (4 Oxygen Molecular oxygen is relatively insoluble in aqueous media; as Smith (1973) reminds us, its solubility in water is scarcely greater than that of calcium carbonate. With oxygen being present in air at a partial pressure of 0.209 atm, water in equilibrium with air at 1 atm and at 20" will contain only c. 9 mg/l of dissolved oxygen. Because solutes tend to diminish even this solubility, the dissolved oxygen concentration in nutrient bacteriological media exposed to air at 30" may be c. 6-8 mg/l (Brown, 1970). Oxygen is significantly more soluble in organic solvents (generally seven to eight times as soluble as in aqueous media), so that if one were merely to consider the physical solubility of oxygen in the various compartments of a bacterial cell, one would conclude that there is a likelihood of its being concentrated in the lipophilic cell membranes. The chemist recognizes that the oxygen molecule is a powerful oxidizing agent, the two redox couples of traditionally greatest interest to the biologist being those in which the products are water and hydrogen peroxide respectively. 02+4Hf+4e-+2H20 Eo' at pH 7 = +0-815 V 0 ~ +2 H++2 e-+H2 0 2 Eo' at pH 7= $0.270 V. The oxygen molecule is paramagnetic due to two of its valence electrons being unpaired, a property which confers on oxygen the ability to behave as a diradical. This makes oxygen a powerful reagent in initiation and addition reactions and opens up an entire vista of oxygen-free radical pathology (Demopoulos, 1973). But there is a further implication of the electronic configuration of the oxygen molecule. In 'normal' oxygen the two unpaired valence electrons are located in separate orbits and are of parallel spin@@. This is the lowest energy state of the molecule which is known as its ground or triplet state and is represented by the signature 3 2 g . Molecular oxygen in this triplet state may be energized to yield singlet oxygen (102*) wherein the two unpaired electrons are antiparallel in spin. There are thus two forms of singlet oxygen of unequal energies, that form in which the antiparallel, unpaired electrons are in identical orbit being represented as 'Ag, whilst that more highly energized form in which the antiparallel, unpaired electrons are in separate orbits @@ being represented as

00

I&*.

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J. G . MORRIS

It should be of particular interest to bacteriologists that one classical chemical means of making singlet oxygen is the reaction of a hypohalite ion such as hypochlorite with hydrogen peroxide, but singlet oxygen may also be produced in living cells by other systems e.g. by photoexcitation of triplet oxygen in the presence of suitable sensitizers, or the spontaneous dismutation of superoxide anion. Whenever and however it is produced, because of its exceptional reactivity it poses a threat to the integrity of cellular components. However, in aqueous media it is a very short-lived species, and some living cells contain substances which by acting as quenchers of singlet oxygen could minimize its structural and other damaging effects. Possibly again the more acute threat is to those hydrophobic lipid/protein regions within the cell (e.g. membranes) wherein singlet oxygen would not be solvated and could be longer lasting. Though interest in singlet oxygen was first shown by those biologists studying photodynamic and radiation damage to living cells, more widespread concern in this highly reactive species has been shown since it has been suggested that it may play some part in the ageing of cells or even in carcinogenesis (Cusachs & Steele, 1967; Khan & Kasha, 1970). (b) Peroxide anion

If singlet oxygen is a relative newcomer to the bacteriologist's list of potentially dangerous derivatives of oxygen, the peroxide anion is one whose place on this list is well established. Formed by the acceptance of two electrons by the oxygen molecule (thus completing the valence octet) the peroxide anion is not a free radical but is nevertheless chemically very reactive. There are many reports of the bactericidal potency of hydrogen peroxide; if confirmation were necessary, it is given by the use of H202 to minimize bacterial contamination of spacecraft components (Wardle & Renninger, 1975). So much has also been written concerning the widespread formation of hydrogen peroxide in flavoprotein catalysed reactions, its accumulation by catalase and/or peroxidase deficient microbes, and of the toxicity of organic peroxides formed in culture media, that I am sure I need do no more than remind you that the potential hazards posed by the peroxide anion are not lessened by the discovery of more novel toxic by-products of biological oxygen utilization. (c) Superoxide anion and hydroxyl radical

One species which has lately achieved particular prominence is the superoxide anion 0 2 . - which, being the product of univalent reduction of the oxygen molecule, is a highly reactive free radical. This species is synthesized in aerated aqueous solution by numerous procedures effecting the 1 electron reduction of molecular oxygen e.g. ultrasonication, pulse radiolysis, photo-illumination of suitable dyes, reduction by ferrous ions in the presence of phosphate, and also in the course of the interaction of molecular oxygen with various cellular constituents including reduced flavins, flavo-

