Antimicrobial Agents and Membrane Function

Antimicrobial Agents and Membrane Function FRANKLIN M. HAROLD Division of Research, National Jewish Hospital and Department of Microbiology, University of Colorado School of Medicine, Denver, Colorado, U . S. A . That which one man gains by discovery is a gain of other men. And these multiple gains become invested capital, the interest in which is all paid to every owner, and the revenue of new discoveryis boundless. It may be wrong t o take another man’s purse, but it is always right t o take another man’s knowledge, and it is the highest virtue to promote another man’s investigation. John Wesley Powell, Director, U.S. Geological Survey, 1886.

I. Introduction . 11. Structure and Functions of Microbial Membranes . A. Permeability Barriers B. Transport Systems . C . Electron Transport and Generation of ATP . . D. Membrane, Wall and Nucleus : An Integrated Unit 111. Compounds which Disorganize Lipoprotein Membranes . A. Organic Solvents . B. Detergents . C. Reversible Membrane Disorganization? . ‘$ D. Peptide Antibiotics . E. Basic Polypeptides and Proteins F. Polyene Antibiotics and Membrane Sterols . $ IV. Proton Conduction and Uncoupling of Oxidative Phosphorylation . V. Alkali Metal Ionophores . A. Valinomycin . B. Enniatins C. Gramicidins D. Macrotetralides: Nonactin and its Homologues . E. Nigericin, Monensin and other Carboxylic Polyethers . F. Other Ionophores . VI. Inhibitors of Energy Transfer and of the Respiratory Chain . A. ATPase and Energy Transfer . B. Inhibitors of the Respiratory Chain . C. Interaction of Heavy Metals with the Membrane . VII. Bacteriocins: Antibiotics which Interact with Specific Membrane Receptors . VIII. Summary and Prospect . IX. Acknowledgements . References .

.

45

46 47 47 48 49 51 53 54 55 57 58 60 61 63 68 69 74 74 76 78 80 81 81 86 90 91 93 95 96

46

FRANKLIN M. HAROLD

I. Introduction By 1940 it was clearly recognized that certain antibiotics and synthetic antimicrobial agents act at the level of the cytoplasmic membrane. Most of these, including the antibiotic tyrocidine (Hotchkiss, 1944) and the quaternary ammonium compounds (Domagk, 1935) brought about gross disruption of the osmotic barrier; they were indeed useful disinfectants, but offered little promise of revealing the molecular details of membrane structure or function. This bleak prospect has been transformed by the pioneering efforts of many investigators. Among the landmarks are the studies of Lardy and his associates on the use of antibiotics in the analysis of oxidative phosphorylation, the work of Lampen, Kinsky and Van Deenen on the interaction of polyene antibiotics with sterols and, more recently, the far-reaching discovery of ion conduction by Chappell, Mitchell, Mueller and especially Pressman. It has thus become clear during the past decade that antibiotics will prove to be as valuable in the analysis of membrane functions as they have in unravelling the complexities of macromolecule synthesis. The purpose of this review is to consider the interactions of antibiotics (and antimicrobial agents generally) with cellular membranes, and the application of these reagents to the study of membrane physiology in micro-organisms.However, much of the experimental material currently available refers to mitochondria and to artificial membrane systems, perhaps because biochemists were particularly alert to the selective effects of many antibiotics upon membrane functions. It proved necessary to limit the scope of this review in several respects. Antimicrobial agents which inhibit the synthesis of the cell wall and the replication of DNA were excluded even though the membrane participates in these processes. This article is restricted to those functions which appear to be intrinsically associated with membranes : impermeability to small molecules, active transport and the generation of metabolic energy. Because of my own interests examples were chosen from the bacteria more often than from the fungi. Finally, I have tried to select from the profusion of pharmacological agents which affect membranes, those compounds which promise to be of particular value in microbiology. These restrictions still leave a literature both voluminous and scattered, and I can but offer my apologies to those investigators whose contributions were overlooked. Some selection of references was unavoidable ; review articles and recent research papers were cited whenever possible, sometimes at the expense of prior reports. TWOgeneral sources of information on antibiotics deserve special mention. A monumental work by Korzybski, Kowszyk-Gindifer and Kurylowicz ( 1967) compiles chemical data and

ANTIMICROBIAL AGENTS AND MEMBRANE FUNCTION

47

applications for every known antibiotic. But for those who wish to use antibiotics as chemical probes in the dissection of physiological processes, the indispensible reference work is the treatise edited by Gottlieb and Shaw (1967). Throughout the preparation of this review I have drawn heavily upon their work for information, insight, and models of useful scholarship. 11. Structure and Functions of Microbial Membranes Eucaryotic cells, such as fungi and algae, differ fundamentally from the procaryotic bacteria in the organization of membranous elements. I n the former, the plasma membrane serves as the main osmotic barrier and energy generation is the function of specialized organelles, mitochondria and chloroplasts. I n procaryotic cells, the division of labour is much less obvious. Not only transport and permeability but also oxidative and photosynthetic phosphorylation are attributes of the plasma membrane or, at least, of membranous elements which cannot yet be clearly separated from the plasma membrane (Salton, 1967; Lascelles, 1968). I n addition the plasma membrane is intimately involved in the biosynthesis of all cellular elements external to it, such as cell-wall mucopeptides, lipopolysaccharides, teichoic acids and exoenzymes ; it is the locus to which flagella are attached, and it apparently ensures the equal partitioning of the genome among daughter cells at division. The multiplicity of known and suspected functions of the bacterial cytoplasmic membrane suggests an intricate mosaic ; we may well discover that, far from being relatively simple, bacterial membranes are actually among the most complex.

A. PERMEABILITY BARRIERS Despite the complexity of some microbial envelopes, the plasma membrane appears to be in all cases the main osmotic barrier. I n Mycoplasma species, the membrane is directly exposed to the medium. I n Grampositive bacteria and in fungi, the cell wall shields the membrane but impedes the passage of only quite large molecules (Schemerand Gerhardt, 1964). The envelope of Gram-negative bacteria is more elaborate, and includes a lipopolysaccharide layer external to the plasma membrane. This also appears as a “unit membrane” in electron micrographs, and clearly constitutes a permeability barrier to certain compounds (Section II.D, p. 51), but the properties of sphaeroplasts and of plasmolysed cells leave no doubt that even here the main osmotic barrier resides at the inner, cytoplasmic membrane (Salton, 1967). Bacteria frequently contain internal membranous organelles. Some of these, such as the vesicular chromatophores (Lascelles, 1968), may constitute osmotic compartments in the intact cell. Whether mesosome4

48

FRANKLIN M. HAROLD

are segregated from cytoplasm and medium by a permeability barrier is not clear (Salton, 1967), and for the present we may retain the comfortable assumption that bacteria generally lack internal structural compartments. Microbial membranes contain proteins and lipids in roughly equal proportions. Characterization of membrane proteins is just beginning but considerable information on the phospholipids is available (see reviews by Gel’man et al., 1967; Goldfine, 1968; Kates, 1966; Lennarz, 1966). As is well known, sterols are major constituents of eucaryotic membranes but are absent from bacteria, except possibly for trace amounts (Schubert et al., 1968; de Souza and Nes, 1968). The role of sterols in membrane structure is not clear, but is usually considered to be the stabilization of phospholipid arrays ; in bacteria, carotenoids may play an analogous role. The traditional Danielli model of membrane structure continues to be a valuable guide to the design of experiments, and inspired the successful effort to prepare artificial, purely lipid membranes. Of these, the black bilayer membranes approximate most closely the properties of living membranes with respect to water permeability, electrical characteristics and perhaps overall structure. Phospholipid sphaerules, consisting of multiple concentric lipid shells within which solutes may be trapped, are another useful model albeit less realistic (see reviews by Lucy, 1968; Rothfield and Finkelstein, 1968; Tien and Diana, 1968). Nevertheless there is growing doubt that a phospholipid bilayer is an adequate representation of membrane structure. The evidence for and against the existence of subunits, and the relationship of the electron microscopists’unit membrane to the complexitiesof theliving membrane, have been thoroughly discussed in many recent reviews (Chapman and Wallach, 1968; Gel’man et al., 1967; Korn, 1966; Rothfield and Finkelstein, 1968; Salton, 1967; Wallach and Gordon, 1968). As is the case with other cellular membranes, observations on bacteria suggest that the triple-layered unit membrane seen in electron micrographs does not, in fact, correspond to a trilaminar phospholipid bilayer (Grula et ul., 1967) and that the conformation of membrane proteins is different from that predicted by the Danielli model (Lenard and Singer, 1966).

B. TRANSPORT SYSTEMS There is general agreement that the plasma membrane is fundamentally quite impermeable to most metabolites and nutrients of biological interest. Passage of nutrients across the membrane is mediated by specific transport systems; these may be coupled to a source of metabolic energy and can then accumulate their substrate in the cyDo-

ANTIMICROBIAL AGENTS AND MEMBRANE FUNCTION

49

plasm against substantial concentration gradients. The specificity of action and of genetic control which is characteristic of bacterial transport systems (Kepes and Cohen, 1962; Stein, 1967) is clearly a function of specific protein components. Proteins which bind transport substrates with high affinity have been isolated from the cytoplasmic membrane and from the periplasmic space, and their role in membrane transport is under intensive investigation (Pardee, 1968). Much less progress has been made with the mechanism of energy coupling. I n fermentative bacteria, as in red blood cells, ATP and phosphoenolpyruvate which can be derived from glycolysis are almost certainly the ultimate energy donors for “active” transport (Pardee, 1968; Skou, 1965; Stein, 1967). This is not necessarily true for aerobic bacteria. Studies on mitochondria have provided excellent evidence that ion transport can be energized, not only by ATP, but also by energy-rich states or intermediates which are generated by respiration prior t o the formation of ATP (Lehninger et al., 1967). Whether such entities can participate in membrane transport by micro-organismsis not known, nor is it clear how metabolic energy is employed to drive active transport. Students of membrane transport have come to distinguish two kinds of energy-requiring transport systems (Mitchell, 1967a; Stein, 1967): (1) Primary transport systems, in which translocation of the substrate is directly coupled to an enzymic process. The sodium- and potassiumdependent ATPase of mammalian cell membranes which mediates accumulation of K+ and extrusion of Na+ (Skou, 1965)remains the most familiar example. Enzymes performing similar functions appear to exist in bacteria as well (Section VI.A, p. 81). (2) Secondary or gradientcoupled transport makes use of concentration gradients estabIished by the primary transport systems to drive the accumulation of other metabolites. The accumulation of sugars and amino acids by the mammalian intestine, which requires sodium and is indirectly driven by the sodium pump, is a well established case. This concept is less familiar to microbiologists, yet it appears a priori likely that the accumulation of many nutrients in bacteria depends, not directly upon the splitting of ATP, but upon the utilization of gradients (H+, Na+ or K+, perhaps) established by a limited number of primary transport systems.

C. ELECTRON TRANSPORT AND GENERATION OF ATP ATP formation at the substrate level can occur in solution, but thus far at least, ATP generation linked to the respiratory chain appears to be obligatorily associated with membranous structures. Eucaryotic micro-organismshave mitochondria and chloroplasts which are structurally quite analogous to those of higher organisms and need not be

50

FRANKLIN M. HAROLD

discussed here. The situation in the bacteria is much more ambiguous.

It is well established that dehydrogenases and the electron carriers of

respiration are found in the membrane fraction after disintegration of the cell (Gel’manet al., 1967; Salton, 1967; Smith, 1968). Some investigators have argued that the respiratory enzymes are localized in the mesosome but the balance of the evidence presently available does not favour this view. It may also be significant that the stalked particles seen in membranes of certain aerobic bacteria (Abram, 1965; Gel’man et al., 1967; Mufioz et al., 1969) and which are probably analogous to the PI particles of the inner mitochondrial membrane, are not confined to the mesosome. Morphological criteria do not permit us to differentiate the respiratory system from the plasma membrane, but chemical separation may be possible. Salton et al. (1968) have recently described the isolation from Micrococcus lysodeikticus of a membrane fraction which is depleted of lipids but still forms a continuous sheet and contains the bulk of the cytochromes and of succinate dehydrogenase. The enzymes and electron carriers of bacterial respiration have been the subject of recent reviews (Gel’man et al., 1967; Smith, 1968) which also summarize what is known concerning the mechanism of oxidative phosphorylation in bacteria. I n the last analysis, the hypotheses proposed for bacteria can be reduced to those now being vigorously debated by students of mitochondria. A quick sketch of this complex subject is necessary here, since so many of the antimicrobial agents to be considered below affect oxidative phosphorylation. 1. Chemical Coupling

I n the traditional view, free energy released at certain sites in the electron-transport chain is trapped in the form of energy-rich intermediates. A sequence of reversible chemical transformations, which includes both non-phosphorylated and phosphorylated intermediates, links the redox reactions of the respiratory chain to the ultimate product, ATP. Some, at least, of these hypothetical intermediates can themselves serve as energy donors for energy-requiring processes such as ion transport. The crux of the matter is the nature of the chemical intermediates, which have thus far eluded isolation and chemical characterization (see reviews by Chance et al., 1967; Pullman and Scliatz, 1967; Slater, 1966). 2. Chemi-Osmotic Coupling

The chemi-osmotichypothesis was deveIoped by Mitchell (see Mitchell,

1966,1967b for recent summaries)in an attempt to provide an alternative

interpretation which would not depend upon hypothetical chemical entities. He proposed that the electron-transport chain is so arranged as to generate H+ and OH- on opposite sides of the mitochondrial inner

ANTIMICROBIAL AGENTS AND MEMBRANE FUNCTION

51

membrane. The membrane is assumed to be relatively impermeable to protons and to ions generally. Consequently a proton-gradient is generated across the membrane, consisting of two components : a difference of pH value and/or a membrane potential. It is this protonmotive force which is called upon to synthesize ATP by reversing the ATPase reaction. The enzyme is assumed to be localized in the membrane in such a way that the active centre is accessible to OH- from one side, to Hf from the other side, and to water as such from neither side. Such a system could synthesize ATP from ADP and Pi by, in effect, withdrawing OH- to the “outside” (acidic and positively charged) and H+ to the “inside’) (alkaline and negatively charged). 3. Conformutional Coupling

It is also possible that the intermediates which intervene between the electron carriers and the first stable chemical product, ATP, are in fact energized conformational states of the mitochondria1 membrane itself. The most persuasive evidence in favour of this concept has come from Green’s laboratory (Green et al., 1968; Harris et ul., 1968). Electron micrographs depict most graphically the effect of substrates and inhibitors upon 6he gross structure of the cristae membranes. As the authors point out, structural transformations elicited by ATP are familiar from the contraction of muscle ;the reverse process should not be implausible. Much effort has been expended in attempts to decide among these alternatives, which are as pertinent to photosynthesis as they are to oxidative phosphorylation (Jagendorf, 1967;Vernon, 1968).Some of the arguments turn upon the effects of various antimicrobial agents and will be considered below, but a general survey of this sophisticated controversy would be out of place here. Significantly, the positions taken by the principals increasingly embody elements derived from the alternative hypotheses; perhaps in the end neither thesis nor antithesis will prevail, but a constructive synthesis.

D. MEMBRANE, WALLAND NUCLEUS: AN INTEGRATED UNIT The Gram stain divides bacteria into two broad classes which differ in many structural and physiological features. One of these is their response to antimicrobial agents. It has long been recognized that Gram-positive organisms are sensitive to anionic detergents and related compounds, to ion-conducting antibiotics, and to a miscellany of antibacterial agents such as actinomycin, whereas Gram-negatives are relatively resistant (for examples see sections 111, p. 53; Iv, p. 63; and V, p. 68). Gram-negative bacteria can be rendered sensitive b y conversion to sphaeroplasts, suggesting that the cell wall prevents access

52

FRANKLIN M. HAROLD

of the drug to the membrane. However, removal of the mucopeptide layer is not required. Treatment of Gram-negative bacteria with EDTA together with an organic cation (tris is most commonly used) suffices to render many Gram-negative bacteria sensitive to drugs which they normally resist (Brown and Richards, 1965 ; Leive, 1965 ; MacGregor and Elliker, 1958; Voss, 1967; Weiser et aZ., 1968). The effect of EDTA on Escherichia coli has been thoroughly studied by Leive (1965, 1968). EDTA and tris caused release of a large fraction of the cell-wall lipopolysaccharide and rendered the cells sensitive to actinomycin ; the cells also became relatively permeable to various substrates which are normally excluded. The morphological effects of EDTA are beautifully depicted in the electron micrographs of Birdsell and Cota-Robles (1967). I n the presence of tris-EDTA, the outer membrane which still surrounds lysozyme sphaeroplasts ruptures and peels back. Large sections of the inner, cytoplasmic membrane are exposed, but coils of the outer layer remain attached to one end. Such lysozyme-EDTA sphaeroplasts are highly sensitive to low concentrations of Brij 59, a non-ionic detergent to which the intact cells are largely resistant (Birdsell and Cota-Robles, 1968). A similar situation obtains in Pseudomonas. EDTA is toxic to some, but not all pseudomonads (Wilkinson, 1967). Cox and Eagon (1968) demonstrated release of lipopolysaccharide with formation of osmotically sensitive “osmoplasts”. These have no obvious abnormalities in permeability properties but lose pre-induced transport systems and are unable to form new ones (Eagon and Asbell, 1966; Asbell and Eagon, 1966). Evidently the lipopolysaccharide layer constitutes a permeability barrier whose disruption exposes the membrane to agents from which it was previously shielded. I n a deeper sense, we must regard wall and membrane as closely integrated components of the cell envelope such that the structure and function of each depends upon the other. Let us recall, for instance, that protoplast formation is accompanied by extrusion of mesosomes and loss of the capacity to divide, apparently because of the altered structural configuration of the envelope. The biosynthesis of cell-wall mucopeptide and lipopolysaccharides are functions of the membrane which will not be surveyed here, but a brief comment is desirable on those antibiotics whose effects are pleiotropic. Bacitracin is a good example. Weinberg (1967), in a lucid summary of the literature, records multiple biochemical effects of this antibiotic including inhibition of protein synthesis and of cell-wall formation as well as leakage of various constituents from the cells. He concluded that the primary target of bacitracin is the cell membrane. Very recently, Siewert and Strominger (1967) found bacitracin to be a specific inhibitor of one step in mucopeptide biosynthesis: it blocks

ANTIMICROBIAL AGENTS AND MEMBRANE FUNCTION

53

dephosphorylation of the lipid-pyrophosphate carrier. Sublethal concentrations of the antibiotic induce morphological changes in both cell wall and membrane of Mycobacterium phlei, including the loss of mesosomes (Rieber et al., 1969). It seems reasonable to conclude that bacitracin binds to the membrane, presumably to a specific site, and induces pleiotropic effects on both cell-wall synthesis and membrane structure, illustrative of their interdependence. Vancomycin is another example of an antibiotic which inhibits cell-wall synthesis by blocking a chemical reaction that involves membrane constituents (Jordan and Reynolds, 1967), and has diverse effects on membrane transport and oxidative phosphorylation. Finally novobiocin, an antibiotic now thought to interfere primarily with DNA polymerase (Smith and Davis, 1967), may bind to a siteon themembrane which could account for its multiple secondary effects on cell-wall synthesis and membrane processes (Brock, 1967).