OXYGEN A N D THE OBLIGATE ANAEROBE

237

proteins, quinones, thiols, iron sulphur proteins and tetrahydropteridines. Superoxide anion is also produced by the action of enzymes including aldehyde oxidase, xanthine oxidase and numerous flavin dehydrogenases. It can act as a potent reducing or oxidizing agent and can serve as an initiator of free radical chain reactions. In aqueous media, superoxide anions are the longest lived of all oxygen-derived free radicals and whilst in aerobic bacteria they may be productively utilized in controlled hydroxylation reactions, there is ample evidence of the damage they can do in uncontrolled reactions with numerous vital cell components. In aqueous solutions superoxide anions interact with each other and are thus removed by dismutation reactions,

+

HOZ* + HO2. -+H202 0 2 k = 7 6 x lo5 dm3mol-ls-I HOz. +02.-+Hf-+Hz02+02 k = 8 - 5 x lo7 dm3m0l-~s-l 02--+02--f2H+-+H202+O2 k = < 1 x lo2 dm3mol-1s-1 which even at pH 7 leads to destruction of superoxide anion at a quite rapid rate (k c. 2 x lo5 dm3mol-1s-1 at pH 7.12; Behar et al., 1970). However this spontaneous dismutation leads to production of singlet oxygen. Furthermore, production of hydrogen peroxide could prove additionally hazardous since, in a process known as the Haber-Weiss reaction, superoxide anion reacts with HZOZto form yet another most dangerous free radical, namely the hydroxyl radical : Oz.-+H202+H++Oz+HzO+HO. Aside from the toxicity directly attributable to hydroxyl radical which is known to account for a considerable part of the extensive cellular damage wrought by ionizing radiation, interaction of hydroxyl radical with superoxide anion could augment the yield of singlet oxygen : 0 2 * - + H O *-+OH-+lOz* Hence in systems producing superoxide anion we have the additional potential of producing three more hazardous derivatives of oxygen viz : hydrogen peroxide, hydroxyl radical and singlet oxygen. No wonder that all organisms (bacteria and eukaryotes alike) that have evolved the means to generate or productively consume oxygen, have simultaneously evolved a means of disposing of superoxide anion by a harmless route. This defence mechanism discovered by Fridovich and his colleagues (Fridovich, 1972) is the possession by all obligate and facultative aerobes of one or more members of a family of enzymes known collectively as superoxide dismutase. (d) Superoxide dismutase

Several types of metalloprotein enzymes have now been discovered whose function it is to catalyse the ‘safe’ dismutation of superoxide anion to yield hydrogen peroxide and (triplet) oxygen. The catalysed reaction proceeds at least ten thousand times more rapidly than the uncatalysed and potentially hazardous spontaneous dismutation of superoxide anion at pH 7, and this high rate of catalysis is sustained over a wide pH range (pH 5 to 10; k = 2 . 3 x 109 dm3mol-1s-1). The nature and properties of the various superoxide dismutases have been fully reviewed by Fridovich (1974a, b ; 1975) so that I need only remind you that whereas the most common

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J. G . MORRIS

superoxide dismutases in the cytoplasm of eukaryotic cells are cyanide-sensitive, Cu-Zn proteins, the bacterial superoxide dismutases are generally cyanide-insensitive, Mn or Fe proteins. Thus the finding of a distinct Mn-protein superoxide dismutase in the mitochondria of eukaryotic cells carrying the Cu-Zn enzyme in their cytosol, and the structural resemblance between this mitochondria1 enzyme and the prokaryotic superoxide dismutases has been seen as confirmation of the prokaryotic origin of the protomitochondrion (Fridovich, 19746). Yet the clearcut distinction between eukaryotic and prokaryotic superoxide dismutases has been eroded by the discovery of a Cu-Zn enzyme (bacteriocuprein) in a bacterium viz. Photobacterium leiognathi (Puget & Michelson, 1974) and the discovery of cyanide-insensitive superoxide dismutases in some facultatively anaerobic protozoa (Lindmark & Muller, 1974). One last remark concerning the mode of action of the superoxide dismutases. There has been considerable speculation as to whether these enzymes serve to quench singlet oxygen however this is formed. Though the controversy on this point is not entirely ended (Weser, Paschen & Younes, 1975) there seems at present no reason to suppose that their necessary function in all aerobic cells is other than to effect the safe disposal of superoxide anions.