111. Compounds which Disorganize Lipoprotein Membranes Exposure of bacteria to certain compounds, including organic solvents and detergents, destroys the osmotic barrier. This is readily recognized by release from the cells of small metabolites such as K+, phosphate, amino acids and sugars, and is generally lethal. At the same time, internal enzymes may be rendered accessible to substrates which do not normally pass across the membrane: assay of /3-galactosidase in the presence of toluene is a familiar application. Although it is customary to speak of the “destruction” of the osmotic barrier, the physical integrity of the cytoplasmic membrane is not necessarily impaired. For example, after exposure of E . coli to toluene, there is no gross change in membrane morphology, and enzymes remain sedimentable with the cells (Jackson and DeMoss, 1965). The immediate loss of selective permeability to small molecules reflects, then, not disintegration of the cytoplasmic membrane but a structural disorganization which modifies its permeability to a greater or lesser degree. What is the nature of this disorganization? The structural integrity of a membrane depends upon the orderly arrangement of both proteins and lipids, but its impermeability to small, water-soluble molecules must be attributed primarily to the lipid phase. This serves as a barrier because the hydrocarbon interior largely excludes water. Disorganization of a membrane by solvents or detergents implies a structural change such that this hydrophobic barrier is breached. Re-orientation of lipid molecules in a film or micelle may occur in a variety of ways so as to produce discontinuities and channels in the hydrophobic barrier. Phospholipids in water can exist in a variety of

54

FRANKLIN M. HAROLD

phases: a lamellar phase consisting of phospholipid sheets with a hydrocarbon interior and polar head groups facing the water; cylindrical configurations, in which polar groups line the interior which is filled with water while hydrocarbon chains occupy the space between cylinders; globular micelles, and others (Lucy, 1968; Luzzati, 1968; Stein, 1967). Transitions from one phase to another are known to occur in phospholipid-water mixtures, and may be the physical basis of many phenomena described below.

A. ORGANICSOI.VENTS Chloroform and toluene are traditionally employed to keep solutions sterile and to disrupt permeability barriers. The time-course of events following exposure of E . coli to toluene was described by Jackson and DeMoss (1965). As little as 1.5 pl. of toluene per ml caused rapid cell death and loss of selective permeability. The general structure of the cells was unaffected, enzymes remained sedimentable and even the respiratory chain appeared to remain largely intact. Subsequent changes including temperature-dependent loss of protein, disaggregation of ribosomes and breakdown of RNA, may involve autolytic enzymes. Alcohols probably provide the best insight into the interaction of solvents with lipid membranes. n-Butanol and other alcohols disrupt certain lipoprotein membranes with release of water-soluble proteins (seeWallach and Gordon, 1968, for references),but at the concentrations employed in bacterial physiology (0.4 M ) such drastic effects are not evident (Gilby and Few, 1960b).More probably, alcohols disorganize the lipid structure by penetrating into the hydrocarbon region. I n their study of protoplast lysis, Gilby and Pew (1960b) found that equal degrees of lysis were produced by concentrations of alcohols having equal thermodynamic activities. The concentrations of alcohol in the lipid phase appears to be the critical quantity, and lysis may occur at a concentration which produces a surface pressure of about 34 dynes/ om. (Pethica, 1958). However, short-chain alcohols produce quantitatively greater changes in membrane organization than do the higher homologues (Bangham et al., 1965). Lysis of red blood cells and of Bacillus megaterium protoplasts (Kinsky, 1963; Fitz-James, 1968) by low concentrations of vitamin A may be a related phenomenon. Vitamin A penetrates and expands the surface area, of lecithin-cholesterol monolayers ;massive quantities of the vitamin accumulate in the film, apparently due to formation of a complex with lecithin. Penetration of vitamin A can be prevented by raising the surface pressure above 34 dynes/cm. (Bangham et al., 1964).It would be of interest to determine whether vitamin A accumulates in bacterial membranes, and with which component i t interacts.

ANTIMICROBIAL AGENTS AND MEMBRANE FUNCTION

55

B. DETERGENTS The use of detergents as disinfectants reaches back into the 1930s. As a general rule, cationic detergents are bactericidal to both Grampositive and Gram-negative organisms, anionic detergents primarily to Gram-positive, and non-ionic detergents have little effect on either. Although anionic detergents do attack lipopolysaccharide layers, it is the cytoplasmic membrane which is the primary target. Prolonged exposure to detergents leads to breakdown of macromolecules and other autolytic changes. The literature has been reviewed by Newton (1958), Salton (1968) and by Schulman et al. (1955).

CH3(CHz)loCHz--O--SO3N&

Sodium doclwyl sulphate

[

I

I;

C H ~ ( C H ~ ) I ~ C H ~ - N - C H ~Ur-

I

CH3 CH3

Cetyltrimethylammonium bromide

I I

CH2 CHz--N+(CH3)31-

ND 212, an azasteroid

Chlorhoxidine

FIG.1. Chemical structures of detergents.

Anionic detergents, exemplified by sodium dodecylsulphate (Fig. I ) , not only lyse protoplasts but solubilize isolated plasma membranes, suggesting gross disruption of the lipoprotein framework (Gilby and Few, 1960a; Razin and Argaman, 1963). Activity is a function both of the chain length and of the nature of the polar group (Gilby and Few, 1960a; Salton, 1968). Anionic detergents are employed in fractionating membranes into their constituent parts; in some cases, at least, reaggregation occurs when the detergent is removed by dialysis, with formation of sheet-like structures resembling the original membranes (Razin et al., 1965; Grula et al., 1967). However, early hopes that the dissociation yields membrane subunits have been abandoned. The detergents cleave protein from lipid and re-association upon dialysis appears to be quite random (Grula et al., 1967; Razin and Boschwitz, 1968; Rodwell et al., 1967; Salton, 1967).

56

FRANKLIN M. HAROLD

Whereas intact bacteria are highly resistant to non-ionic detergents, protoplasts and isolated membranes are solubilized more readily. I n fact, non-ionic detergents such as Nonidet and Brij-59, appear to be reagents of choice for dissociating isolated membranes into fragments which retain enzymic activities (Birdsell and Cota-Robles, 1968; Salton et aZ., 1967). Cationic detergents exemplified by cetyltrimethylammonium bromide (Fig. 1) lyse protoplasts but do not disaggregate isolated membranes. Gilby and Few (1960a)proposed that the positively charged head associates with the phosphate groups of phospholipids, while the non-polar portion of the detergent penetrates into the hydrophobic interior of the membrane. Thus, both the basic head group and the alkyl chain influence the potency. The resulting distortion of the membrane could increase its permeability, exposing the protoplasts to osmotic lysis. The interaction of detergents with purely lipid artificial bilayer membranes has been studied by Seufert (1965). Anionic, non-ionic and cationic detergents all lowered the electrical resistance by several orders of magnitude. I n addition, anionic and non-ionic, but not cationic, detergents produced a resting potential if the membrane separated compartments of differing salt concentrations. The specific increase in permeability to cations was attributed to re-arrangement of the bilayer to produce localized water-filled pores lined with fixed negative charges ; these would preferentially pass cations. One wonders whether sublethal concentrations of detergents induce similar specific permeability changes in biomembranes. A broad range of structures can be loosely classified as cationic detergents. Examples include azasteroids, steroid analogues which containnitrogenin the nucleus (Smith et al., 1964; Varicchio et al., 1967); substituted guanidines (Weinberg, 1968)including the very potent bactericidal agent, chlorhexidine (Fig. 1; Davies et al., 1954; Davies and Field, 1968; Hugo and Longworth, 1964a, b, 1966); and the triphenylmethane dyes which were among the earliest chemotherapeutic agents (Browning, 1964). All share the combination of a positively-charged polar nitrogen group with a hydrophobic region. I n sufficient concentrations they disorganize membranes just as cetyltrimethylammonium bromide does, with release of osmoljtes and penetration of dyes excluded by the intact cells. In many cases, including cetyltrimethylammonium bromide (Salton, 1968; Schulman et al., 1955)it is possible to show quantitative correspondence between killing and the release of solutes, so that disruption of the membrane can safely be taken to be the lethal event. Sometimes, however, viability is decreased by concentrations of drug which cause little leakage (for examples, see Hugo and Longworth, 1964a; Varicchio et al., 1967),suggesting that the lethal event may be a

ANTIMICROBIAL AGENTS AND MEMBRANE FUNCTION

57

more subtle one ;we shall consider chlorhexidine again, in Section VI.A, p. 81. The relationship between chemical structure and activity has been studied in detail for the guanidines (Weinberg, 1968), dyes (Browning, 1964) and azasteroids (Smith et al., 1964; Varicchio et al., 1967) but has as yet given little insight into the reaction between drug and membrane ; this would appear to be a promising field for further inquiry. The response of micro-organismsto detergents is subject to some degree of genetic control. I n addition to the major differences between Grampositive and Gram-negative bacteria (Section II.D, p. 51), individual genes have been found to modify the sensitivity of E. coli to anionic detergents. Nakamura (1968) has mapped a gene which increases resistance to sodium dodecylsulphate and also to phenyl alcohol; conversely, some colicin-tolerant mutants are particularly sensitive to detergents (de Zwaig and Luria, 1967). Genetic alterations in membrane proteins have been postulated but not demonstrated, and it may be well to keep the lipopolysaccharide layer in mind (Section II.D, p. 51).

C. REVERSIBLE MEMBRANE DISORGANIZATION? The preceding sections have stressed the lethal effects of detergents and solvents, but there is every reason to expect, and to seek, reagents which distort membrane structure reversibly. Narcotics and local anaesthetics are thought to act in this manner upon mammalian cell membranes (Cuthbert, 1967). Sublethal concentrations of organic solvents may have reversible effects on membrane permeability. Phenethyl alcohol, structurally related to toluene, reversibly increases membrane permeability of E. coli (Silver and Wendt, 1967) and of Neurospora crassa (Lester, 1965); the membrane may in fact be the primary target of this reagent which has received much study as an inhibitor of macromolecule synthesis. The most promising of the compounds which increase membrane permeability reversibly appear to be the steroid diamines. Irehdiamine (Fig. 2) and the related malouetine are plant alkaloids which first attracted the attention of molecular biologists as inhibitors of bacteriophage growth in E. coli. Silver and Levine (1968a, b) subsequently found M induce rapid efflux of thiomethylthat concentrations around galactoside and K+ and also inhibit their uptake. The effects of irehdiamine on transport could be reversed by removal of the drug or by Mg2+,but loss of viability was apparently irreversible. These studies are incomplete and questions persist regarding the effects of the steroids on energy metabolism and on specific transport systems, but they are a valuable lead.

58

FRANKLIN M. HAROLD

\/NHz

FIU.2. Chemical structure of irehdiamine, a steroid diamine.

Levallorphan, an analogue of morphine which inhibits RNA synthesis in E . coli (Simon and Van Praag, 1964)and induces breakdown of ATP (Greene and Magasanik, 1967),is also a possible candidate for reversible effects on membrane structure (E. J. Simon, personal communication).

D. PEPTIDE ANTIBIOTICS 1. Tyrocidines The observation that Bacillus species in mixed culture antagonize the growth of other Gram-positive bacteria goes back to the very dawn of bacteriology. The reason was found in 1940 when Dubos and Hotchkiss isolated tyrothricin (Hotchkiss, 1944) and thereby opened the antibiotic era. Tyrothricin proved to be composite, including members of the gramicidin (Section V.C, p. 74) and tyrocidine families. Hunter and Schwartz (1967b) have prepared a comprehensive review on tyrocidines. Tyrocidines kill sensitive organisms by disruption of the osmotic barrier ; small metabolites are released, but the cytoplasmic membrane is not solubilized. More prolonged exposure brings about breakdown of ribosomes and of nucleic acids (Mach and Slayman, 1966). Tyrocidines also lyse protoplasts, indicating direct action on the membrane. Unlike the related gramicidins, tyrocidines increase membrane permeability generally and are not specific cation conductors (Pressman, 1965; Graven et aH., 1966b). The chemical structure of the tyrocidines (Fig. 3) was clarified by Craig and his associates (for early references see Hunter and Schwartz, 1967b; Ruttenberg et al., 1965, 1966, Ruttenberg and Mach, 1966). They form a family of related cyclic peptides; the left half is invariant, but substitutions occur in the right half. A special case is the misnamed gramicidin-S, which resembles the tyrocidines in biological activity ; its structure is that of a dimer of the invariant half. All the biologically active tyrocidines bear a net positive charge. The evident similarity between the effects of tyrocidines and cetyltrimethylammonium bromide led Hotchkiss to conclude (1944) that “Tyrocidine is a bacterial detergent, unique only in its origin and complex chemical structure”. The relationship between activity and chemical

ANTIMICROBIAL AGENTS AND MEMBRANE FUNCTION

L-Leu

P

59

(~)L-DAB

--f

I

D-Phe

(~)L-DAB

t L-Pro -+L-Phe

7 u-Phc

\u-l’hc

L-Thr

(~)L-DAB

5

MOA Tyrocirline A

Polymyxin B1

WIG. 3. Chemical structures of some polypeptide antibiotics. Asp, indicates an aspartate residue ;DAB, a diaminoisobutyrate residue ;Glu, a glutamate residue ; Lue, a leucine residue; MOA, a 6-methyloctanoate residue; O m , an ornithine residue; Pho, a phenylalanine residue; Pro, a proline residue; Thr, a threonine residue; Tyr, a tyrosine residue; and Val, a valine residue. --f indicates a C-N linkage.

structure is far from clear. The tyrocidines are strongly surface-active, and tend to form aggregates which resemble lipid micelles. Aggregation may be related to antibiotic activity; an open-chain analogue of tyrocidine having the same amino-acid sequence does not aggregate and also is not an antibiotic (Ruttenberg et al., 1966). However, gramicidin-S, which also does not form aggregates, is very similar to the tyrocidines in its biological effects. Katchalsky and his associates (1964) have described synthetic peptides related to gramicidin-S. The corresponding linear decapeptide had about one tenth of the activity of the cyclic molecule, as had a random copolymer of the constituent amino acids in the proper ratios and steric configurations. Curiously, a copolymer of D-ornithine and L-leucine was more active against E. coli than gramicidin-S itself. It appears that basic residues are essential to antimicrobial activity ; hydrophobic residues serve t o anchor the molecule to the membrane, but the role of the peculiar mixture of D and L amino acids and of the overall conformation of the molecule remains to be clarified.

60

FRANKLIN M. HAROLD

2. Polymyxins Bacillus polymyza and related strains elaborate a class of structurally

I

allied antibiotics consisting of heptapeptide rings with a long side-chain terminating in methyloctanoic or in iso-octanoic acids (Fig. 3). Their activities and applications have been thoroughly reviewed by Sebek (1967). It is noteworthy that the polymyxins are in general more active against Gram-negative bacteria than against the Gram-positives. An impeccable series of experiments led Newton (see Newton, 1956, for a summary) to conclude that polymyxin B binds to the cytoplasmic membrane and breaches the osmotic barrier. Subsequent effects include inhibition of respiration, cytological changes especially in the nuclear region (Wahn et al., 1968; also Sebek, 1967) and release of ribosomal RNA (Nakajima and Kawamata, 1966). Massive quantities of the antibiotic are adsorbed by the cells, mostly to internal receptors made available by breakdown of the permeability barrier. Wahn et al. (1968) reported that E. coli cells treated with polymyxin display numerous blebs or projections all over their surface. It would now appear that these are not, as one might think, due to cytoplasm escaping through breaks in the membrane, but result from interaction of polymyxin with the cell wall (Koike et al., 1969). Morphological effects of polymyxin on the membrane were not visible. It is not clear why polymyxins act preferentially on Gram-negative bacteria. Association with the lipopolysaccharide layer may be a factor, but is evidently not obligatory as polymyxins do bind strongly to the plasma membrane itself (Newton, 1956; Koike et al., 1969). Little is known concerning the significance of either the specific amino-acid constituents or of the lipid side-chain. At the molecular level, the mode of action of polymyxins is probably to be sought in a fairly generalized reaction with phospholipids. Polymyxin B preferentially penetrates monolayers of phosphatidylethanolamine ; the increase in surface area or pressure may induce re-orientation of membrane lipids and breakdown of the permeability barrier (Few, 1955; Schulman et al.,1955; Newton, 1956).

E. BASICPOLYPEPTIDES AND PROTEINS '

Many basic polypeptides, including the histones and protamines, exhibit antimicrobial activities. The synthetic polylysines have received the most study (Katchalsky et al., 1964); they are bacteriostatic for E. coli at low concentrations but higher levels are bactericidal, inhibit respiration and induce leakage of amino acids. It is presumed that they disorient the cytoplasmic membrane as the cationic detergents do. However, polylysines agglutinate bacteria and alter their electrophoretic

ANTIMICROBIAL AGENTS AND MEMBRANE FUNCTION

61

mobility, and one wonders whether this surface-binding might not secondarily distort the membrane and render it leaky. Polylysine does bind to the exposed cytoplasmic membranes of protoplasts, enhancing their resistance to osmotic shock (Harold, 1964). In view of the relatively specific effects of polylysine on membrane phenomena in mitochondria (Johnson et al., 1967) and chloroplasts (Dilley, 19GS), its effects on protoplast permeability should be explored. In yeast, a variety of basic polypeptides and proteins induce gross membrane leakiness (Yphantis et al., 1967). The bactericidal action of serum proteins and of complement may also involve destruction of the permeability barrier, but this subject is outside the scope of this article.

F. POLYENE ANTIBIOTICS AND MEMBRANE STEROLS Thus far we have discussed compounds which penetrate into phospholipid membranes but apparently do not bind to any one receptor molecule. In the case of the polyene antibiotics there is very strong evidence that their effects on membranes result from specific association with sterols. Consequently polyenes have no antibacterial activity and, strictly speaking, come within the scope of this review only by grace of certain

OH

OH

OH

OH

OH

FIG.4. Chemical structure of filipin, a polyene antibiotic.

1Mycoplasmn species which incorporate sterols into their membrane and are thereby rendered sensitive to polyenes (Weber and Kinsky, 1965). As the literature through 1966 has been thoroughly surveyed in several recent reviews (Kinsky et uE., 1966; Kinsky, 1967; Lampen, 1966), only the main conclusions need be summarized here. The polyene antibiotics are a large and diverse class of compounds, which share certain structural features, namely a system of conjugated double bonds, and the general geometry of a ring. Few of their structures have been established; the tentative structure of one that is widely used, filipin, is shown in Fig, 4,

62

FRANKLIN M. HAROLD

but even this is called into question by the recent discovery (Bergy and Eble, 1968) that filipin is a complex of at least four related compounds. Polyenes are known to differ with respect to the size of the ring, the number of carboxyl groups and double bonds and the presence (and structure) of a carbohydrate moiety. Nevertheless, all appear to act upon the cytoplasmic membrane : the membrane is not destroyed, but is rendered leaky to small metabolites. Inhibition of growth, glycolysis and other processes follow secondarily. Polyenes cause graduated degrees of damage to yeast membranes, as measured by leakage. The most limited damage is done by succinyl perimycin, a synthetic derivative of the polyene antibiotic perimycin. This induces only the loss of K+ from yeast and all its effects, including the inhibition of growth, are reversed by high concentrations of K+ (Borowski and Cybulska, 1967). Other polyenes produce more generalized leakage. Nystatin induced loss of K+ and, more slowly, of cellular constituents such as phosphate, but inhibition of glycolysis could be annulled by K+ and NHL. Nystatin did not cause leakage of sorbose, except at high concentrations. Finally, filipin induced rapid leakage of most small metabolites such as K+, phosphate, sorbose and amino acids; inhibition of glycolysis could not be reversed even by a mixture of ions and cofactors, and several intracellular enzymes were exposed (ATPase, pyruvate decarboxylase) but even here the membrane was not physically disrupted. There is a general correlation between the size of the ring and the degree of membrane damage, the smaller rings having the more profound effects. However, by the criterion of fungicidal activity, the heptaene antibiotics are the most potent even though they have large rings and induce the least leakage The evidence for the role of sterols as specific receptors for polyene binding has been summarized (Kinsky et al., 1966; Kinsky, 1967; Lampen, 1966) and it seems superfluous to re-iterate it here. Suffice it to mention that at least one polyene antibiotic, filipin, does react with artificial phospholipid membranes which do not contain sterols (Sessa and Weissmann, 1967; Weissmann and Sessa, 1967). This, however, is seen only at very high concentrations of the antibiotic, some three orders of magnitude above the growth-inhibitory level, and reflects the nonspecific effects of a minor component of the filipin complex (Sessa and Weissmann, 1968).There is no doubt that sterols are the specificreceptors at physiological concentrations of the antibiotics, near 1 M . Sterols are clearly a necessary condition for polyene sensitivity, but apparently not a sufficient one. To what extent a membrane is perturbed by polyene antibiotics depends on the proportion of sterols to phospholipids (Kinsky et al., 1966; Demel et al., 1968; Kinsky et al., 1968; Sessa and Weissmann, 1968; Weissmann and Sessa, 1967).