The Superoxide Dismutase Theory of Obligate Anaerobiosis This theory states that it is their total lack of superoxide disniutase that is the prime cause of the aero-intolerance of obligate anaerobes. There can be no doubt that in all organisms which normally utilize oxygen, superoxide dismutase plays a crucial protective role. This has been conclusively denionstrated by Fridovich and his colleagues in a series of elegant experiments in which they exploited the observation that several micro-organisms could increase their contents of superoxide dismutase in response to exposure to elevated concentrations of oxygen. For example, Streptococcus fuecalis grown under 20atni of oxygen contained 16 times more superoxide dismutase than did anaerobically grown cells, but possessed no catalase. Escherichia coli B exposed to 5 atm of oxygen synthesized 25 times its normal content of superoxide dismutase but no more catalase (Gregory & Fridovich, 1973a). In each case the cells rich in superoxide dismutase were considerably more oxygen duric than were cells less well endowed with this enzyme. Some comparison of the efficacies of superoxide dismutase and of catalase in affording protection against hyperbaric oxygen was possible using Bacillus subtilis, for this organism was found to increase its content of catalase but not of superoxide dismutase when exposed to elevated concentrations of oxygen (Gregory & Fridovich, 19736). Yet cells containing the increased catalase content fared no better on exposure to 20 atm of oxygen than did cells containing the catalase but identical superoxide dismutase levels. Escherichia coli B was found to contain two superoxide dismutases (I) a constitutive Fe-protein enzyme in the periplasm, and (2) a Mn-protein enzyme in the cytosol, whose synthesis was induced by oxygen. Independent manipulation of the levels of these enzymes (Gregory, Yost & Fridovich, 1973) showed that cells rich in the periplasmic enzyme were particularly resistant to the bactericidal action of exogenously generated superoxide anions, whilst only those cells rich in the cytoplasmic Mnenzyme showed enhanced resistance to hyperbaric oxygen and to streptonigrin (an antibiotic whose action probably involves intracellular generation of superoxide

239

OXYGEN AND THE OBLIGATE ANAEROBE

anion). Similar results were reported with Saccharomyces cerevisiae var. ellipsoideus whose superoxide dismutase and catalase contents were increased by growth under oxygen to give organisms more tolerant of hyperbaric oxygen (Gregory, Goscin & Fridovich, 1974), and again with the cyanobacterium Anacystis nidulans whose growth in the presence of air induced the synthesis of its superoxide dismutase and conferred enhanced resistance to photo-oxidative death (Abeliovich, Kellenberg & Shilo, 1974). In their survey of the superoxide dismutase contents of various bacteria which led to the enunciation of the ‘superoxide dismutase theory of obligate anaerobiosis’, McCord et al. (1971) found no superoxide dismutase in any of the aero-intolerant species they examined (Table 1). It seemed, therefore, that the somewhat hazy line TABLE1 Superoxide dismutase and catalase contents of a variety of micro-organisms* Superoxide dismutase (units/mg) Aerobes: Escherichia coli Salmonella typhimurium Halobacterium satinariuin Rhizobium japonicum Micrococcus radiodurans Saccharomyces cerevisiae Mycobacterium sp. Pseudomonas sp. Strict anaerobes: Veillonetlaalcalescens Clostridium pasteurianum, sticklandii, lentoputrescens, cellobioparum, barkeri Clostridium acetobutylicum Clostridium sp. (strain M.C.) Butyrivibrio jibrisolvens N2C3t Aerotolerant anaerobes: Butyribacterium rettgeri Streptococcus faecalis Streptococcus mutans Streptococcus bovis Streptococcus mitis Streptococcus lactis Zymobacterium oroticum Lactobacillus plantarum

1.8 1.4 2.1 2.6 7.0 3.7 2.9 2.0

Catalase (units/mg) 6.1 2.4 3.4 0.7 289 13.5 2.7 22.5

0 0

0 0

0 0 0 0

0 0.1 10.1

1.6 0.8 0.5 0.3

0.2 1.4 0.6 0

-

0 0 0 0 0 0 0 0

* From McCord, Keele & Fridovich (1971). t N2C3 is an unclassified cellulolytic Gram-negativerod isolated from the rumen of an African zebu steer.

between aero-tolerant and aero-intolerant species could be more satisfactorily defined in biochemical terms viz. presence or absence of superoxide dismutase. However, we have found some small superoxide disniutase activity (Table 2) in a few aero-intolerant bacterial species (Hewitt & Morris, 1975). We have so far been unable to induce enhanced synthesis of the enzymes in the species of Clostridium which contain nieasureable if low concentrations of superoxide dismutase. What we have found however