ANTIMICROBIAL AGENTS AND MEMBRANE FUNCTION

63

Insight into the molecular basis of the interaction between polyenes and membrane sterols has come particularly from the work of Kinsky, Van Deenen and their associates on monolayers and phospholipid bilayer membranes. The polyenes penetrate lipid monolayers which contain cholesterol and increase the surface pressure. The magnitude of this effect on monolayers correlates well with the degree to which various polyenes disrupt fungal membranes. The increase in surface pressure is large, quite out of proportion to the number of antibiotic molecules which penetrate into the monolayer, and hence it is argued that the polyenes induce a re-orientation of the sterol molecules, a molecular domino effect as it were (Kinsky et al., 1966; Demel et al., 1968).Visual evidence for this phenomenon was obtained by electron microscopy. Artificial membranes exposed to filipin show numerous “pits”, 125 A in diameter, which may be deposits of the polyene-cholesterol complex. Such pits were also seen in membranes of erythrocytes lysed by filipin (Kinsky et al., 1966, 1967a, b). Studies with lipid bilayer membranes are beginning to shed some light on the nature of the permeability changes induced by the polyenes. Whereas filipin ultimately disrupts the bilayers, nystatin and amphotericin B lower electrical resistance and increase membrane permeability, particularly to anions. It is unlikely that the antibiotics serve as lipid-soluble anion carriers ; more probably they induce the formation of pores or channels, a reaction which must involve the sterols (Finkelstein and Cass, 1968; Andreoli and Monahan, 1968). Whether the pits seen in electron micrographs correspond to these pores or channels, remains to be determined.

IV. Proton Conduction and Uncoupling of Oxidative Phosphorylation The concept that ions traverse membranes in association with lipidsoluble carriers is traditional in membrane physiology, but only recently has it been recognized that certain pharmacological agents exert their effects by serving as artificial ion carriers. To my knowledge, this possibility was first envisaged by Mitchell (1961a) in his proposal bhat uncouplers of oxidative phosphorylation render the mitochondria1 membrane permeable to protons. The general significance of this insight became apparent following the discovery by Pressman (Moore and Pressman, 1964; Pressman, 1965)that valinomycin and other antibiotics promote uptake of Kf by mitochondria, and has since been vigorously explored by many investigators. It would be difficult to exaggerate the importance of these discoveries to membrane physiology ; the realization that ion transport and energy generation are inextricably linked is but

64

FRANKLIN M. HAROLD

one of the benefits. The present section will consider proton-conducting agents, reserving the alkali-metal ionophores for Section V, p. 68. Historically, the concept of proton conduction is rooted in the discovery that certain compounds (of which 2,4-dinitrophenol is the most familiar) uncouple oxidative phosphorylation from respiration, and in fact arose out of efforts to explain the mechanism of this uncoupling. Dozens of uncouplers are now known, many of them excellent inhibitors of growth and uncouplers of oxidative phosphorylation in bacteria. Among these we should mention the salicylanilides which have been used

-

clo" OH

c1

Tetrachlorosalicylanilide

/Cl c1 Pentachloropheiiol

N=C--C--C=N I

NH I

Carbonylcyanide Tetrachlorotrifluoromethylbenzimidazole tnn-chlorophenylhydrazone .

FIG.5. Chemical structures of uncouplers of oxidative phosphorylation : Tetrachlorosalicylanilide (TCS); carbonylcyanide rn-chlorophenylhydrazone(CCCP) ; pentachlorophenol (PCP) and tetrachlorotrifluoromethylbenzimidazole(TTFB).

for many years as disinfectants in medicine and industry (Hodes and Stecker, 1968; Hamilton, 1969; Woodroffe and Wilkinson, 1966a, b). Halogenated salicylanilides are potent uncouplers of oxidative phosphorylation in mitochondria (Whitehouse, 1964; Williamson and Metcalf, 1967) and in bacteria (Hamilton, 1968). Other compounds which uncouple oxidative phosphorylation in both mitochondria and micro-organisms include penhiwhlorophenol, derivatives of carbonylcyanide phenylhydrazone containing chlorine (CCCP) or fluorine (FCCP), and tetrachlorotrifluoromethylbenzimidazole (Asano and Brodie, 1965; Beechey, 1.966; Bragg and Hou, 1968; Cavari et al., 1967; Heytler, 1963; Heytler and Prichard, 1962; Weinbach, 1957). Structures of some of these compounds are shown in Fig. 5. Uncouplers are routinely used in the analysis of energy-dependent processes in micro-organisms such as active transport and macromolecule synthesis. At the same time, the mechanism of uncoupling

ANTlMlCROSIAL AGENTS AND MEMBRANE BUNCTlON

65

continues to be a touchstone for any hypothesis designed t o explain the mechanism of oxidative phosphorylation. Although our primary interest here centres on the effect of uncouplers on bacterial energy metabolism, the available evidence is almost entirely derived from mitochondria ; we shall assume in what follows that the same principles apply to both. In the traditional view, coupling between electron transport and phosphorylation involves chemical intermediates common to the two pathways. 2,4-Dinitrophenol and related compounds may bring about hydrolysis of such intermediates and thereby dissociate respiration from phosphorylation (Slater, 1966). I n the chemi-osmotic hypothesis (Mitchell, 1966, 1967b) such intermediates do not exist and ATP synthesis depends upon a gradient of p H value and of electrical potential across the membrane (Section II.C, p. 49). It is an essential postulate of the hypothesis that the mitochondrial membrane is relatively impermeable to protons and, indeed, to ions generally. Mitchell (1961a) pointed out that 2,4-dinitrophenol and many other uncouplers are lipid-soluble acids which could facilitate passage of protons across the membrane and thus collapse the proton gradient. Mitchell and Moyle also produced the first evidence that a number of familiar uncouplers including 2,4-dinitrophenol, CCCP and FCCP specifically catalyse passage of protons across the membranes of mitochondria and of bacteria (Mitchell, 1961b, 1966; Mitchell and Moyle, 1967a). The basis for their conclusion deserves a brief examination as it involves principles which we shall encounter again in subsequent sections. Upon addition of a pulse of hydrochloric acid t o an unbuffered suspension of mitochondria the p H value falls abruptly, then rises slowly as H+ passes into the interior of the organelle. The rate of titration of the inner compartment is limited by the permeability of the mitochondrial membrane, and can therefore be greatly accelerated by disrupting the membrane with detergents. Compounds such as 2,4-dinitrophenol and FCCP likewise accelerate titration of the inner compartment but do not disrupt the membrane and are considered to catalyse diffusion of protons (or OH-). Quantitative measurement of proton permeability in the presence and absence of a putative proton conductor requires a more sophisticated approach (Mitchell and Moyle, 1967a). Movement of protons into the mitochondria introduces positive electrical charges ;unless compensatory ion movements take place, a membrane potential builds up (inside positive) which inhibits further proton movements. Valinomycin, a highly selective I<+ conductor (Section V.A, p. &?), permits K+ to flow out and thereby relieves the electrical restrictions on proton movements. By this procedure it was determined that FCCP increased the proton conductance of the mitochondrial membrane by a factor of 160, sufficient 3

66

FRANKLIN M. HAROLD

t o account for uncoupling in terms of the chemi-osmotic hypothesis (Mitchell and Moyle, 1967a, b ; see also Mitchell, 1966, 1967b for discussions of uncoupling by proton conductors). I n photosynthesis, also, there is evidence for a close relationship between proton gradients and phosphorylation. Chloroplasts and chromatophores take up protonsinthelight ;the resultingproton gradient is dissipated by many uncouplers of photophosphorylation (Mitchell, 1966, 1967b; Jagendorf, 1967; Kaplan and Jagendorf, 1968; Schwartz, 1968).Finally, uncouplers of oxidative phosphorylation facilitate proton movements across membranes of cells which do not carry out oxidative phosphorylation, including red-blood cells (Harris and Pressman, 1967), Streptococcus fueculis (Harold and Baarda, 1968b) and anaerobic E . coli (Pavlasov&and Harold, 1969). I n all cases it appeared that only proton translocation was accelerated, not that of other ions. Perhaps the most compelling evidence comes from studies on phospholipid bilayer membranes. It has been established that a number of familiar uncouplers, including 2,4-dinitrophenol, CCCP and tetrachlorotrifluoromethylbenzimidazole, greatly increase the electrical conductance of such membranes to protons, and protons only (Bielawski et ul., 1966; Hopfer et ul., 1968; Liberman and Topaly, 1968). I n principle, proton conductors could act either as lipid-soluble carriers or else pass protons along the 7r-orbitals of molecules which themselves remain relatively fixed in the membrane. Liberman and Topaly (1968) concluded from their observations that a carrier mechanism was involved. Despite the evidence summarized above, by no means all investigators accept the thesis that uncoupling of oxidative phosphorylation is due to proton conduction. Chance et ul. (1967) have questioned the entire concept of a proton-impermeable mitochondria1 membrane. Weinbach and Garbus (1968a, b) have described experiments which suggest that uncouplers induce conformational changes in membrane proteins and they hold these responsible for uncoupling. On balance, it seems t o me that the reality of proton conduction has been established beyond reasonable doubt, but it should be stressed that this does not, ips0 fucto, validate the chemi-osmotic hypothesis. It is conceivable, for example, that proton conductors facilitate access of protons to labile, energy-rich intermediates of oxidative phosphorylation (Hopfer et ul., 1968). Alternatively, uncouplers may facilitate passive entry of protons which the organelle must then extrude by a proton pump driven by high-energy intermediates. A proton cycle would thus be set up, the net effect of which is the dissipation of metabolic energy and uncoupling of ATP synthesis (Harris et ul., 1967a).Whatever proves to be the correct explanation it is still true (with some limitations, Section V.E, p. 78) that “facilitating proton diffusion through the membranes has

ANTIMICROBIAL AGENTS AND MEMBRANE FUNCTION

67

now become a sufficient reason for a chemical being an uncoupler, whether one accepts the chemi-osmotic hypothesis or not” (Jagendorf, 1967). A complication should be mentioned which applies particularIy to the effect of uncouplers on bacteria. I n mitochondria, low concentrations of uncouplers prevent phosphorylation but respiration itself is not inhibited and may even be stimulated. I n bacteria, compounds such as 2,4-dinitrophenol, pentachlorophenol and CCCP tend to inhibit respiration as well as phosphorylation, especially with certain substrates (see, for example, Asano and Brodie, 1965; Cavari et al., 1967; Smith, 1968). This effect, which can also be observed in mitochondria exposed to relatively high concentrations of uncouplers, is the basis of a hypothesis put forward by van Dam and Slater (1967).They suggest that uncoupling, as well as inhibition of respiration, are ultimately due to dissipation of energy by transport of the uncoupler itself. However, the inhibition of respiration may yet find an explanation as a secondary consequence of proton movements (see below). By whatever mechanism, proton conductors do uncouple oxidative phosphorylation and interfere with. production of ATP. It is therefore not surprising that uncouplers inhibit energy-dependent processes such as motility and active transport (for examples see Faust and Doetsch, 1969; Hamilton, 1968; Kepes and Cohen, 1962; Pardee, 1968; Stein, 1967; Winkler and Wilson, 1966) and reasonable to attribute the inhibition simply to lack of ATP. There is, however, a discrepancy. It has been known for some time that uncouplers inhibit phosphate uptake in yeast even under anaerobic conditions (Windisch and Heumann, 1960; Riemersma, 1968).Anaerobic growth of E.coli is sensitive to uncouplers (Kovai: and Kuiela) and other examples of this kind range from microorganisms t o the toad bladder (Galeotti et al., 1968; Klahr et al., 1968). Some insight into the basis of this unexpected phenomenon came from studies with a strain of Streptococcus faecalis which lacks cytochromes and appears to produce ATP exclusively via glycolysis. A number of uncouplers including tetrachlorosalicylanilide (TCS), CCCP, pentachlorophenol and also a novel antibacterial agent, tetramethyldipicrylamine (Meyer et al., 1967) inhibited energy-dependent transport of K+, phosphate and of certain amino acids. The uncouplers did not inhibit glycolysis, ATP synthesis, ATP turnover or even the utilization of ATP for the synthesis of macromolecules. It was therefore concluded (Harold and Baarda, 1968b) that the uncouplers specifically prevent the utilization of metabolic energy for active transport. I n a subsequent study (PavlasovB and Harold, 1969) this conclusion was extended to the accumulation of ,B-galactosides by E . coli under anaerobic conditions : TCS and CCCP had no effect on the ATP pool, nor did they inhibit the

68

FRANKLIN M. HAROLD

transport system as such, but they prevented accumulation of thiomethyl galactoside against a concentration gradient. A substantial body of evidence, (Harold and Baarda, 1968b, Pavlasovb and Harold, 1969 and unpublished results) warrants the conclusion that inhibition of active transport is a consequence of the fact that uncouplers facilitate passage of protons across the membrane. These experiments call into question the tacit assumption that uncouplers inhibit energy-dependent processes secondarily by interfering with ATP synthesis. Even under aerobic conditions, uncoupling of oxidative phosphorylation and inhibition of active transport may be consequences of a single primary effect, proton conduction. We may even legitimately ask whether inhibition of bacterial motility by uncouplers (Paust and Doetsch, 1969) is due to lack of ATP or hints a t more subtle phenomena. We do not know why proton conductors interfere with active transports even when glycolytic ATP is the ultimate energy donor. It is possible that a gradient of p H value across the cytoplasmic membrane is part of the machinery by which metabolic energy is coupled to membrane transport : many active transports may, in fact, be secondary translocations driven by a primary proton pump which maintains the p H gradient (Mitchell, 1963, 1966, 1967a; Chappell and Crofts, 1965, Harold and Baarda, 1968b; Pavlasovb and Harold, 1969). Other explanations are by no means excluded, and can be formulated in terms quite analogous to those employed above to explain the uncoupling of oxidative phosphorylation (Harold and Baarda, 196Sb). Be this as i t may, proton-conducting uncouplers raise insistent questions not only concerning the mechanism of oxidative phosphorylation but for membrane processes in general.

V. Alkali Metal Ionophores I n 1944 Hotchkiss reviewed what was known a t the time concerning the mode of action of the antibiotics tyrocidine and gramicidin. He concluded that tyrocidine disrupts the cytoplasmic membrane of sensitive cells and subsequent work has borne him out (Section III.D, p. 58). Gramicidin appeared to act in a different manner. It did not induce leakage of cell constituents but affected respiration and phosphate uptake much as did the recently discovered 2,4-dinitrophenol. Hotchkiss recognized that alkali-metal ions were involved in the effects of gramicidin, but the time was not yet ripe for further insight. Interest in gramicidin then languished, except for the demonstration that it does indeed uncouplq; oxidative phosphorylation in mitochondria and bacteria, and also photophosphorylation (for references see Hunter and Rchwartz, 1967c).

ANTIMICROBIAL AGENTS AND MEMBRANE FUNCTION

69

A decade later, interest in polypeptide uncouplers revived as a result of Lardy's search for antibiotics which affect oxidative phosphorylation. One antibiotic, valinomycin, appeared to be the most potent uncoupler yet discovered (McMurray and Begg, 1959).Attempts to explain certain peculiar features of uncoupling by valinomycin led to the discovery (Moore and Pressman, 1964) that uncoupling required the presence of K+ and was related to the induction of K+ uptake by mitochondria. Within a year, gramicidin as well as a number of other antibiotics of very different structure had been found to induce cation uptake (Chappel1 and Crofts, 1965; Pressman, 1965). An amusing account of the discovery of cation conduction has been given by Pressman (1969). Studies in many laboratories have since revealed that the primary effect of these antibiotics is to facilitate passage of alkali-metal ions across lipid membranes. The antibiotics differ strikingly in specificity, ranging from valinomycin which is highly selective for Kf to the promiscuous gramicidins. These antibiotics are the first compounds to exhibit the solubility and specificity characteristics that ion carriers in natural membranes would be expected to possess (Pressman et al., 1967; Pressman, 1968),and they are thus both models and guides to future research on ion transport.

A. VALINOMYCIN Valinomycin, an antibiotic produced by certain strains of Xtreptomyces, inhibits the growth of a variety of fungi and Gram-positive bacteria.

Valinomycin

Enniatin

FIG.6. Chemical structures of certain depsipeptide antibiotics. Hydroxyisoval,

indicates a hydroxyisovaleric acid residue ; N-methylval, an N-methylvaline residue; and val, a valine residue.

Its structure, determined by Shemyakin and his associates and confirmed by synthesis (Shemyakin et al., 1965; Shemyakin, 1965) is shown in Fig. 6. It is a cyclodepsipeptide consisting of three repetitions of the

70

FRANKLIN M. HAROLD

sequence (D-valine-L-lactic acid-L-valine-D-hydroxyisovaleric acid). Like all of the antibiotics to be considered in this section, valinomycin is soluble in ethanol but virtually insoluble in water. Studies on its structure and mode of action through 1965were reviewed by Hunter and Schwartz (1967a). More current surveys of this rapidly moving subject will be found in Pressman’s articles (1968, 1969).We shall begin here with the effects of valinomycin on artificial lipid membranes and then consider interaction of the antibiotic with biological systems of increasing complexity. Valinomycin greatly increased the K+ permeability of both phospholipid spherules (Chappell and Crofts, 1966 ; Chappell and Haarhoff, 1967) and of black bilayer membranes. The latter system proved most informative. Valinomycin, in presence of K+,increased the electrical conductivity of the membranes by five orders of magnitude and also elicited an electrical potential across membranes separating compartments containing different concentrations of K+. Both phenomena are characteristic of the passage of charged particles across the electrical barrier, presumably a complex of the neutral valinomycin molecule with K+. Valinomycin did not induce anion permeability and discriminated sharply among the various cations ; K+was preferred t o Na+ by a factor of a t least 400. When present at equal concentrations, the sequence of ionic conductivities is : H+ > Rbf > Kf > Cs+ > Na+ > Li+. However, a t physiological p H values the concentration of H+ is very low and the antibiotic then acts as a highly selective carrier for K+ (Andreoli et al., 1967 ;Lev and Buzhinsky, 1967; Liberman and Topaly, 1968; Mueller and Rudin, 1967). The selectivity of valinomycin is independent of the composition of the lipid membrane and must thus reside in the antibiotic molecule itself (Mueller and Rudin, 1967; Pressman, 1968). A priori, several mechanisms were envisaged by which valinomycin could facilitate K+ movements. Valinomycin is a ring-shaped molecule ; several investigators suggested that cations are inserted into the hole and considered various factors, including the diameters of the ring and of the ion, which could determine the stability of the complex. The ring could act as a carrier or else a stack of valinomycin molecules could make up a K+-selective pore (Chappell and Crofts, 1965; Mueller and Rudin, 1967; Andreoli et al., 1967). Recent evidence favours the concept that valinomycin acts as a lipidsoluble carrier which shuttles K+ across membranes in the form of a valinomycin-K+ complex (Pressman et al., 1967; Pressman, 1968; Stein, 1968; Liberman and Topaly, 1968; Tosteson, 1968). The most compelling argument is the finding that valinomycin facilitates K+ diffusion across a bulk phase of lipid or organic solvent. The concentration dependence indicates that each valinomycin molecule carries a single K+ ion; Na’ fails to form a lipid-soluble complex with valinomycin,

ANTIMICROBIAL AGENTS AND MEMBRANE FUNCTION

71

but Na+ and other cations do displace K+ from the antibiotic (Pressman et al., 1967; Pressman, 1968; Tosteson, 1968; Tosteson et ul., 1968). The three-dimensional structure of the valinomycin-K+ complex has just been established by use of optical rotatory dispersion, nuclear magnetic resonance and other spectroscopic techniques (Ivanov et ul., 1969; Haynes et ul., 1969). It is a kind of clathrate, with the K+ enfolded 1 within a cage that is held together by induced dipoles between K+ and ?' the oxygen atoms of valinomycin. The non-polar side chains are directed outwards to form a hydrophobic surface, resulting in a lipid-soluble molecule which can equilibrate with K+ a t the lipid-water interface. Shemyakin and his associates (1965) have synthesized numerous analogues and derivatives of valinomycin in an effort to establish the structural features required for antibiotic activity. Linear analogues were inactive, as were rings containing more or fewer repeating units. The hydroxyisovaleric acid moieties were essential, but considerable latitude was allowable in the hydrophobic amino acids. There was excellent correlation between antibiotic activity, the induction of K+ transport in mitochondria (Shemyakin et al., 1965; Pressman, 1965) and K+ conduction across phospholipid membranes (Mueller and Rudin, 1967), indicating that the antibiotic activity of valinomycin may well be a consequence of K+ translocations. With this background let us now turn t o a consideration of the biological effects of valinomycin. The interpretation is simplest for Streptococcus fueculis which generates ATP by glycolysis and lacks oxidative phosphorylation. Valinomycin was bacteriostatic for Xtrep. fuecalis and inhibition of growth could be reversed by raising the K+ content of the medium. The antibiotic specifically induced loss of K+ from the cells, by stoichiometric exchange for other cations present in the medium, especially Na+. I n agreement with findings made in other systems, valinomycin greatly incrcased membrane permeability to K+ but not to other ions or metabolites. It did not interfere in any way with the generation of ATP by glycolysis or with its utilization (Harold and Baarda, 1967). Cessation of growth is due to inhibition of protein synthesis, which requires K+ (Lubin, 1964; Harold and Baarda, 1967; 1968a). The effects of valinomycin on erythrocytes can also be understood in terms of facilitated K+ diffusion (Harris and Pressman, 1967; Tosteson et ul., 1967; Tosteson et ul., 1968). Valinomycin inhibits the growth of various aerobic bacteria and fungi (Hunter and Schwartz, 1967a; Shemyakin et ul., 1965), but I am aware of only a single paper describing its physiological effects. Pressman (1967) demonstrated that, in both Azotobucter and Mycobucterium phlei, valinomycin induced K+ uptake and stimulated respiration, just as in mitochondria. It was suggested that inhibition of growth is due to dissipation

'

72

FRANKLIN M. HAROLD

of metabolic energy resulting from the induced K+ uptake, an interpretation derived from the extensive work of Pressman and his associates with mitochondria. This topic is both complex and controversial but a summary is essential since any future research on the effects of valinomycin on aerobic bacteria must take the mitochondrial system as its point of departure. There seems to be fairly general agreement on the basic facts. Valinomycin, at nanomolar concentrations, initiates net K+ uptake by mitochondria. This is an energy-requiring process which can be supported either by oxidation of a substrate or by exogenous ATP. UncoupIers of oxidative phosphorylation, including the proton conductor CCCP, reverse the direction of K+ movement, such that K+ runs out into the medium. Valinomycin also increases theflux of 42Kacross the membrane in both directions, i.e. it increases permeability of the mitochondrial membrane to KS (Chappell and Crofts, 1965, 1966; Harris et al., 1966, 1967a, 1967b; Hofer and Pressman, 1966; Pressman, 1968; Mitchell, 1967b; Mitchell and Moyle, 1967a, b). The net uptake of K+ is accompanied by ejection of H+ and/or the uptake of anions, and sometimes by swelling ; the magnitudes of these processes depend upon conditions (Chappell and Crofts, 1965, 1966; Pressman, 1965; Harris et al., 1966). Although originally described as an uncoupler of oxidative phosphorylation, this is clearly not the primary effect of valinomycin, since conditions can be found under which phosphorylation keeps pace with the increased respiratory rate (Hofer and Pressman, 1966; Harris et al., 1967a).Pressman, Harris and their associates (Cockrell et aZ., 1966; Harris et al., 1966; Pressman, 1968)have determined that as many as six K+ ions can be translocated per ATP molecule hydrolysed. Indeed, it has been possible to couple Kf efflux induced by valinomycin to the synthesis of ATP (Cockrell et aZ., 1967). Two fundamentally different hypotheses have been offered to rationalize the experimental results. These are shown schematically in Fig. 7, and are obviously related to the chemical coupling (a)and chemi-osmotic (b) hypotheses of oxidative phosphorylation. (a) Pressman and his collaborators (Harris and Pressman, 1969; Harris et aZ., 1967b; Pressman, 1968, 1969; Pressman et al., 1967)believe that the mitochondrial membrane contains an electrogenic cation pump. This pump tends to drive K+ into the organelle againsti its concentration gradient, at the expense of metabolic energy derived either from ATP or from the hypothetical energy-rich intermediate of oxidative phosphorylation. Now, mitochondria do not normalIy accumulate K+. Pressman has suggested that the physiological role of the pump is the transport of anions and that, in untreated mitochondria, K+ has no access to the pump. The effect of valinomycin is to breach the permeability barrier;

ANTIMICROBIAL AGENTS AND MEMBRANE FUNCTION

73

this delivers K" to the pump which now carries out net K+ accumulation. Compensatory movements of protons, anions or both maintain electroneutrality. A cyclic flux of K+ is thus set up by valinomycin-in through the pump, out by several possible pathways which do not seem to be well defined-which would consume and dissipate the energy-rich intermediates and therefore uncouple oxidative phosphorylation. A. Respiratory Chain

4

X-I

ATP

Cations+ ___f

H+

B.

ATPase

Respiratory Chain

+----

+----

H+

H+

Cations+

___3

FIG.7. Possible schemes depicting the relationship of cation transport t o energy goneration (see text for explanation). After Mitchell ( 1969).

(b) Mitchell's view (l966,1967b, 1969; Chappell and Crofts, 1965) is in essence the converse of that just outlined. The primary gradient is established by pumping protons out of the mitochondrion, either by operation of the respiratory chain itself, or by means of a proton pump. Since the mitochondria1 membrane is relatively impermeable to ions, proton movements are restricted by formation of a membrane potential -(inside negative). Valinomycin simply renders the membrane permeable to K+, and no specific interaction with a n ion pump is required: Kf accumulation results from movement of the ion down the electrochemical gradient produced by the proton pump. Uncoupling of oxidative phosphorylation is ascribed to the dissipation of the proton-motive force by K" movements. Both Mitchell's and Pressman's hypotheses have difficulties, which have been skilfully exposed by Pressman and Mitchell, respectively, as well as by others (for recent position statements see, Caswell, 1968; Harris and Pressman, 1969; Mitchell, 1967, 1969; Mitchell and Moyle, 1969; Poe, 1968; Pressman, 1968,1969; Slater, 1967). Clearly, what is a t issue here is the very nature of energy coupling in oxidative phosphorylation and the time does not seem ripe for final judgement.

74

BRANKLTN M. HAROLD

B. ENNIATINS The enniatins are a family of cyclodepsipeptide antibiotics which inhibit the growth of Gram-positive bacteria and fungi. Both in structure (Fig. 6; Shemyakin et al., 1963; Shemyakin, 1965) and in their mode of action they resemble valinomycin but are less potent. Enniatins facilitate K+ diffusion across lipid bilayer membranes (Mueller and Rudin, 1967); an increase in permeability to thiourea has been reported (Lippe, 1968) but the effect is small. A stoichiometric complex of enniatin B with K+ has been isolated (Shemyakin et al., 1967a; Wipf et al., 1968). This is presumably the carrier, but its threedimensional structure is not yet known (for discussion see Lardy et al., 1967; Mueller and Rudin, 1967; Pressman, 1968). The sequence of ionpreference for complex formation is : Rb+ > K+ > Csf > Naf (Mueller and Rudin, 1967; Henderson et al., 1969). Shemyakin and his associates ( 1 963,1967a, b) have prepared numerous analogues and derivatives of the enniatins, which give some insight into the structural requirements for antibiotic activity. A regular sequence of amino acids and hydroxyamino acids is critical, as is the size of the ring. The enniatins which are active antibiotics induce K+ uptake by mitochondria (Lardy et al., 1967; Pressman, 1968) suggesting that, as with valinomycins, the induction of K+ permeability is a t the root of the antimicrobial effect. However, no studies on the mode of action of enniatins on bacteria appear to have been published. I n our hands (F. M. Harold and J. R . Baarda, unpublished observations) the effects of enniatin A (5 pg.ml.) on Strep. fuecalis were very similar to those of valinomycin. C. GRAMICIDINS The gramicidins are a family of closely related linear peptides. I n all, the terminal amino group is formylated and the carboxyl group is masked by ethanolamine, and substitutions in various positions give rise to the individual members. Gramicidin A, for example, has the sequence HCO -L-Val-Gly -L-Ala-D-Leu-L-Ala-D-Val-L-Val-D-Val- L - Try - D - LeuL-Try-o-Leu-L-Try-o-Leu-L-Try-NHCH,CH,OH. The structure and mode of action of gramicidins have been reviewed by Hunter and Schwartz (1967~). Although it had been known for twenty years that gramicidins uncouple oxidative phosphorylation, progress a t the molecular level had to await the discovery (Chappell and Crofts, 1965; Pressman, 1965) that gramicidins induce energy-dependent net uptake of cations by mitochondria. They also increase the permeability of the mitochondria1

ANTIMICROBIAL AGENTS AND MEMBRANE FUNCTION

75

membrane to cations (Chappell and Crofts, 1965, 1966; Harris et al., 1967a). However, whereas valinomycin is highIy selective for K+, the gramicidins induce the uptake of Na+, Li+ and NH4+ as well as K+. Gramicidins increase the cation permeability of artificial lipid membranes, with little discrimination between cations (Chappell and Crofts, 1966; Henderson et al., 1969; Mueller and Rudin, 1967; Tosteson et al., 1968). Selectivity for K+ over Na+ was only by a factor of six, compared with 400 for valinomycin (Mueller and Rudin, 1967). The molecular basis of the interaction between gramicidins and cations is unknown. Models can be constructed in which one or perhaps two linear polypeptide molecules coil up around the cation (Lardy et al., 1967; Mueller and Rudin, 1967 ; Tosteson et al., 1968) but there appears to beno information as to carrier versus pore conduction. The increased cation permeability of the cytoplasmic membrane is sufficient t o account for the inhibition of growth of Strep. faecalis by lop7 M-gramicidin D. The antibiotic induces rapid loss of K+ from the cells by exchange for external Na+, Li+ or even NH4+ and thus halts protein synthesis for lack of Kf (Harold and Baarda, 1967). In addition, gramicidin partly inhibits glycolysis and the active transport of various metabolites. This apparently results from loss of K+ by exchange for H+; gramicidin thus acts to some degree as a proton conductor (Harold and Baarda, 1968b). A similar explanation was earlier advanced by Harris et al. (1967a) to explain aspects of the uncoupling of oxidative phosphorylation by gramicidin. The effects of gramicidin on erythrocytes can be understood in the same way (Bielawski, 1968; Scarpa et al., 1968), and account for the early observation that gramicidin induced slow, progressive haemolysis when injected into animals (Hotchkiss, 1944). There are surprisingly few data on the effect of gramicidin on energy metabolism of aerobic bacteria, apart from the original observations of Hotchkiss (1944) that K+ was required for the stimulation of respiration H) in Staphylococcus by this antibiotic. High concentrations ( inhibited motility of Pseudomonas fiuorescens but stimulated oxygen uptake (Faust and Doetsch, 1969). The relationship of cations to the mode of action of grainicidin is more explicit in the findings of Pressman and his associates (Pressman, 1965; Harris et al., 1967a) and of Chappell and Crofts (1965, 1966) with mitochrondria. I have already summarized the divergent interpretations which these two groups give to their results (Section V.A, p. 69). Yet a third hypothesis has been offered by Palcone and Hadler (1968), who regard the cation uptake induced by gramicidin as a consequence of a primary effect of the antibiotic on oxidative phosphorylation, rather than the other way around.

76

FRANKLIN M. HAROLD

D. MACROTETRALIDES : NONACTIN AND

ITS

HOMOLOGUES

Certain streptomycetes produce yet another class of ion-conducting antibiotics, loosely referred to as the “nactins”. These are cyclic tetralactones which, like valinomycin, form lipid-soluble complexes with K+. The original member of this series appeared to lack antibiotic activity and was named nonactin. Subsequently, homologues containing additional methyl groups were isolated and designated monactin, dinactin and trinactin. Moreover, the “nactins” turned out to be potent inhibitors of Gram-positive bacteria after all (Meyers et al., 1965; see also the review by Shaw, 1967b). The structure of these compounds is shown in Fig. 8. The demonstration that the nactins, like valinomycin, induce ion transport and uncouple oxidative phosphorylation led to their recognition as ioiiophores (Graven et d.,1966b, c).

FIG.8. Chemical structure of monactin, a macrotetralide antibiotic.

The primary ion-conducting properties of the macrotetralides have been explored in considerable detail. Dinactin and monactiii induced both electrical conductivity and ion-diffusion potentials in phospholipid bilayers with a K+/Na’ selectivity ratio of 37 (Mueller and Rudin, 1967). A diffusible complex appears to be formed, containing K+ and the antibiotic in equimolar amounts. This carrier conducts Kf across a bulk lipid phase as well as across a membrane (Eisenman et al., 1968; Tosteson, 1968). Curiously, monactin and dinactin appear to complex sodium as well as K+; failure of the antibiotics to translocate Na+ as measured by electrical conductivity must thus be attributed to the differing properties of the two complexes (Tosteson, 1968). The conformation of the K+ complex of monactin was determined by X-ray crystallography (Kilbourn et al., 1967). As shown in Fig. 9, the molecule in the crystalline state is not planar but folds up into the general shape of a “tennis-ball seam”, with the K+ encaged in the middle. The polar oxygens are directed inwards while the lipophilic surface permits the complex t o traverse lipid barriers and equilibrate with K+ a t the interface (Eisenman et al., 1968; Tosteson, 1968).

ANTIMICROBIAL AGENTS AND MEMBRANE FUNCTION

77

Very little work has been done on the antimicrobial effects of the nactins. Monactin inhibited growth of &rep. faecalis by inducing exchange of cellular K+ for Na+. The antibiotic had no direct effects on the generation and utilization of glycolytic ATP. A technical convenience is that, unlike valinomycin, monactin was readily removed by washing the cells, which restored their original impermeability to ions (Harold and Baarda, 1968a). The effects of monactin on red blood cells are also due simply to increased K+ flux (Tosteson et al., 1968).

FIG.9. Xpacc-filling model of the monactin-K omitted to show the central cavity).

complex (the cation has beon

I am not aware of any work with aerobic bacteria, but the effects of the nactins on mitochondria have been investigated in detail. The nactins induce ion transport, preferentiall? of K+; this is energy-dependent and may be accompanied by swelling. Under some conditions they uncouple oxidative phosphorylation and induce ATP hydrolysis (Graven et al., 1966b, c, 1967; Henderson et ak., 1969). The effects of the nactins are clearly similar to those of valinomyciii and their interpretation must be considered in much the same terms (Section V.A, p. 69).

78

FRANKLIN M. HAROLD

E. NIGERICIN,MONENSIN AND

OTHER

CARBOXYLICPOLYETHERS

Harned et al. (1951) originally described the antibiotic nigericin as a lipid-soluble monocarboxylic acid active against Gram-positive but not Gram-negative bacteria. Most remarkably, inhibition was reversed by addition of excess K+ to the medium. The structure of nigericin, established very recently by Steinrauf et al. (1968),is shown in Fig. 10. Studies in Lardy’s laboratory provided the first clue to its mode of action: nigericin was found to counteract the effect of valinomycin, i.e. to release K+ from the mitochondria (Graven et al., 1966a; Lardy et al., 1967).

FIG.10. Carboxylic polyether antibiotics.

Several antibiotics are now known which, like nigericin, are monocarboxylic acids. O f these the most important is monensin, a new antibiotic of considerable clinical promise (Haney and Hoehn, 1967). Its structure (Agtarap et al., 1967; Agtarap and Chamberlin, 1967) is shown in Fig. 10. Others of this general type include dianemycin, X-206 and X-537 ; scattered references indicate that their mode of action is similar to that of nigericin (Henderson et al., 1969; Pressman et al., 1967; Pressman, 1968, 1969). Nigericin induced increased permeability to both K+ and protons in various phospholipid membrane systems (Mueller and Rudin, 1967 ; Henderson and Chappell, 1967; Henderson et al., 1969; Tosteson et al., 1968) but this was not accompanied by electrical effects such as ionic potentials or increased conductivity. The antibiotic forms lipid-soluble complexes with Rb+ and K+, and can carry these even across a bulk solvent phase, but only a t high pH values (Pressman et al., 1967 ; Press-

ANTIMICROBIAL AQENTS AND MEMBRANE BUNCTION

79

man, 1968, 1969).The proposed explanation is as follows : K+ binds only to the dissociated form of the antibiotic, and can traverse lipid barriers in this state; at lower pH values, the undissociated antibiotic acts as a proton carrier. Nigericin therefore catalyses electrically neutral exchange of K+ for H+ (and of K+ for other cations) which cannot be detected by electrical measurements. By contrast, let us recall that valinomycin forms a Ki- complex which bears a net positive charge; valinomycin therefore induces both ionic potentials and increased conductivity (Pressman, 1968, 1969; Pressman et al., 1967). Various members of this group differ significantly in ion selectivity. For example, nigericin prefers K+ to Na+, whereas monensin and dianemycin prefer Na+ (Pressman, 1968; Henderson et al., 1969). The structural basis of the selectivity may reside in the properties of the complex of cation and antibiotic, which is not a salt but an electrically neutral clathrate-in fact, a Zwitterion: it appears that only under conditions in which the carboxyl group is dissociated does the molecule fold over to enclose the cation. The protonated form has an entirely different conformation, perhaps that of an open chain (Pressman, 1968, 1969; Agtarap et al., 1967). These considerations are central to an understanding of the metabolic effects of nigericin and monensin. I n erythrocytes (Harris and Pressman, 1967 ; Pressman et al., 1967), nigericin induced electrically neutral exchange of K+ for H+. The same was observed in Strep. faecalis with nigericin (Harold and Baarda, 1968a) and monensin, albeit the latter prefers Na+ to K+ and rejects Rb+ (Estrada-0 et al., 1967b; Henderson et al., 1969; Pressman, 1969; F. M. Harold and J. R. Baarda, unpublished observations). The loss of K+, by exchange for either H+ or Na+, is a sufficient explanation for the inhibition of growth by nigericin and for its reversal by excess K+. However, unlike the other cation-conducting antibiotics, nigericin also strongly inhibits net uptake of K+, phosphate and alanine by Strep. faecalis. Since nigericin induces K+-H" exchange, we may regard it as a proton conductor of a special kind, and it is this entry of protons which accounts for the inhibition of transport (Harold and Baarda, 1968b). Whether catalysis of both cation-cation and cation-proton exchange can account for the complex effects of nigericin and monensin on mitochondria is not yet certain. Graven et al. (1966a) originally found that nigericin counteracts the effects of valinomycin and gramicidin, causing release of K+. This cation efflux was not energy-dependent, nor did it require prior exposure of the mitochondria to valinomycin (Graven et al., 1966a; Lardy et al., 1967; Pressman et al., 1967). I n addition, nigericin inhibits phosphate uptake and the oxidation of certain NADH2linked substrates and, also unmasks ATPase (Estrada-0 et al., 1967b).