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J. G . MORRIS

TABLE2 Specific activities of superoxide dismutase in crude extracts of (a) aerobically grown E . coli and (b) several anaerobic bacteria

Organism Escherichia coli B Escherichia coli K-12, mutant AB 1157 Chlorobium thiosulfatophilum NCIB 8346 Chromatium sp. NCIB 8348 Desulfotomaculum nigrificans NCIB 8395 Desulfovibrio desulfuricans NCIB 8307 Clostridiumpasteurianum ATCC 6013 Clostridiumperfringens NCIB 11105

Specific activity of superoxide dismutase (units/mg of protein)* 44.0 36.8 14.0 0.6 2.6 0.6 0.5

15.6

* Superoxide dismutase activity was measured spectrophotometrically in undialysed, crude extracts (from Hewitt & Morris, 1975). (Hewitt & Morris, unpublished) is that Clostridium tertium though capable of growth in air at 1 atm possesses, if anything, a lesser content of superoxide dismutase than does our strain of Clostridium perfringens Type A which is incapable of growing in air at 0 . 1 atm. Also that CI. tertium grown in air apparently contains no more superoxide dismutase than when grown anaerobically. I would at present conclude that superoxide dismutase is indeed required by all normally oxygen-consuming aerobes and that some superoxide dismutase may be possessed even by aero-intolerant bacteria. These may in consequence demonstrate a lesser sensitivity to oxygen than were the enzyme totally absent. However, this last point is by no means proved, and we are unable to conclude that were it only possible to provide any aero-intolerant organism with a sufficiency of superoxide dismutase, it would thereby be rendered aero-tolerant. Indeed this is most unlikely in the case of those strict anaerobes which grow only in media of low Eh.

Which Derivative of Oxygen is Potentially most Hazardous? Which of hydrogen peroxide, superoxide anion, hydroxyl radical (or even singlet oxygen) is the primary lethal agent for a given organism exposed to oxygen in a given culture medium, will doubtless depend on so many factors that it is perhaps of little consequence that apparently conflicting reports already abound in the literature. Thus whilst the periplasmic Fe-protein superoxide dismutase of E. coli B has been reported to confer protection against exogenous superoxide anion (Gregory et al., 1973), using the superoxide-generating drug dialuric acid, Dechatelet, Shirley, Goodson & McCall (1975) have concluded that the contribution of superoxide as exogenous bactericidal agent towards E. coli B is probably minimal compared with that of hydrogen peroxide. Nowhere is the situation more confused than in the interpretations placed on the bactericidal mechanism operative in post-engulfment killing by polymorphonuclear leukocytes. There is evidence for the involvement of a

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myeloperoxidase-HzOz-halide ion mixture, but its bactericidal action has been variously attributed to iodination of the bacterial cell wall (Klebanoff, 1967), scission of peptide bonds in the bacterial cell wall (Selvaraj et al., 1974), generation of toxic aldehydes by deamination of amino acids (Straws et al., 1971), generation of superoxide anion (Babior et al., 1973), generation of singlet oxygen (Allen, Yevich, Orth & Steele, 1974), generation of hydroxyl radical (Selvaraj el al., 1974); the possibly more direct role played by hydrogen peroxide has been recently re-emphasized (Klebanoff, 1974; DeChatalet et al., 1975). Analogies have been drawn with the lactoperoxidase HzOz-thiocyanate bactericidal mixture present in raw milk and saliva, with singlet oxygen production again being proposed, though production of a low molecular weight, dialysable inhibitor has recently been reported in this system (Bjorek, Rosen, Marshall & Reiter, 1975).