80

FRANKLIN M. HAROLD

The effects of rnonensin are essentially similar, except for different cation specificity (Estrada-0etal., 1967a).Pressman (l968,1969;Pressmanetal., 1967) believes that the basis for these effects is to be sought in the induction of neutral, K'-H+ exchange a t random sites on the mitochondrial membrane with release of Kf and entry of protons. A very different interpretation has been considered by Lardy and his associates (Lardy et al., 1967; Estrada-0 et al., 1967b). The ion exchanges induced by nigericin have been used as a test of the chemi-osmotic hypothesis. Conditions can be found under which nigericin and monensin induce extensive Kf-H+ exchange yet do not uncouple oxidative phosphorylation (Graven et al., 1966a; Estrada-0 et al., 1967a; Pressman et al., 1967). According to the chemi-osmotic hypothesis, ATP synthesis requires a proton gradient across the membrane and therefore nigericin, which induces H+ entry, should act as an uncoupler (Pressman P t al., 1967). The same argument has been applied bo photophosphorylation. Nigericin completely blocks proton uptake by chromatophores of Rhodospirillum rubrum, yet does not uncouple photophosphorylation (Shavit et al., 1968; Thore et al., 1968). This was held to be incompatible with the chemi-osmotic hypobhesis which calls for a proton gradient as the driving force for photosynthetic ATP formation. Jackson et al. (1968) confirmed the observations but interpret them in quite another way. Induction of K+-H+ exchange by nigericin would obliterate a pH gradient but, being electrically neutral, would not affect a membrane potential ; whether or not uncoupling is observed would then depend on whether a p H gradient or a membrane potential is the primary form of energy storage. Such a potential should, however, be collapsed by valinomycin which permits electrogenic K+ movements ; the combination of nigericin plus valinomycin did, indeed, uncouple photjophosphorylation (Jackson et al., 1968; Thore et al., 1968). I n fine, the failure of nigericin to uncouple oxidative and photophosphorylation is not necessarily incompatible with the chemi-osmotic hypothesis (see also Henderson et al., 1969).

P. OTHERIONOPHORES The list of known and potential ionophorous agents is by no means exhausted and we should mention some of the others because they may have attained prominence by the time this review appears in print. 1. Monazomycin is a basic antibiotic of unknown structure, which induces net uptake of Kt, Na+ and Li+ by mitochondria (Lardy et al., 1967; Estrada-0 et al., 1967b) and also uncouples oxidative phosphorylation. Nothing appears to be known concerning its effects on microorganisms.

ANTIMICROBTAL AGENTS AND MEMBRANE FUNCTION

81

2. Polyethers. Pedersen (1968) has synthesized a series of polyethers which exhibit varying degrees of selectivity in complexing cations (Eisenman et nl., 1968; Pedersen, 1968; Pressman, 1968; Tosteson, 1968) and which facilitate passage of ions across lipid phases and membranes. These are structurally related to the macrotetralide nactins. Some of these induce ion uptake in mitochondria ; others, inactive in themselves, inhibit the induction of K+ uptake by valinomycin (Lardy, 1968). Although nothing has been reported on the effects, if any, of such compounds on micro-organisms, they hold the promise of tailor-made ion conductors and should be kept in mind. 3. A lamethicin is a cyclic polypeptide produced by Trichoderma viride (Meyer and Reusser, 1967). It is surface active and inhibits growth of various Gram-positive bacteria albeit only a t rather high concentrations. Pressman (1968) mentions that it induces cation uptake by mitochondria. Of greatest interest is the discovery by Mueller and Rudin (1968) that alamethicin induced electrical excitability in artificial phospholipid bilayer membranes. Little is known concerning its effects on microorganisms ; experiments in this laboratory (unpublished) suggested that i t may disrupt the permeability barrier of Strep. faecnlis.

VI. Inhibitors of Energy Transfer and of the Respiratory Chain The compounds considered in the preceding sections interact primarily with the lipid moiety of membranes. Some penetrate into the membrane to distort its structure, others form lipid-soluble complexes with specific ions. The mode of action of these compounds could be reduced to physical and chemical phenomena which account for most (if not all) of their biological effects. We now turn to inhibitors which interact with individual proteins, structural or catalytic, to block specific steps in the metabolic pathways associated with membranes. These include the coupling of electron transport t o ATP generation, the utilization of metabolic energy for active transport or other functions, and the respiratory chain itself.

A. ATPase

AND

ENERGY TRANSFER

ATPases are quite universally associated with biological membranes and participate in energy transfer and transformation by these organelles. The ATPase of the inner mitochondria1 membrane, for example, requires Mg2+ and is localized in spherical particles attached to the basal membrane by a stalk. It seems to be generally accepted that this enzyme

82

FRANKLIN M. HAROLD

catalyses the terminal step in oxidative phosphorylation, the synthesis of ATP itself (see reviews by Mitchell, 1966; Racker, 1967; Pullman and Schatz, 1967). The same enzyme, but acting in reverse, mediates the utilization of exogenous ATP to energize reversed electron flow, transhydrogenase or ion transport when substrate oxidation is blocked (Section II.B, p. 48). An analogous ATPase occurs in chloroplasts (Moudrianakis, 1968). The other process in which ATPase has been definitely implicated is ion transport. ATPases which require Mg2+and are stimulated by Na+ and K+ are commonly found in cytoplasmic membranes of mammalian cells and in the microsome fraction. There is overwhelming evidence, recently reviewed by Skou (1965), Stein (1967) and Albers (1967), that these enzymes mediate the translocation of Naf and K+. Other translocations may be secondarily driven by this enzyme through gradient coupling (section II.B, p. 48). The membrane fraction isolated from bacteria also has ATPase activity; how many enzymes are invoived is not clear. I n some cases the ATPase activity is associated with stalked particles, and may be part of the machinery for generating ATP by oxidative phosphorylation (Gel’man et al., 1967; Mufioz et al., 1968, 1969), but this cannot be the function of ATPase in fermentative bacteria. In general, ATPases of bacterial membranes are only slighbly stimulated by Na+ and K+ or not stimulated at all (see Abrams, 1965; Harold et aZ.,1969a, for references). All the same there is good evidence that, in bacteria also, ATPase is involved in ion transport. This section will survey compounds which inhibit ATPase and related energy-transfer reactions. The best known of these, oligomycin, was discovered as a result of Lardy’s search for antibiotics which affect oxidative phosphorylation (Lardy et al., 1958); although of limited application to bacterial systems, oligomycin is the prototype t o which all other inhibitors must be compared. 1. Oligomycin, Rutamycin and Related Antibiotics. The oligomycin complex of antibiotics is produced by Streptomycesspeciesas isrutamycin, a related but distinct compound. Oligomycins and rutamycin inhibit the growth of many filamentous fungi and some yeasts but have no antibacterial action. Several other antibiotics have recently been added to this group. Peliomycin, ossamycin and venturicidin cIosely resemble rutamycin in their biological effects (Walter et al., 1967); peliomycin strongly inhibits growth of Micrococcus lysodeikticus (Price et al. , 1963) and may thus prove particularly useful in the study of bacterial ATPase. Like other antibiotics which act upon membranes, all are insoluble in water but soluble in organic solvents. Their structures have not yet been determined.

ANTIMICROBIAL AGENTS A N D MEMBRANE BUNCTION

83

It became clear very early that oligomycin affects respiration since only aerobic organisms are sensitive to this antibiotic. I n a recent illustration of this generalization, Parker et al. (1968) showed that oligomycin inhibits growth of yeast on ethanol, which must be respired, but is much less inhibitory to growth on glucose which can be fermented; the antibiotic also increased the frequency of mutants deficient in respiratory functions. The effects of oligomycin and related antibiotics have been studied most extensively in animal mitochondria, but yeast mitochondria respond in much the same way (Shaw, 1967a; Schatz, 1968). The summary which follows leans heavily on the comprehensive review by Shaw (1967a). I n intact mitochondria, oligomycin and rutamycin inhibit both phosphorylation and respiration but do not alter the P / O ratio; uncouplers restore respiration but not, of course, phosphorylation. I n damaged or fragmented mitochondria, oligomycin tends to block phosphorylation but not respiration. Thus these antibiotics do not inhibit electron transport as such but the energy-transfer steps which couple ATP synthesis to the redox reactions. Inhibition of respiration is a secondary effect which reflects respiratory control rather than the site of action of the inhibitor. These findings led to the discovery that oligomycin and rutamycin inhibib reactions closely related to ATP synthesis, including ATP/ADP exchange and ATP hydrolysis (Lardy et al., 1958; Shaw, l967a). The molecular basis of these inhibitions is under active investigation in many laboratories. Briefly, oligomycin and rutamycin bind to a component of the inner mitochondria1 membrane which is distinct from the ATPase itself; the enzyme is sensitive t o the antibiotic when attached to the membrane but becomes resistant when solubilized. Sensitivity to oligomycin involves one or more proteins which are part of the stalk by which the ATPase particle is attached t o the membrane. Inhibition of ATPase activity is thus a secondary phenomenon, due perhaps to a transmitted effect on the conformation of the enzyme (Bulos and Racker, 1968; Kagawa and Racker, 1966; Kopaczyk et al., 1968; MacLennan and Tzagaloff, 1968). For reviews see Pullman and Schatz (1967) and Racker (1967). The precise mechanism by which oligomycin inhibits phosphorylation and respiration is still quite controversial, and the schemes which have been proposed (reviewed by Shaw, 1967a; Lee and Ernster, 1968; Roberton et ab., 1968; Mitchell, 1966) reflect the authors’ divergent views of the basic nature of energy coupling in oxidative phosphorylation. For the purposes of the present discussion, the generalized scheme of Pig. 11 will suffice, based on the views of Lee and Ernster (1968) and of Roberton et al. (1968).

84

FRANKLIN M. HAROLD Huccinstc

Flavoprotein S Piericirliri

NAUHn

YlnV(J-

; i l)rotcin

i-1 -

enzyme

Q

Antimycin Heptylqui noline oxide

T---

5.

1.

+Cyt. a, a3

C'yt. b w Cyt. C1

A

7

I

~

A D P + P i ATP 1011 trnrisport, other functions Oliyomycin Dicyclohoxylcarbodiiniidc ~

x-P aurovertiit

Dio !J? AT1'

FIG.11. Elcctron transport and oxidative phosphorylation (in terins of tho chemi-

cal-coupling hypothesis), showing the sites of action of the inhibitors piericidin, antimycin, heptylquinoline oxide, oligomycin, dicyclohexylcarbodiimide aurovertin and Dio 9.

Certain energy-linked functions including ion transport, transhydrogenase and reversed electron flow can be energized by oxidizable substrates even in the presence of oligomycin. Since the antibiotic blocks ATP formation, this is evidence that processes such as ion transport can be supported by energy-rich intermediates or states, of unspecified nature, preceding ATP itself. When electron transport is blocked, either by lack of a suitable substrate or by an inhibitor of the respiratory chain, ion transport can be energized by ATP; under these conditions it is sensitive to oligomycin, in keeping with the scheme (Fig. 11;Lehninger et al., 1967; Pullman and Schatz, 1967; Shaw, 1967a; Roberton et al., 1968; Lee and Ernster, 1968). A most interesting development is the recognition that oligomycin inhibits ion transport, not only in mitochondria but also in tissues which depend upon the Na+- and K+-stimulated ATPase. This enzyme is inhibited by the antibiotic, albeit a t higher concentrations than are required in mitochondria (Blake et al., 1967; Inturrisi and Titus, 1968; Whittam et al., 1964). I n some cases, at least, oligomycin inhibited the K+-dependent dephosphorylation of the enzyme, but not the Na+dependent phosphorylation (Pahn et al., 1968). The unique importance of antibiotics of the oligomycin type is precisely that they block energy transductions in a variety of membranes, and thus reveal the unity of

Oa

ANTIMICROBIAL AGENTS AND MEMBRANE PUNCTION

85

structure and function which underlies the diversity. On the other hand, bacterial ATPases are generally resistant to oligomycin. Brief mention should be made of aurovertin, a fungal metabolite toxic t o animals but which has little effect on either fungi or bacteria. It is of interest in the present context because, like oligomycin, aurovertin inhibits ATP synthesis by oxidative phosphorylation. Its site of action may be the ATPase (F,) particle itself, since it can inhibit even the solubilized enzyme (see the discussions in Lee and Ernster, 1968; Shaw, 1967a; Roberton et al., 1968). However, some of the effects of aurovertin on mitochondrial function are not readily reconciled with this hypothesis.

FIG.1%.Clieniical structure of dicyclohexylcarbodiimide.

2 . Dicyclohexylcarbodiimide. Dicyclohexylcarbodiimide (DCCD), a synthetic reagent of known structure (Fig. 12), is used in organic chemistry as a dehydrating agent. Curiously, the effects of this compound on mitochondrial processes are almost identical with those of oligomycin and rutamycin : it inhibits respiration and coupled phosphorylation, as well as energy-linked functions supported by ATP. Dicyclohexylcarbodiimide inhibits mitochondrial ATPase by reaction with a protein component of the basal membrane, and thus does not inhibit the solubilized enzyme (Beechey et al., 1967; Bulos and Racker, 1968 ; Roberton et al., 1968). Dicyclohexylcarbodiimide apparently forms a covalent bond with its receptor site, which is thought to be a proteolipid and may be identical with the rutamycin receptor (Beechey et al., 1967; Bulos and Racker, 1968; Knight et al., 1968; Roberton et al., 1968). Inhibition of oxidative phosphorylation presumably accounts for the effects of dicyclohexylcarbodiimide on growth and respiration of yeast (Kovad et al., 1968) but cannot explain its effects on fltrep. faecalis which does not carry out oxidative phosphorylation. Growth of this homofermentative organism was inhibited by 0.1 mM-dicyclohexylcarbodiimide. The drug also inhibited energy-dependent accumulation of Kf by exchange for Na+ and Hfas well as the transport of alanine and phosphate ;it decreased the rate of glycolysis by inhibiting ATP degradation, but apparently did not interfere with the synthesis of ATP. These physiological effects were correlated with studies on the ATPase of membranes of Strep. faecalis. Dicyclohexylcarbodiimide irreversibly inhibited the membrane-bound enzyme, but not the solubilized enzyme (Harold et al., 1969a). Thus dicyclohexylcarbodiimide, like oligomycin,

86

FRANKLIN M. HAROLD

promises to be a valuable reagent not only in the study of oxidative phosphorylation but for energy-dependent membrane functions in general. 3. Dio 9 is a n antibiotic of unknown structure active against Grampositive bacteria. It is another compound which has, so far, been employed largely in the study of energy transfer in mitochondria and chloroplasts. Depending upon conditions, Dio 9 either inhibits or uncouples respiration and phosphorylation (Guillory, 1964), and also initiates pronounced swelling of mitochondria (Fisher and Guillory, 1968). Dio 9 also inhibits energy-conservation reactions in chromatophores of Rhodospirillum rubrum (Fisher and Guillory, 1967). The very complex effects of this antibiotic are presumably related to inhibition (and sometimes stimulation) of ATPase. It is particularly noteworthy that Dio 9 inhibits both solubilized and membrane-bound ATPase (Fisher and Guillory, 1967; Schatz et al., 1967; Schatz, 1968). Dio 9 inhibited growth of Strep. faecalis a t 5 pg./ml. Its gross physiological effects were similar to those of DCCD; it blocked cation transport and alanine uptake, and inhibited the ATPase. However, as in the other systems, it inhibited both the solubilized ATPase and the membranebound enzyme, suggesting that it may act directly on the enzyme protein itself. To what extent its antibiotic activity is explained by the inhibition of ATPase remains to be ascertained (Harold et ab., 1969b). 4. Biguanides are potent synthetic antimicrobial agents, of which chlorhexidine (Fig. 1, p. 55) is probably the most widely used as a disinfectant in industry and medicine. The biguanides are surface-active and disorganize the cytoplasmic membrane (Hugo and Longworth, 1964a, b ; Weinberg, 1968). However, chlorhexidine is bactericidal a t concentrations below those required to induce leakage of cell constituents (Hugo and Longworth, 1964a). Certain guanidine derivatives, including alkylguanidines, phenethylbiguanide and decamethylenediguanide, are inhibitors of energy transfer in mitochondria and chloroplasts (Guillory and Slater, 1965; Gross et al., 1968). Prompted by this clue, Harold et al. (1969b) explored the effects of low concentrations of chlorhexidine and of a related compound, vantocil IB, on Strep. faecalis. They proved to be good inhibitors of both cation transport and of the membrane-bound ATPase at concentrations which did not lyse the cells, and this metabolic effect may contribute to their antimicrobial action.

B. INHIBITORS OF THE RESPIRATORY CHAIN The respiratory chain of bacteria is similar in principle to that of mammalian and fungal mitochondria, though there are considerable

ANTIMICROBIAL AGENTS AND MEMBRANE FUKCTION

87

diffcrences in detail. Gel’man et al. (1967) and Smith (1968) have reviewed the information currently available ; the electron-transport chain of bacterial photosynthesis is less well established (Vernon, 1968). Like mitochondria of higher organisms, the bacterial respiratory chain contains dehydrogenases for various substrates and cytochromes of the 6, c and a types in that order ; quinones participate in electron transport and may be intimately involved in oxidative phosphorylation. Although the phosphorylation etliciency of extracts is generally low, intact bacteria have two or even three coupling sites. However, it has not yet been possible to fractionate the bacterial respiratory chain into complexes analogous to those obtained from mitochondria, and thus the location of the coupling sites is less well defined. Finally, although the mode of action of many respiratory inhibitors is similar in bacterial and mammalian systems, substantial differences exist in some cases-not only between bacterial and mammalian systems but from one bacterial species to another. For example, bacterial respiration is generally insensitive to antimycin, but particles from Bacillus subtilis are strongly inhibited and bacterial photophosphorylation is exceedingly sensitive to antimycin I (see the review by Rieske, 1967). There may thus be variation in .the I detailed structure or accessibility of the sites subject to these inhibitors. The available information on inhibitors of electron transport comes largely from studies with mammalian mitochondria (Fig. 11, p. 84). The present survey of these compounds will be a cursory one, not to minimize their importance but because the insights derived from this work bear upon the intimate details of electron transport rather than upon the structure and function of membranes in general. 1. Piericidin and Rotenone. Rotenone, a toxic substance of plant origin, has been employed for years as an inhibitor of NADH, oxidation and coupled phosphorylation in the region of the first coupling site, but is generally not an effective inhibitor of bacterial electron transport. j There is therefore much interest in the novel antibiotic, piericidin A. A product of Xtreptomyces, piericidin was first recognized as an insecticide with little antimicrobial activity (Tamura et ul., 1963). It was subsequently found to inhibit both bacterial and mitochondria1 respiration at the rotenone site. i The structure of piericidin A (Takahashi et al., 1965) is shown in Fig. ’ 13. The resemblance between the antibiotic and coenzyme Q suggested that piericidin might interfere with quinone function and this appears t o be the case. I n mammalian mitochondria, very low concentrations of piericidin inhibit oxidation of NADHB and reduction of coenzyme Q (Fig. 11, p. 84) ; higher concentrations of the antibiotic block electron transport between succinate and coenzyme Q as well (Hall et al., 1966; Jeng et al., 1968). Indeed, piericidin appears to be the most potent ~

‘

1

88

FRANKLIN M . HAROLD

inhibitor known to act in the region of the first coupling site of oxidative phosphorylation (Vallin and Low, 1968).Piericidin binds to mitochondria with very high affinity for a specific site, apparently identical with that to which rotenone binds (Horgan and Casida, 1968; Horgan et al., 1968a, b ; Coles et al., 1968; Jeng et al., 1968; Hatefi, 1968). Binding is apparently not covalent, and the antibiotic can be recovered by extraction with solvents and other treatments (Horgan et al., 1968a, b ; Coles ef al., 1968). OH

Piericidin A

~HCHO Antimycin A (R = hexyl)

Heptylquinoline oxide

R (R=heptyl)

4,

O

FIG.13. Chemical structures of some inhibitors of electron transport.

Bacterial respiration is generally, but not always, less sensitive to piericidin than is mitochondria1 respiration, but the sites of inhibition appear to be very similar (Jeng et al., 1968; Knowles et al., 1968;Kosaka and Ishikawa, 1968; Snoswell and Cox, 1968).I n several cases ubiquinone or related compounds were shown to overcome the inhibition of respiration, perhaps by displacement of the antibiotic. Respiration of intact cells is insensitive to piericidin, presumably because the antibiotic fails to reach the sensitive sites. 2 . A n t i m y c i n and A l k y l Quinoline Oxides. The antimycins are a family of closely related antibiotics produced by Streptomyces species ; the structure of antimycin A, the only member extensively used, is shown in Fig. 13. The antibiotic is highly inhibitory to many fungi and mammalian

ANTIMICROBIAL AGENTS AND MEMBRANE FUNCTION

'

89

cells but ddes not inhibit the growth of bacteria. An excellent and coniprehensive review by Rieske (1967)forms the basis of this section. Antimycin is the respiratory inhibitor par excellence, and its mode of action has been much studied. It binds specifically and stoichiometrically to a site which may form the junction between cytochromes b2 and c1 (Fig. 1 1 , ~84). . The antimycin-sensitive site, like that to which piericidin binds, is of particular interest since it is also thought to be the coupling site for phosphorylation (Rieske, 1967; Rieske et al., 1967). The growth of yeasts such as Torulopsis is inhibited by antimycin when ethanol serves as substrate, but not when glucose is provided; results of this kind first suggested that antimycin acts specifically at the level of mitochondria1 respiration (Rieske, 1967 ; Butow and Zeydel, 1968). A mutant of Torulopsis has been described in which respiration, both of intact cells and of mitochondria, is relatively resistant to antimycin. Binding of antimycin to the particles was also decreased. These mutants are reminiscent of yeasts which are naturally resistant to antimycin, such as Saccharomyces ; these lack the antimycin-sensitive site and indeed have only two coupling sites for oxidative phosphorylation. It will be of interest to see whether mutation to antimycin resistance involves loss of this coupling site. Bacterial cells as well as membrane fragments are generally resistant to antimycin, although a few sensitive systems have been found (Gel'man et al., 1967; Rieske, 1967; Smith, 1968). Photophosphorylation by particles of Rhodospirillum rubrum is particularly sensitive to antimycin (Rieske, 1967; Vernon, 1968). It is thought that the coupling site in the cytochrome b-c region of the bacterial respiratory chain has a structure slightly different from its mitochondria1 counterpart, but is not algether absent, because bacterial respiration is generally sensitive to another class of antibiotics which act at the same site as does antimycin, namely the alkyl quinoline oxides. Lightbown and Jackson (1956) originally isolated the alkyl quinoline N oxides from culture filtrates of certain Pseudomonas strains by virtue of their capacity to antagonize the action of streptomycin. Their chemical structure, determined by Cornforth and James (1956), is shown in Fig. 13. Alkyl quinoline oxides inhibit oxygen uptake by intact cells of Bacillus subtilis and Staphylococcus spp., and this presumably accounts for their incompatibility with streptomycin. Cells of Escherichia coli were resistant, but particles derived from the envelope were sensitive. Heptyl quinoline N oxide has proven to be a versatile inhibitor of respiratory and photosynthetic electron transport, both in mitochondria and bacteria. Its site of action is probably identical with that of antimycin (Lightbown and Jackson, 1956; Rieske, 1967; Rieske et al., 1967). This statement is well supported by the finding (Butow and Zeydel, 1968) that mutants

90

FRANKLIN M. HAROLD

selected for resistance to antimycin were resistant to heptyl quinoline oxide as well. Not all the cellular effects of antimycin can be ascribed to the inhibition of electron transport. I n Bacillus megaterium, one of the few bacterial species that is relatively sensitive to antimycin, the antibiotic did not markedly depress respiration but blocked active transport of a-methylglucoside and of a-amino-isobutyric acid ;higher concentrations induced lysis of protoplasts, apparently by disorganization of the cytoplasmic membrane (Marquis, 1965). 3. Other inhibitors of electron transport. Numerous other compounds inhibit electron transport in micro-organisms, but their sites of action are as yet ill defined. Among these is flavensomycin, an antifungal compound which also inhibits the growth of certain bacteria; it inhibits electron transport prior to the antimycin-sensitive step, probably between the dehydrogenases and coenzyme Q (Gottlieb, 1967 ; Gottlieb and Inoue, 1967). Other antibiotics which may prove to be of interest to students of electron transport are U19718 (Reusser, 1968) and also pyocyanine, patulin and usnic acid (reviewed in Gottlieb and Shaw, 1967).

C. INTERACTION OF HEAVY METALSWITH

THE

MEMBRANE

The antibacterial properties of mercury compounds were applied by Arab physicians in the middle ages, and until the advent of sulphonamides in the 1930s mercury compounds were the chief antiseptics available. Heavy metals other than mercury are not sufficiently toxic to micro-organisms for use as antimicrobial agents, but concentrations in the millimolar range sometimes exhibit pronounced effect on membrane transport in bacteria and fungi. The physiological effects of mercury derivatives are generally ascribed to interaction with sulphydryl groups, though the metal does bind to other groups of biological importance (Passow et al., 1961). Inhibitory effects of Hg2+ on both cells and enzymes are usually reversed by sulphydryl compounds such as cysteine. To be sure, Hg2+ blocks sulphydryl groups of intracellular enzymes but proteins exposed at the outside of the permeability barrier are the most sensitive, and many of the effects of mercury derivatives on intact microbial cells can be understood in terms of the inhibition of membrane processes. It is possible to cite but a few of many known examples. Addition of MHgCI, instantaneously stops glycolysis by Strep. faecalis ; the inhibition is completely reversed by cysteine and is apparently due to a block in the transport of glucose into the cells. Para-Chloromercuribenzoate inhibits transport of /3-galactosides in E. coli by interaction with a sulphydryl

ANTIMICROBIAL AGENTS AND MEMBRANE FUNCTION

91

group which forms part of the active site of the galactoside-transporting M protein (Fox and Kennedy, 1965). The effects of other divalent metal ions, UOZ2+,Ni2+ and Co2+, on membrane functions have received detailed study in yeast. This work has been reviewed (Rothstein and Van Steveninck, 1966) and a very brief summary may suffice here. Uranyl ion appears to block specifically the transport of sugars into cells ; energy-linked transport is more sensitive than facilitated diffusion. Ni2+and CoZf have similar but not identical effects. At least two kinds of binding sites for UOZzfhave been detected, differing in specificity. The binding sites with the highest affinity for metal ions were identified as inorganic polyphosphate, which is thought to participate in sugar transport as a phosphate donor. Other metal ions also affect transport processes. The trivalent La3+ion stops metabolism of Strep. faecalis apparently as the result of interference with membrane functions (Wurm, 1951). It is a potent inhibitor of ion translocation in mitochondria (Mela, 1968). Zinc and cadmium, which induce active accumulation of K+ (Brierley and Settlemire, 1967) and of Mg2+ (Brierley, 1967) by mitochondria, and uncouple oxidative phosphorylation, inhibit transport in bacteria (Eagon and Asbell, 1969). This would appear to be a promising field for further research. Finally, mention should be made of the remarkable observation of Novick and Roth (1968), that genes which control the resistance of Staphylococcus to lead, zinc, cadmium and mercury are located on the penicillinase plasmid. The physialogy of resistance to metal ions is almost totally unexplored.

VII. Bacteriocins: Antibiotics which Interact with Specific Membrane Receptors Bacteriocins are macromolecular antibiotics produced by certain strains of bacteria and active only against other strains of the same species. Many appear to be simple proteins, and even in those that are structurally complex the specificity resides in proteins. It is their narrow spectrum of antimicrobial activity that sets the bacteriocins apart from other antibiotics, and this is ascribed to their interaction with highly specific cellular receptors. The bacteriocins have been the subject of several recent reviews (Holland, 1967; Nomura, 1967a, b; Reeves, 1965). This section will be confined to the interaction of bacteriocins with specific membrane receptors and the role of the membrane in the remarkable physiological consequences of this interaction ; most of the relevant reports deal with the bacteriocins of E. coli, the colicins. Colicins are classified by the pattern of receptor specificity and also of immunity. Colicins designated by the same letter, for example the E group, share the same receptor. A sensitive cell may adsorb many

92

FRANKLIN M . HAROLD

colicin molecules, and have hundreds of receptor sites per cell. Nonetheless, the killing action of most colicins follows single-hit kinetics, indicating that even a single colicin molecule has a finite probability of causing a lethal event (Nomura 1967b; Shannon and Hedges, 1967). Colicins produce diverse biochemical effects. Colicins of types E l , I and K inhibit the synthesis of DNA, RNA and protein but do not inhibit respiration. Colicins E2 inhibit DNA synthesis and induce DNA degradation, whereas E 3 colicins inhibit synthesis of protein but not of DNA and RNA (Romura, 1967a, b ; Reeves, 1968). It is curious that colicins E2 and E3, which share a common receptor, produce different metabolic effects. Nomura (1967a, b) showed that cells exposed to colicins of various types can be rescued by treatment with trypsin, which presumably digests the colicin. Thus the colicins remain at their receptor site, external to the permeability barrier, and act from there. ' The biochemical processes set in motion by the adsorption of colicins are far from clear. Colicin E2 may induce a DNAase (Holland, 1968; Holland and Threlfall, 1969). Colicin E 3 brings about the inactivation of ribosomes. Most pertinent to this survey, colicins E l , K and I appear to block energy metabolism. These colicins do not inhibit respiration but result in drastic lowering of $he cellular ATP level. The synthesis of all macromolecules (DNA, RNA, protein and polysaccharide) ceases, as do active transport and motility (Nomura, 1967a, b ; Levisohn et al., 1968; Fields and Luria, 1969a, b). The effects of colicins El and K on transport systems are particularly interesting (Fields and Luria, 1969a), Accumulation of thiomethylgalactoside, which is an energy-dependent process, was abolished. However, there was no gross breakdown of the permeability barrier and the transport system continued to equilibrate galactosides across the membrane. This is reminiscent of the effects of 2,4-dinitrophenol and consistent with an earlier suggestion that colicins E l and K uncouple oxidative phosphorylation. However, the situation appears t o be more complex. Levisohn et aH.(1968)found that the inhibition of ATP turnover by colicins is less complete than that caused by 2,4-dinitrophenol, and various non-nucleotide phosphorus compounds accumulate. Indeed, pyruvate and various phosphorylated intermediates of glycolysis are excreted by colicin-treated cells (Fields and Luria, 1969b). Moreover, the effects of colicins depend to a considerable degree upon the presence of oxygen. One consequence of colicin binding appears to be inhibition of pyruvate dehydrogenase but this is clearly not the primary lesion. Fields and Luria (1969b) suggest that colicins may promote leakage of protons through the membrane. Their results are compatible with this hypothesis but it fails to account for the role of oxygen since protonconducting compounds act anaerobically as well (PavlasovB and Harold, 1969).

~

ANTIMICROBIAL AGENTS AND MEMBRANE FUNCTION

93

How can a single colicin molecule, adsorbed to a receptor located a t the outer surface of the plasma membrane (Nomura, 1967a, b ; Srnarda and Taubeneck, 1968), induce DNA breakdown or disrupt energy metabolism? Nomura proposed that each colicin has a “biochemical target”, and affects it by means of stimuli sent through a specific transmission system, presumably located in the cytoplasmic membrane. Indeed, all colicin-sensitive processes are directly or indirectly associated with the membrane. This hypothesis predicts that mutants resistant to colicins may arise in a t least two general ways. Loss of the receptor results in mutants that can no longer adsorb the colicin. Alterations in the transmission system should produce mutants which still adsorb the colicin but have become tolerant. Colicin-tolerant mutants have been isolated in several laboratories (Holland, 1968; Nomura and Witten, 1967; de Zwaig and Luria, 1967). Some of these mutant cells are fragile and hypersensitive to detergents ; others are hypersensitive to dyes such as methylene blue. It was suggested that colicin-tolerant mutants have suffered loss or modification of a membrane protein; identification of such proteins and definition of their role in membrane function is an urgent task. A physical model intended to rationalize the role of the membrane in colicin action has been proposed by Changeux and Thii?ry (1967). They regard the membrane as a structure composed of repeating subunits which interact co-operatively with each other ; binding of a single colicin molecule would change the conformation of one or a few subunits, followed by a molecular domino effect by which the entire membrane shifts into a new pattern. Proteins attachedto the inner surface of‘the membrane would thus find themselves in an altered environment, and this may lead to the arrest of functions mediated by such proteins. It should be noted that bacteriocins are a mixed lot, and not all the antibiotics so classified appear to act via specific receptors. For instance, megacjn A, the best known of the bacteriocins produced by Grampositive bacteria, disrupts the cytoplasmic membrane of many strains of B. megaterium (see Holland, 1967, Nomura, 1967b forreferences tothe original papers) ; megacin A is now thought to be a phospholipase.

VIII. Summary and Prospect

I have attempted to classify the antimicrobial agents which act upon membranes according to their primary effect at the molecular level. At least three general modes of action can be distinguished a t present. (1) Compounds which associate with membrane components and disorganize lipoprotein structures. The detergents are familiar representatives of this class, as are the surface-active antibiotics tyrocidine and polymyxin. Polyenes are not particularly surface-active but forni

94

FRANKLIN M. HAROLD

complexes with sterols. Most of these compounds do not destroy the physical integrity of membranes, but rather disorganize the permeability barrier which depends upon hydrogen bonds and hydrophobic interactions. The precise changes in molecular architecture cannot yet be specified. (2) The cation-conducting compounds have remarkable and diverse effects on the functions performed by biological membranes, but these can largely (perhaps entirely) be ascribed to the formation of lipidsoluble complexes with alkali-metal ions or protons. To these compounds we owe the recognition that ion transport is not only a central function of living membranes but an integral part of energy generation. The ionophores are both research tools and models which afford insight into membrane transport generally. All the ionophores reported thus far conduct cations. Future research may reveal anion conductors (according t o Bodanszky and Bodanszky ( 1 968) the structure of the new antibiotic stendomycin would be compatible with such a role) and indeed lipid-soluble carriers for non-ionic compounds. (3) The targets of many antimicrobial agents are, or include, specific proteins which serve structural or catalytic functions. This category includes quite non-specific reagents, such as Hg2+,but interest centres on inhibitors of the various energy transductions which membranes carry out. I n oxidative and photosynthetic phosphorylation, the free energy of redox reactions is converted into ion gradients and ATP. Membrane transport utilizes ATP to establish concentration gradients of ions and other metabolites; ATP can also be used to reverse the normal direction of the respiratory chain, and finally the potential energies of ion gradients and ATP are sometimes mutually interconvertible. Among the many inhibitors which block specific steps in energy metabolism, oligomycin and other inhibitors of “ATPase’) deserve special mention because of the central role of this enzyme in mediating the interconversion of chemical and potential energy. These few categories are not intended to be either final or exhaustive. The colicins, for example, bind to highly specific receptors a t the outer surface of the membrane and remain there, yet a single molecule may block active transport or protein synthesis. It appears that the membrane can function as a unit such that perturbations may be transmitted from one end of a cell to the other, but virtually nothing is known regarding the molecular basis of this communication process. Finally, some known antibiotics are not easily accommodated by this Procrustean bed. Further research will no doubt reveal that some of the compounds relegated to this paragraph do not primarily affect membrane processes a t all; others may be assigned to established categories. Yet,

ANTIMICROBIAL AGENTS AND MEMBRANE FUNCTION

95

it is by no means unlikely that an obscure compound will afford insight into a principle of membrane function which is still unrecognized. Readers in search of a project may find the foIIowing of potential interest : albomycin and other sideromycins, flavensomycin, patulin and usnic acid (all reviewed in Gottlieb and Shaw, 1967); azalomycin (Sugawara, 1967,1968) ; pinosylvin methylether, thujaplicin and other antimicrobial constituents of wood (Lyr, 1966; Aggag and Schlegel, 1966); nisin, a polypeptide antibiotic produced by lactobacilli (see White and Hurst, 1968, for earlier references) ; and showdomycin, a novel antibiotic of known structure which may interact with sulphydryl groups at the cell surface (Hadler et al., 1968a, b). One obvious category is, so far, almost unrepresented. I am not aware of any antimicrobial agent which primarily inhibits membrane synthesis. Perhaps the best example is diphenylamine which blocks carotenoid synthesis, and inhibits the growth of certain Gram-positive bacteria (Salton and Schmitt, 1967). A major question, which can be raised but not answered, is the role of antibiotics in the organisms which produce them. Of what use or significance are valinomycin, monactin, nigericin, antimycin, piericidin, oligomycin and many other remarkable inhibitors which we have surveyed to Xtreptomyces spp. ? And why do streptomycetes produce the great majority of the membrane-active antibiotics? It has been argued (Brock, 1966) that antibiotics are agents of chemical warfare, secreted by the producing organism to suppress the growth of competing species. This view has failed to gain general acceptance. Most investigators regard the antibiotics as secondary metabolites, whose production is due to breakdown of certain control mechanisms (Woodruff, 1966; Bu’lock, 1965), but which confer no selective advantage and, indeed, serve no 1 functionin themselves. I find this view unsatisfactory (seealso Bodanszky i and Perlman, 1969) and believe that the remarkable specificity of struc- ’ ture and function displayed by a compound such as valinomycin requires . a teleonomic explanation. Finally, antimicrobial agents which act upon membranes offer an amusing instance of compartmentation in science. The torrent of papers describing the application of the ionophores to mitochondria contrasts with a bare trickle of microbiological studies in this field. This review will have accomplished its purpose to the extent that it helps disorganize such artificial barriers to the diffusion of knowledge.