Summary There is no doubt that production of both hydrogen peroxide and superoxide anion poses a threat to all bacteria which encounter molecular oxygen, a threat that is amplified by the possible interaction of these substances to produce hydroxyl radical, which is one of the most potent oxidants known to man. Any production of singlet oxygen could also cause cellular devastation. Thus maximum protection would be afforded to that organism which contained (a) a catalase and/or peroxidase to dispose of hydrogen peroxide, (b) a superoxide dismutase to accelerate removal of superoxide anion by a self-destructing mechanism which although producing hydrogen peroxide avoids formation of singlet oxygen, and (c) some means of harmlessly quenching singlet oxygen arising from sources other than superoxide anion. Evidently not all of these defence mechanisms are available even to all aero-tolerant organisms, e.g. Bacillus popilliae is an obligate aerobe which lacks catalase (Costilow & Keele, 1972), but it seems likely that superoxide dismutase is indispensable to all aero-tolerant organisms that normally produce or consume oxygen. (This cautious wording recognizes the possible existence of a totally oxygen-indifferent organism, aero-tolerant because of its non-consumption of oxygen. This was proposed in the case of a strain of Lactobacillus plantarum ATCC 8014 which reportedly contained no catalase, peroxidase or superoxide dismutase but yet was extremely tolerant of oxygen (Gregory & Fridovich, 1974). But more recently, Yousten, Johnson & Salin (1975) have reported that their catalase-negativeL. plantarum ATCC 8014 was able to utilize oxygen and possessed some superoxide dismutase and a NADH peroxidase). Bacteria that are totally devoid of a catalase/peroxidase and superoxide dismutase would be placed at maximal risk by all those intracellular processes, both enzymic and non-enzymic, which yield hydrogen peroxide and superoxide anions once oxygen gained access to their cellular contents. The majority of strict anaerobes probably fall into this category though some aero-intolerant species may possess catalase and/or peroxidase activity (Smith, 1967; Morris, 1975) whilst a few may contain low levels of superoxide dismutase (Hewitt & Morris, 1975). It is interesting in this regard that the aero-tolerant mutant strain of Clostridium perfringens 19P isolated by Zhvadovh et al. (1 974) contained a non-flavoprotein, NADH peroxidase; its superoxide dimutase content, if any, was not measured. Yet again, it is worth emphasizing the significant differences in oxygen sensitivity and its causes, that may exist between otherwise

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similar strains of the same bacterial species-a point well illustrated by the 12 strains of BiJdobacterium examined by de Vries & Stouthamer (1969).

What of the Future? The catalase theory, and its successor the superoxide dismutase theory, have been major milestones in the quest for a satisfactory explanation of obligate anaerobiosis. However, neither has marked the end of this quest, for the broad spectrum of oxygen sensitivity displayed even amongst aero-intolerant organisms has yet to be explained. Furthermore, we now recognize that no unitary hypothesis predicated on the total absence from the organisms of any single protective agent, can wholly explain the toxic effects of oxygen on all obligate anaerobes in all media. Much effort will hereafter be devoted to identification of primary targets of oxygen damage. With the possibility that due to its greater solubility in lipophilic media, molecular oxygen could be particularly concentrated in cell membranes, wherein oxygen free radicals could be longer lived, it is likely that effects of oxygen on membrane functions in anaerobes will be accorded particular attention. Such studies and investigations of other cellular targets of attack by oxygen-free radicals and peroxides, will be complemented by studies on aerobes subjected to hyperbaric oxygen, or to photodynamic and radiation damage, or to oxygen damage during freezing and/or desiccation (Swartz, 1971). There is already evidence of specific oxygen damage to the membranes of frozen and/or lyophilized bacteria e.g. changes in membrane permeability and at the membrane associated DNA initiation complex (Israeli, Kohn & Gitelman, 1975). The anaerobiologist will therefore have to become even more receptive to ideas flowing from the fields of free radical chemistry, radiation biology and membrane biochemistry. Before attempts to isolate and study aero-tolerant mutant strains of obligate anaerobes can hope to reap their full rewards, progress will have to be made on the genetics of some obligate anaerobes-at least sufficient to allow of controlled gene transfer and the mapping of mutant lesions. Until that day arrives, we shall have to be content with the converse mapping of lesions conferring aero-intolerance on mutants of aero-tolerant bacteria amenable to genetic analysis. In this regard we have recently (Hewitt & Morris, unpublished) isolated obligately anaerobic mutants of E. coli K12 which, unlike the temperature-sensitive mutants reported by Fridovich (1 974a), are not afflicted in either of their superoxide dismutases. Possibly the greatest challenge of all, is that posed by the ecology of obligately anaerobic bacteria and their relations with aerophiles. Clinical interest in anaerobic infections and the anaerobic human gut microflora, complemented by evaluative studies of hyperbaric oxygen therapy, plus the enduring interest in the microbiology of the rumen and of anaerobic sewage digestion, will ensure that progress is made in investigations of wholly anaerobic communities. Such studies must however be broadened to include mixed communities of aero-intolerant and aero-tolerant microorganisms so that we may more satisfactorily explain the anachronistic distribution of obligate anaerobes throughout a predominantly aerobic biosphere. Work in our laboratory was supported by the Science Research Council.

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