IX. Acknowledgements Major J. W. Powell is quoted from “Beyond the Hundredth Meridian” by Wallace Stegner, with kind permission of the author and of the

96

FRANKLIN M. HAROLD

publisher, Houghton-Mifflin Co. Thanks are due to Dr. J. D. Dunitz for permission to reproduce Fig. 9, and to Drs P. Mitchell and B. C. Pressman for permission to read unpublished manuscripts. Ethel Goren, Rudolf Love and Nadia de Stakelburg helped generously in the preparation of this article. Original research from my laboratory was supported in part by U.S. Public Health Service Grant AI-03568 from The Institute of Allergy and Infectious Diseases. REFERENCES Abram, D. (1965). J . Bact. 89,856 Abrams, A. (1965).J . biol. Ghem. 240, 3675. Aggag, M. and Schlegel, H. G. (1967). I n “Wirkungsmechanisnien von Fungiziden und Antibiotika”, Biologische Gesellschaft der DDR, Sektion Mikrobiologie. pp. 17-24. Akademie Verlag, Berlin. Agtarap, A. and Chamberlin, J. W. (1967). Antimicrob. Agents Chemother. pp. 359-362. Agtarap, A., Chamberlin, J. W., Pinkerton, M. and Steinrauf, L. K. (1967). J . Am. chem. Soc. 89, 5737. Albers, R. W. (1967). A . Rev. Biochem. 36, 727. Andreoli, T. E. and Monahan, M. (1968). J . gen. Physiol. 52, 300. Andreoli, T. E., Tieffenberg,M. andTosteson, D. C. (1967).J . gen.PhysioZ. 50,2527. Asano, A. and Brodie, A. F. (1965). J . biol. Chem. 240, 4002. Asbell, M. A. and Eagon, R. G. (1966). J . Bact. 92,380. Bangham, A. D., Dingle, J. T. and Lucy, J. A. (1964). Biochem. J . 90, 133. Bangham, A. D., Standish, M. M. and Miller, N. (1965). Nature, Lond. 208, 1295. Beechey, R. B. (1966). Biochem. J . 98,284. Beechey, R. B., Roberton, A. M., Holloway, C. T. and Knight, I. G. (1967). Biochemistry, N . Y . 6,3867. Bergy, M. E. and Eble, T. E. (1968). Biochemistry, N.Y. 7, 663. Bielawski, J. (1968). E u r . J . Biochem. 4, 181. Bielawski, J., Thompson, T. E. and Lehninger, A. L. (1966). Biochem. biophys. Res. Commun. 24, 948. Birdsell, D. C. and Cota-Robles, E. H. (1967). J . Buct. 93, 427. Birdsell, D. C. and Cota-Robles, E. H. (1968). Biochem. biophys. Res. Commun. 31,438. Blake, A., Leader, D. P. and Whittam, R. (1967). J . Physiol., Lond. 193, 467. Bodanszky, M. and Bodanszky, A. (1968). Nature, Lond. 220, 73. Bodanszky, M. and Perlman, D. (1969). Science, N . Y . 163,352. Borowski, E. and Cybulska, B. (1967). Nature, Lond. 213, 1034. Bragg, P.D. and Hou, C. (1968). Gum. J . Biochem. 46, 631. Brierley, G. P. (1967). J . biol. Chem. 242, 1115. Brierley, G. P. and Settlemire, C. T. (1967).J . biol. Ghem. 242, 4324. Brock, T. D. (1966). “Principles of Microbial Ecology”. Prentice Hall, Englewood Cliffs, N.J. Brock, T. D. (1967). I n “Antibiotics”, (D. Gottlieb and P. D. Shaw, eds.), Vol. I, pp. 651-665. Springer Verlag, New York. Brown, M. R. W. and Richards, R. M. E. (1963). Nature, Lond. 207, 1391. Browning, C. H. (1964). In “Experimental Chemotherapy”, (R. J. Schnitzer and F. Hawking, eds.), Vol. 11, pp. 1-36. Academic Press, New York.

ANTIMICROBIAL AGENTS AND MEMBRANE FUNCTION

97

Bu’lock, ,J. 11. (1965). “Biosynthesis of Natural Products”. McGraw--Hill, New Y orlc . Bulos, B. arid Kacker, E. (1968). J . bid. Chem. 243, 3891. Rutow, It. A. and Zeydel, M. (1968). J . b i d . Chem. 243, 2546. Caswell, A. H. (1968). J.biol. Chem. 243, 5827. Cavari, R. %., Avi-Dor, Y . and Qrossowicz, N. (1967). Hiochem. J. 103, 601. Charicc, B., Leo, C. P. a n d Mela, L. (1967). F e d n Proc. Fedn. A m . Socs exp. Biol. 26, 1341. Changeux, J . 1’. a n d ThiBry, J. (1967). J . theor. Bid. 17, 315. Chapman, D. a n d Wallach, D. F. H. (1968). I n “Biological Membranes”, (D. Chapman, ed.), pp. 125-202. Academic Press, New York. Chappell, J. B. a r i d Crofts, A. R . (1965). Biochem. J. 95, 393. Chappell, J. R. a n d Crofts, A. R. (1966). 17% “Rrgulation of Mct’abolicProcesses in Mitochondria”, (J.M. Tager, S. P a p a , FC. Quagliarcllo arid E. C. Slater, eds.), pp. 293 -314. Elsevicr, Amsterdam. Chappell, J . B. arid Haarhoff, K. N. (1967). In “Biochemistry of Mitochondria”, (E. C. Slater, %. Kaiiiiige a n d 1,. Wojt,czak, eds.), pp. 75-91. Academic Press, I~orldorl. Cockrcll, It. S., Harris, K. S. and Pressman, B. C. (1966). Biochemistrg, N . Y . 5, 2326. Cockrcll, 1%. R., Harris, K. J. arid Pressman, B. C. (1967). Nature, Lond. 215, 1487. Coles, C. J . , Griffiths, D. E., Hutchinson, D. W. arid Sweetman, A. J. (1968). Riochem. biophgs. Res. Commuix. 31, 983. Cornforth, J. W. arid James, A. T. (1956). B‘iochem. J . 63, 124. Cox, 8. T. a n d Ea,gon, R. C . (1968). Cam. J. &‘icrobiol. 14, 913. Cuthbort, A. W. (1967). Pharmac. Rev. 19, 59. van Dam, K. a n d Slater. B:. C. (1967). Proc. natn. Acad. Sci.U.S.A. 58, 2015. l h v i e s , A. arid Field, B. S. (1968). Biochem. J. 106, 46P. Da,vics, G. K., Francis, J., Martin, A. R., Hose, 3’.L. arid Bwairi, G. (1954). Br. J . Pharwt,ac. Ch,emother. 9, 192. I h n o l , 1%. A., Crombag, P.J. L., V a n Deenen, L. L. M. and Kinsky, 8 . C. (1968). 13%oclui~n. biophys. Acta 150, 1. Dillcy, K. A. (1968). Biochemistry, N . Y . 7, 338. Domagk, G. (1935). Dt. m e d . TVschr. 61, 829. Eagon. 1%.C . a n d Asbell, M. A. (1966). Biochem. b%oph?js.Kes. Commun. 24, 67. Eagon, R . G. and Asbell, M. A. (1969). J. Nuct. 97, 812. imari, G., Ciarii, S. M. arid Szabo, G. (1968). Fedn Proc. lf‘edn. Am. Socs exp. 01. 27, 1289. I%trada-O, S., Graven, S. N . a i d Lardy, H. A. (1967a).J. b i d . Cherub. 242, 2925. E s t r a d a - 0 , S., Rightmirc, B. and Lardy, H . A. (1967b). Antirnicroh. Agents Chernother. 2 79. F a h i , S., Koval, G. J. arid Albers, R. W. (1968). J. h i d . Chem. 243, 1993. Falcone, A. B. and Hadler, H. I. (1968). Archs Biochem. Biophys. 124, 115. F:mst,. M. A. a n d Dootsch, R. N. (1969). J. Bact. 97, 806. Fow, A. V. (1955). Biochim. biophys. Actu 16, 137. Fields, K. L. a n d Liiria, S. E. (1969a). J . Bact. 97, 57. Ficlds, K. I,. arid Luria, S. R. (1969b).J. Ruct. 97, 64. A. arid Cass, A . (1968). .7.gen. I’hysiol. 5 2 , 145s. FiiikeIst,~~in, Fisher, K . Ti. a n d Guillory, R. J. (1967). Biochim. biophys. A c t a 143, 654. Fisher, R. K. and Cuillory, R. J. (1968). Biochim. hiophys. Acta 162, 182. Fitz-James, P. C. (1968). I n “Microbial Protoplasts, Spheroplasts and L-Forms”, (L. B. Gum, ad.), p p . 239-247. Williams a n d Mrilkins, New York. 4

98

FRANKLIN M. HAROLD

Fox, C. F. and Kennedy, E. P. (1965). Proc. natm. Acad. Sci. U . S . A . 54, 891. Galcotti, T., Kova6, L. arid Hcss, B. (1968). Nature, Lond. 218, 194. Gcl’man, N. S., Lukoyanova, M. A. and Ostrovskii, D. N. (1967). “Respiration and Phosphorylation of Bacteria”. English Translation, Plenum Press, New York. Gilby, A. It. and Few, A. V. (1960a). J . gem. Microbiol. 23, 19. Gilby, A . R. and Few, A. V. (1960b). J . gen. Microbiol. 23, 27. Goldfinc, H. (1968). A. Rev. Uiochem. 37, 303. Gottlicb, D. (1967). In. “Antibiotics”, (D. Gottlieb and P. D. Shaw, eds.), pp. 617620. Springer Verlag, New York. Gottlieb, D. and Inoue, Y. (1967). J . Bact. 94, 844. Gottlieb, D. and Shaw, P. D. (eds.) (1967). “Antibiotics, Vol. I : Mechanism of Act,ion”. Springer Verlag, New York. Graven, S. N., Estrada-0, S. and Lardy, H. A. (1966a). Proc. natn. Acad. S c i . U.S.A. 56, 654. Graven, S. N., Lardy, H. A., Johnson, D. and Rutter, A. (1966b). Biochemistry, N.Y. 5, 1729. Gravori, S. N., Lardy, H. A. and Rutter, A. ( 1 9 6 6 ~ )Biochemistry, . N . Y . 5, 1735. Craven, S. hT.,Lardy, H. A. and Estrada-0, S. (1967).Biochemistry, N. Y . 6, 365. Green, D. E., Asai, J., Harris, R. A. and Penniston, J. 7’. (1968). Archs Biochem. Uiophys. 125, 684. Greene, R . arid Ma,gasanik,B. (1967). Molec. Pharmac. 3, 453. Gross, E., Shavit, N. and Sari Pietro, A. (1968). Archs Biochem. Biophys. 127, 224. Grula, E. A., Butler, T. F., King, R. D. a n d Smith, G. L. (1967).Can. J . Microbiol. 13, 1499. Guillory, 8 . J. (1964). B2%och%m. biophys. Acta 89, 197. (!uillory, It. J . and Slatcr, E. C. (1965). Biochim. biophys. Acta 105, 221. Hadler, H. I.,Claybourn, B. E. and Tschang, T. P. (1968).Biochem. biophys. Res. Commun. 31, 25. Hadler, H. I., Claybourn, B. E. and Tschang, T. P . (1968). J . Antibiot., Series A , 21, 575. Hall, C., Wu, M., Crane, F. L., Takahashi, H., Tamura, S. and E’olkers, K. (1966). Hiochem. biophys. Res. Commun. 25, 373. Hamilton, W. A. (1968).J . gen. Microbiol. 50, 441. Hamilton, W. A. (1969). I n “Tho Inhibition and Destruction of the Microbial Cell”, (W. B. Hugo, ed.), Academic Press, London. Haney, M. E. arid Hoehn, M. M. (1967).Antimicrob. Agents Chemother. pp. 349-352. Harned, R. L., Hidy, P. H., Corum, C. J. and Jones, K . L. (1951). Antibiotics Chemother. 9, 594. Harold, F. M. (1964). .I. Bact. 88, 1416. Harold, F. M. arid Baarda, J. R. (1967).J . Bact. 94, 53. Harold, F. M. arid Baarda, J. R. (196%). J . Bact. 95, 816. Harold, F. M. arid Baarda, J. R. (1968b).J . Bact. 96, 2025. Harold, F. M., Baarda, J . R., Baron, C. and Abrams, A. (1969a). J . biol. Chem. 244, 2261. Harold, F. M., Baarda, J. R., Baron, C. and Abrams, A. (1969b). Biochim. biophys. Acta 183, 129. Harris, E . J. and Pressman, B. C. (1967). Nature, Lond. 216, 918. Harris, E. J . arid Pressman, B. C. (1969). B%ochim.biophys. Acta 172, 66. Harris, E. J., Cockrell, R. S. and Pressman, B. C. (1966). Biochem. J . 99, 200. Harris, E. J., Hofer, M. P . and Pressman, B. C. (1967a).Biochemistry, N. Y . 6, 1348. Harris, El. J., Catlin, G. and Pressman, B. C. (1967b). Biochemistry, N . Y . 6,1360.

ANTIMICROBIAL AGENTS AND MEMBRANE FUNCTION

99

, 1%. A., Penniston, J. T., Asai, J. and Green, D. E. (1968).Pr.oc. natrz. Acad. S c i . U.S.A. 59, 830. Hatefi, Y. (1968).Proc. n a t n . Acad. S c i . U.S.A. GO, 733.

Haynes, D. H., Kowalsky, A. and Pressman, B. C. (1969).J. biol. Chem. 244, 502. Henderson, P. J. F. and ChappeII, J. B. (1967). Biochem. J. 105, 16P. Henderson,€’. J.F.,McGivan, J. D. andchappell, J. B. (1969).Biochem. J. 111,521. Heytler, P. G. (1963). Biochemistry, N. Y. 2, 357. Heytler, P. G. and Prichard, W. W. (1962).Biochem. biophys. R e s . C o m m u n . 7,272. Hodes, L. J. and Stecker, H. C. (1968). I n “Disinfection, Sterilization and Preservation”, (C. A. Lawrence and S. S. Block, eds.), pp. 453-468. Lea andFebiger, Phi Iadelphia. Hfifer, M. P. and Pressman, B. C. (1966). Biochemistry, N.Y. 5 , 3919. Holland, I. B. (1967). I n “Antibiotics”, (TI. Gottlieb and P. D. Shaw, eds.). Vol. I, pp. 684-695. Springer Verlag, New York. Holland, I. R. (1968). J . molec. Kiol. 31, 267. Holland, 1. B. and Threlfall, E. J . (1969). J. Ract. 97, 91. Hopfer, U., Lehninger, A. L. arid Thompson, T. E. (1968). Proc. n a t n . Acad. Sci. 7I.S.A. 59, 484. Horgan, D. J . and Casida, J. E. (1968). Biochem. J. 108, 153. Horgan. D. J., Singer, T. P. and Casida, J. E. (1968a).J. biol. Chem. 243, 834. Horgari, D. 5.,Ohno, H., Singer, T. P . and Casida, J . E. (1968b).J . biol. Chem. 243, 5967. Hotchkiss, K . D. (1944). A d v . E n z y m o l . 4, 153. Hunter, F.€C.> J r . and Schwartz, L. S. (1967a).In “Antibiotics”, (D. Gottlieb and P. I>. Shaw, eds.). Vol. I, pp. 631-635. Springer Verlag, New York. Hunter, F . H., J r . and Schwartz, L. S. (1967b).I n “Antibiotics”, (D. Gottlieb and P. D. Shaw, eds.), Vol, I, pp. 636-641. Springer Verlag, New York. Hiinter, F. E., Jr. and Schwartz, L. 8. ( 1 9 6 7 ~ )In . “Antibiotics”, (D. Gottlieb and 1’. D. Shaw, ods.). Vol. I, pp. 642-648. Springer Verlag, New York. Hugo, W. B. arid Longwort,h, A. R . (1964a). J . P h a r m . Pharmac. 16, 655. Hugo, W. B. and Longworth, A. R . (1964b). J . P h a r m . Pharmac. 16, 751. Hugo, W. B. arid Longwort>h,A. R,. (1966). .T. P h a r m . Phaymac. 18, 569. Ivarlov, V. T., Laine, I. A., Abdulaev, N. D., Senyavina, L. B., Popov, E. M., Ovchinniliov, Yn. A. and Shemyakin, M. M. (1969). Biochern. biophys. R e s . C‘om,mu?r.34, 803. Inturrisi, (2. E. and Titus, E. (1968). Molec. Phaymac. 4, 691. Jackson, J . R., Crofts, A. R. and von Stedingk, L. V. (1968).E u r . J.Biochem. G,41. Jackson, 1%.W. and DeMoss, J . A. (1965). J . Bact. 90, 1420. Jagendorf, A. T. (1967).F e d n Proc. F e d n . Am. Socs e x p . H i d . 26, 1361. Jeng, M., Hall, C., Crane, F. L., Takahashi, N., Tamura,, S. and Folkers, K. (1968). Biochemistry, N . Y . 7, 1311. Johnson, C. L., Mauritzen, C. M., Starbuck, W. C. and Schwa,rt,z, A. (1967). Biochemistry, N.Y. 6, 1121. Jordan, D. C. arid Reynolds, I?. E. (1967). I n “Antibiotics”, (D. Got,tJieb and P. D. Shaw, eds.). Vol. I, pp. 102-116. Springer Verlag, New York. Kagawa, Y . and Racker, E. (1966). J . b i d . Chem. 241, 2461. Kaplan, J. H. and Jagendorf, A. T. (1968). J. biol. Chem. 243, 972. Katchalsky, E., Sela, M., Silman, H. I. and Berger, A. (1964). I n “The Proteins”, (H. Neiirath, ed.). Vol. 11,pp. 406-602, 2nd ed. Academic Press, New York. Kates, M. (1966). A. R e v . Xicrobiul. 20, 13. Kepes, A. and Cohcn, G. N. (1962). I n “The Bacteria”, (I.C . Gunsalus and R.. Y . Stanier, eds.). Vol. IV, pp. 179-222. Academic Press, New York.

I00

FRANKLIN M. IIAROLD

Kilbormi, 13. T., I)uriitx, J. D. and Pioda, L. A. K . (1967). J . molec. Biol. 30, 559. Kinslcy, S. C. (1963). Awhs Hiochem. Biophys. 102, 180. Kinsliy, S . C. (1967). I ~“Antibiotics”, L (D. Cottlieh arid P. D. Shaw, eds.). Vol. I, pp. 122-141. Springer Vcrlag, New York. Kinsky, S. C., Luse, 8 . A. arid Van Deenen, L. L. M. (1966). E e d n Proc. Fedn. Am. ~Socse q . Bid. 25, 1503. Kinsky, S. C., Demel, R. A. arid Vaii Deencn, L. L. M. (1967a). Biochim.biophys. Acta 135, 835. Kinsky, S . C., Lime, S.A., Zopf, D., Van Deenen, L. L. M. and H a x b y , J. (1967b). Biochim. hiophys. Acta 135, 844. Kinsky, S. C., Haxby, J., Kinsky, C . B., Demel, R . A. and Van Deenen, L. L.M. (1968). 72iochim. biophys. Acta 152, 174. Klihr, S., Bonrgoignic, J. and Bucker, N. S. (1968). Nature, Lowd. 218, 769. Knight, 1. G., Holloway, C. T., Robrrton, A. M. and Reechey, K. B. (1968). B%ochem.J . 109. 271’. Krrowlos, C . J., Daniel, 1:. M., Ericksoii, S. K . arid Redfearn, E. R . (1968).Biochem. J. 106, 49P. Koikc, M., Iida, TC. arid Matsuo, T. (1969). ,I.Bact. 97, 448. Kopaczyk, K., Asai, J ., Allmann, D. W., Oda, T. and Green, D. E. (1968). A r c h B‘iochern. L3iophys. 123, 602. Korn, 13. 1). (1966).S c i e i m , N.Y. 153, 1491. Korzybslii, T., Kowsxyk-Giridifer a,nd Kurylowicz. W. (1967). “Antibiotics: Origin, Nature and Properties”. English translation, Pergamon Press, New YOrli. Kosaka, T. a r i d Ishikaw-a, 8 . (1968). J . Biochem., Tokyo 63, 506. KovaE, L. arid Kniela, 8. (1966). Biochim. biophys. Acta 127, 355. KovaE, L., Caleott>i,T. arid Hess, B. (1968). Biochim. biophys. Actn 153, 715. Lnmperi, J. 0. (1966). .In “Biochemical Studies of Antimicrobial Drugs”, S y m p . Soc. y e n . Microbiol. 16, 11I . Lardy, H . A. (1968). Ii’edn PTOC. Fedn. Am. Socs e x p . Biol. 27, 1278. Lardy, H . A., .Johnson, D. arid McMurray, W. C. (1958). Archs B%ochem.B i o p h y s . 78, 587. Lardy, H . A., Craven, S. N. arid Estrada-0, S. (1967). Fedrc Proc. F e d n . Am. Socs e x p . Lid. 26, 1355. Lrtscellcs, J . (1968). A&c. microbial Physiol. 2, 1. Lee, C . P . and Ernster, L. (1968). E‘w. J . Biochem,. 3,391. L(:hiiingc.r, A. L., (larafoli, E. and Rossi, C. S. (1967). Adv. Enzgmol. 29, 259. Lcive, 1,. (1965).Proc. nntn. Acnd. Sci. U.S.A. 53, 745. h i v e , L. (1968).,I. b i d . Chem. 243, 2373. Lenard, J. and Singer, S. J. (1966). Proc. nmtn. A c a d . Rci. U . S . A . 56, 1828. Lcnriarz, W. J. (1966). A d v . Lipid Res. 4, 175. Lcster, G . (1965).J . Bact . 90, 29. Lev, A. A. and Bnzhinslcy, E. P. (1967). Cytology (U.S.B.R.) 9, 102. Lcvisohri, It., Konisky, J. and Nomura, M. (1967). J . Bact. 96, 811. Liherman, E. A. arid ‘I’opaly, V. P. (1968). Biochim. biophys. Acta 163, 125. Lighthown, J. W. arid Jackson, F. L. (1956). Biochem. J . 63, 130. Lippe, C. (1968). Nature, L o n d . 218, 196. Liibin, M. (1964). F e d n PTOC. Fed%. Am. Socs e x p . B i d . 23, 994. Liicy, J . A. (1968). I n “Biological Membranes”, (D. Chapman, ed.), pp. 233-288. Academic I’ress,New York. Luzz:ttji, V. (1968). I n “Biological Membranes”, (D. Chapman, ed.), pp. 71-124. Academic Press, New York.

ANTIMICROBIAL AGENTS AND MEMBRANE FUNCTION

101

Lyr, H. (1967). I n “Wirliungsmechanismen von Fungiziden urid Antibiotika”, (Biologischc Gesellschaft der DDR, Sektion Mikrobiologie),pp. 27-35. Akademia Verlag, Berlin. MacGregor, D. IL. and Elliker, P. R . (1958). Can. J . Microbiol. 4, 499. Mach, R. and Slayman, C . W. (1966). Biochim. biophys. Acta 124, 351. MacLennan, D. H. and Tzagoloff, A. (1968). Biochemistry, N . Y . 7, 1603. Marquis, R. E. (1965).J . Bact. 89, 1453. McMurray, W. C. and Begg, R. W. (1959). A r c h s Biochem. B i o p h y s . 84, 546. Mcla, L. (1968). A r c h s Biochem. B i o p h y s . 123, 286. Meyer, C. E. and Rousser, F. (1967).Experientia 23, 86. Mcyer, T. S., Moore, C. E. and Frank, P. F. (1967). N a t u r e , Lond. 215, 312. Mcyers, E., Pansy, F. E., Perlman, D., Smith, D. A. and Weisenborn, F. L. (1965). J . Antibiot., Ser. A 18, 128. Mit,chcll, 1’. (196la). N a t u r e , Lond. 191, 144. Mitjchc:ll, P. (196lh).Biochem. J . 81, 24P. Mitchell, 1’. (1963).Biochem. Soc. Symp. 22, 142. Mit>chcll,1’. (1966).B i d . Rev. 41, 445. Mitchell, P. (1967a). I n “Comprehensive Biochemistry”, (M. Plorliiii arid l3. H. Stotz, eds.). Vol. 22, pp. 167-197. American Elsevier, New York. Mit,chcll, P. (1967b). F e d n Proc. F e d n . Am. Socs e q p . Biol. 26, 1370. Mitchell, P. ( 1969). In “Mitochondria-Structure and Function”, 5th. Meeting, Fed. Europ. Biochem. Soc. Academic Press, New York. Mitchell, P. and Moyle, J. (1967a). Biochem. J . 104, 588. Mitchell, P. and Moyle, J. (1967b). I n “Biochemistry of Mitochondria”, (E. C. Slat>cr,Z.Kaniugaand L. Wojtczak, eds.), pp. 53-74. Academic Press, New York. Mitchell, P. and Moyle, J. (1969). Eur. J . Bioclzem. 7, 471. Moorc. C. and Pressman, H. C. (1964). Biochem. hiophys. Res. C o m m u n . 15, 562. Moiidrianakis, E. N. (1968). Fed%.Proc. Fedn. Am. Socs exp. B i o l . 27, 1180. Mueller, P. and Rudin, D. 0. (1967). Biochem. bioph,ys. Res. C o m m u n . 26, 398. Miicller, P. a,nd Rudin, D. 0. (1968). N a t u r e , L o n d . 217, 713. Muiioz, E., Freer, J. H., Ellar, D. J. and Salton, M. R. J. (1968). Biochim. biophys. Acta 150, 531. Mi~iioz,E., Salton, M. 13,. J., Ng, M. H. and Schor, M. T. (1969). Eur. J . Biochem. 7, 490. Nakajima, K. and Kawamat,a, J. (1966). Bfikens J . 9, 115. Nakamiu-a, H. (1968). J . Bmt. 96, 987. Newt>on,B. A. (1956). Bact. Rev. 20, 14. Newton, R. A. (1958). I n “The Strategy of Chemotherapy”, Sy7np. s’oc. g e n . Microbiol. 8, 62. Nomura, M. (1967a). I n “Antibiotics”, (D. Gottlieb and P. D. Shaw, eds.). Vol. I, pp. 69B-760. Springer Verlag, New York. Nomura, M. (1967b). A . Rev.Microbiol. 21, 257. Nomnra, M. and Witten, C. (1967).J . Bact. 94, 1093. Novick, R. P. arid Roth, C. (1968).J . Bact. 95, 1336. I’ardcc, A. B. (1968).Science, N . Y . 162, 632. Parker, J . H., Trimble, I. R. and Mattoon, J. R. (1968). U i o c h e r n . biophys. Res. C o m m u n . 33, 590. Passow, H., Rothstein, A . arid Clarkson, T. W. (1961).Pharmac. Rev. 13, 185. PavlasovB, E. arid Harold, F. M. (1969).J . Hact. 98, 198. Pederscn, C. J . (1968).Fedn D o c . lf7edvj.Am. Socs e x p . B i d . 27, 1305. Pethica, B. A . (1958).J . gen. Microbiol. 18, 473. Poe, M. (1968). A r c h Biochem. Biophys. 128, 725.

102

FRANKLIN M. HAROLD

I’rcssman, B. (2. (1965).Proc. n a t n . A c a d . Sci. U.S.A. 53, 1076. Pressman, B. C. (1967). I n “Wirkungsmechanismen von Fungiziden und Antibiotika”, (Biologische Gescllschaft der DDR, Sektion Mikrobiologie), pp. 3-9. AImdemie Verlag, Berlin. F’ressmari, B. C. (1968). F e d n Proc. F e d n . Am. Socs e x p . B i o l . 27, 1283. Pressman, B. C. (1969).I n “Mitochondria-Structure and Function”, 5th. Meet’ing, Fed. Europ. Biochem. Soc., Academic Press, New York. Pressman, B. C., Harris, E. J., Jagger, W. S. and Johnson, J. H. (1967). Proc. natn. A c a d . S c i . U . S . A . 58, 1949. I’ricc, K. E., Schlein, A., Bradner, W. T. and Lein, J. (1963). Antimicrob. A g e n t s Chemother. pp. 95-99. Pullman, M. E. and Schatz, G. (1967).A. R e v . Biochem. 36, 539. Itackcr, E. (1967).PednProc. Fedn. Am. Socs e x p . Biol. 26, 1335. ltaziri, S. and Argamarr, M. (1963).d . gen. Microbiol. 30, 155. Liazin, S. and Boschwitz, C. (1968).J . gen. Microbiol. 54, 21. Razin, S., Morowitz, H. J. and Terry, T. M. (1965).Proc. natn. A c a d . S c i . U . S . A . 54, 219. ltccves, P. (1965). Ract. R e v . 29, 24. Rccves, P. (1968).J . Bact. 96, 1700. Rensscr, F. (1968). Biochemistry, N . Y . 7, 293. liicber, M., Imaeda, T. and Cesari, I. M. (1969). J . gem. Microbiol. 55, 155. Ricmersma, J. C. (1968). Biochim. biophys. Acta 153, 80. lticske, 5. S. (1967). I n “Antibiotics”, (D. Gottlieb and P. D. Shaw, eds.). Vol. I, pp. 542-584. Springer Verlag, New York. Ricskc, 5. S., Baum, H., Stoner, C. D. and Lipton, S. H. (1967). J . b d . Chem. 242, 4854. Roberton, A. M., Holloway, C. T., Knight, I. G. a n d Beechey, R. B. (1968). Uiochem,. ,7. 108, 445. Modwcll, A . W., Razin, S., Rottem, S. and Argaman, M. (1967). A r c h s Biochem. Riophys. 122, 621. .LZothfiold, L. and Finkelstein, A. (1968).A. R e v . Biochem. 37, 463. Eothst,ein, A . and Van Steveninck, J. (1966). An?$.N . Y . A c a d . S c i . 137, 606. Ruttenborg, M. A., King, T. P. and Craig, L. C. (1965). Biochemistry, N . Y . 4, 11. IJuttenbcrg, M. A., King, T. P. and Craig, L. C. (1966). Biochm istry, N . Y . 5, 2857 12uttcnherg, M. A. and Ma,ch, B. (1966). Biochemistry, N . Y . 5, 2864. Salton, M. 13. J. (1967). A. R e v . Microbiol. 21, 417. Salt>on,M. R. J. (1968).J . gen. Physiol. 52, 227 S. Salton, M. 13. J. and Schmitt, M. D. (1967). Biochim. biophys. Acta 135, 196. Salton, M. R. J., Schmitt, M. D. and ‘Trefts, P. E. (1967). Biochem. biophys. Res. C o m m u n . 29, 728. Salton, M. It. J.,Freer, J. H. a,ndEllar, D. J.(1968).Biochem. biophys. Res. C o m m u n . 33, 909. Scarpa, A., Cccchetto, A. and Azzone, G. F. (1968). N a t u r e , Lond. 219, 529. Schatz, G. (1968).J . biol. Chmvn,. 243, 2192. Schatz, G., Penefsky, H. S. and Racker, E. (1967). J . biol. Chem. 242, 2552. Scherrcr, R. and Gerhardt, P. (1964). N a t u r e , Lond. 204, 649. Schnbert, K., Rose, G., Wachtel, If., Horhold, C. and Ikekawa, N. (1968). E u r . J . Biochem. 5, 246. Schiilmnn, J . H., Pethica, B. A., Pew, A. V. and Salton, M. R. J. (1955). Prog. Biophys. biophj4s. Chem. 5, 41. Schwartz, M. (1968). Nature, L m d . 219, 915.

ANTIMICROBIAL AGENTS AND MEMBRANE FUNCTION

103

Schek, 0. K. (1967). I n “Antibiotics”, (D. Gottlieb and P. D. Xhaw, eds.). Vol. I, p’. 142-152. Spririger Verlag, New York. Scssa, G. and Weissmann, G. (1967). Biochim. biophys. A c t a 135, 416. Sessa, G. arid Weissmann, G. (1968).J . biol. Chem. 243, 4364. Seufert, W. D. (1965). N a t u r e , L o n d . 207, 174. Shannon, H.and Hedges, A. J. (1967). J . Bact. 93, 1353. Shavit, N., Thorc, A., Kcister, D. L. and San Pietro, A. (1968).Proc. n a t n . Acad. S c i . U.S.A. 59, 917. Shaw, P. D. (1967a).I n “Antibiotics”, (D. Gottlieb and P. D. Shaw, eds.). Vol. I, pp. 565-610. Springer Verlag, New York. Shaw, P. D. (1967b). I n “Antibiotics”, (D. Gottlieb and P. D. Shaw, eds.). Vol. I, pp. 649-650. Springer Verlag, New York. Shcmyakin, M. M. (1965). Antimicrob. Agents Chemother. pp. 962-976. Shemyakin, M. M., Ovchinnikov, Yu. A., Ivanov, V. T., Kiryushkin, A. A., Zhdanov, G . L. and Ryabova, I. D. (1963).Experientia 19, 566. Shemyakin, M. M., Vinogratlova, E. I., Feigina, M. Yu. A., Aldanova, N. A., Loginova, N. F., Ryabova, I.D. and Pavlenko, I. A. (1965).Experientia 21, 548. Shrmyakin, M. M., Ovchinnikov, Yu. A., Ivanov, V. T. and Evstratov, A. V. (1967a). N a t u r e , L o n d . 213, 412. Shemyakin, M. M., Ovchinnikov, Yu. A., Ivanov, V. T., Antonov, V. K., Shkrob, A . M., Mikhaleva, I. I., Evstratov, A. V. and Malenkov, G. 0. (1967b).Biochem. hiophys. Res. C o m m u n . 29, 839. Sicwert, G. and Strominger, J. L. (1967). Proc. natn. scad. Sci. U.S.A. 57, 767. Silver, S. and Levine, E . (196th). J . Bact. 96, 338. Silver, S. and Levine, E. (1968b). Bioch,em,. biophys. Res. Gomrnun. 31, 743. Silver, S. arid Wcndt, L. (1967). J . Bact. 93, 560. Simon, E. J. and Van Praag, D. (1964). Proc. natn. A c a d . S c i . U.S.A. 51, 1151. Skou, J. C. (1965). Physiol. Rev.45, 596. Slater, E. C. (1966). I n “Comprehensive Biochemistry”, (M. Florkin and E. H. Stotz, eds.). Vol. 14, pp. 327-396. American Elsevier, New York. Slatcr, E. C. (1967). Eur. J . Biochem. 1, 317. smarda, J. arid Taubeneck, U. (1968). J . gen. Microbiol. 52, 161. Smith, D. H. and Davis, B. D. (1967).J . Bact. 93, 71. Smith, L. (1968). I n “Biological Oxidations”, (T. P. Singer, ed.), pp. 55-122. Intorscience, New York. Smith, R. F.,Shay, D. E. arid Doorenbos, N. J. (1964).J . p h a r m . S c i . 53, 1214. Snoswell, A. M. and Cox, C. B. (1968). Biochirn. biophys. Acta 162, 455. Stein, W. D. (1967). “Thc Movement of Molecules Across Cell Membranes”. Acadcmic Press, New York. St’ein, W. 11. (1968). N a t u r e , L o n d . 218, 570. Steinrauf, L. K., Pinkerton, M. and Chamberlin, J. W. (1968). Biochem. biophys. Res. C o m m u n . 33, 29. de Souza, N. J. and Nes, W. R. (1968). Science, N . Y . 162, 363. Sugawara, S. (1967). J . Antibiot., Series A , 20, 93. Sugawara, S. (1968).J . Antibiot., Series A , 21, 83. Takahashi, N., Suzuki, A. and Tamura, S. (1965). J . Am. chem. Soc. 87, 2066. Tamura, S., Takahashi, N., Miyamoto, S., Mori, R., Suzuki, X., and Nagatsu, J. (1963). Agric. biol. Chem., J a p a n 27, 576. Thore, A., Kcister, TI.L., Shavit, N. and 8an Pietro, A. (1968). Biochemistry, N . Y . 7, 3499. Tien, H. T. and Diana, A. L. (1968). Ghem. P h y s . L i p i d s 2, 55. Tosteson, D. C. (1968). F e d n Proc. B’edn. Am. Socs exp. Biol. 27, 1269.

104

FRANKLIN M. HAROLD

Tostcson, D. C., Cook, P., Andreoli, T. E. aridTieffenberg, M. (1967).J.gen.PhysioZ. 50, 2513. Tostcson, 11. (j., Andreoli, T. E., Tieffenberg, M. and Cook, P. (1968). J. gera. Physiol. 51, 3738. Vallin, I. arid Liiw, H. (1968).E u r . J . Biochem. 5 , 402. Varicchio, F., Doorcnbos, N. J . and Stevens, A. (1967).J . Bact. 93, 627. Vernon, L. 1’. (1968). Hact. Rev.32, 243. Voss, J . (2. (1967). J . gen. Microbiol. 48, 391. Wahri, K., Lutsch, G., Rockstrob, T. and Zapf, K. (1968).A r c h . Mikrobiol. 63, 103. Wallsch, D. F. H. and Gordon, A. (1968). li’edn Proc. Fedn. Am. Socs exp. Biol. 27, 1263. Waltjcr, P., Lnrdy, H. A. and Johnson, D. (1967).J . biol. Chem. 242, 5014. Wcber, M. M. arid Kinsky, S. C. (1965). J . Bact. 89, 306. W c i i i l ~ x h E. , C. (1957).Proc. n!atn. A c a d . S c i . U . S . A . 43, 393. Woiribach, E. (J. and Garbus, J. (1968a). Biochem. J . 106, 711. Wc:iribach, E. C. arid Garbus, J . (1968b).Biochim. biophys. A c t a 162, 500. Wciriberg, E. U. (1967). I n “Antibiotics”, (D. Gottlieb and P. D. Shaw, eds.). Vol. I, pp. 90-101. Springer Verlag, New Yorlr. Wciriberg, E. D. (1968).Ann. N. Y . A c a d . S c i . 148, 587. Wcisor, It.,Asscher, A. W. and Wimpenny, J. (1968). N a t u r e , Lond. 219, 1365. Woissmanir, Q. arid Sessa, GI. (1967).J. biol. Chem. 242, 616. Wkiit>e,R . J. arid Hurst, A . (1968).J. gem. 3ZicrobioZ. 53, 171. Wkiitchousr:, M. W. (1964).Riochem. Pharmac. 13, 319. Whittam, R.,Wheclor, K. P. and Blake, A. (1964). N a t u r e , L o n d . 203, 720. Wilkiiisorr, R. G . (1967). J . gen. M%crobiol.47, 67. Williamsori, 12. L. arid Metcalf, R. L. (1967).Xcience, N.P. 158, 1694. Windisch, F.and Hcumann, W. (1960). Naturwissenschuften 47, 209. Winkler, H . H. arid Wilson, T. H. (1966).J . biol. Chem. 241, 2200. Wipf, H. K., Pioda, L. A. K., Stofanac, Z. arid Simon, W. (1968).Helv. chim. A c t a 51, 377. U’oodroff’e, It. C . S. and Wilkinson, B. E. (1966a).J . gen. Microbiol. 44, 343. Woodrofle, 1%. C . S. arid Wilkirison, B. E. (1966b). J . gen. Microbiol. 44, 353. Wootlriiff, H. B. (1966). I n “Biochemical Studies of Antimicrobial Drugs”, iYym,p. ~’ioc.gen. iWicrobio1. 16, 22. Wurm, M. (1951). ,7. b%ol.Chem. 192, 707. Ypl-innt,is,.I). A., Dainlro, J. L. and Schlenk, F. (1967).J . Bact. 94, 1509. do Z ~ r a i g It. , I?. a r i d Luria, S. E. (1967).J . Bact. 94, 1112.