Printed in U.S.A.. Identification and Characterization of Some Bacterial Membrane. Sulfhydryl Groups Which Are Targets of Bacteriostatic and Antibiotic. Action*.
THEJOURNAL OF BIOLOGICAL CHEMISTRY 8 1984 by The American Society of Biological Chemists, Inc.
Vol. 259, No. 21, Issue of November 10, pp. 13590-13594,1984 Printed in U.S.A.
Identification and Characterization of Some Bacterial Membrane Sulfhydryl Groups Which AreTargets of Bacteriostatic and Antibiotic Action* (Received for publication, April 23, 1984)
Sheldon L. Morris$, Rosemary C. Walsh, and J. Norman HansenQ From the Divisionof Biochemistry, Department of Chemistry, University of Maryland, College Park, Maryland 20742
Covalent modification of sulfhydryl groups which become sensitive toward sulfhydryl agentsduring germination of Bacillus cereus spores exerts a profound bacteriostatic effect, resultingin outgrowth inhibition. The modified spore components are membrane species of 13,000,28,000, and 29,000 daltons. Detergent disruption of the membrane inactivated the sulfhydryl groups. A highly sigmoid inhibition curve (n = 11.8) with diamide suggested the participation of closely neighboring sulfhydryl groups. Substate and substrate analogs of thelactose and dicarboxylic acid permeases protected the sulfhydryl groups against modification. Nisin, a 34-residue peptide antibiotic, inhibited spore outgrowth and sulfhydryl modification at a concentration of about 0.1 PM. Since these sulfhydryl groups have been implicated as involved with the bacteriostaticaction of nitrite, substances directed toward them maybe a useful new classof bacteriostatic agents and antibiotics.
The sulfhydryl group of cysteine participates as a critical residue inmanyenzymatic reactions. Theimportance of sulfhydryl groups in membrane-associated functionsof active transport andoxidative phosphorylation has been recognized for many years (1-6). Sulfhydryl groups associated with critical membranefunctionsarepotentialsitestoward which antibiotic and bacteriostatic agents could be directed. Antibiotics such as nisin (7) and fuscin (8) may act as sulfhydryl agents.Fuscinappearsto be directedtowardmembrane groups (8). The unusual dehydroalanine groups of nisin (7) are potential Michael acceptors which might reactwith membrane sulfhydryl groups. Understanding the nature of sensitive membrane sulfhydryl groups may be useful for the identification or design of new antibiotic agents. We suspected the existence of such membrane sulfhydryl groups as a result of our studies on the mechanism and site of nitrite bacteriostatic action. Thegeneral efficacy and nontoxicity of nitrite as a preservative agentproves the existence *This research was supported by grants to J. N. H. from the Agricultural Research Service, administered by Beltsville, MD (Grant 58-3244-2-439);the Eastern Regional Research Center, Philadelphia, P A (Grant 12-14-1001-1230-WRU-801-1090-20841-4111); and National Institutes of Health Biomedical Research Support Grant RR07042 to theUniversity of Maryland. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $. Present address, Department of Biochemistry, School of Hygiene and Public Health, The Johns Hopkins University, Baltimore, MD 21205. 3 To whom correspondence should be addressed.
of sites in bacteria that are susceptible to an interference which causes bacteriostasis. Because the chemistry and potential sitesof nitrite action arehighly complex, we have been studying simple model systems which may act by the same or similar mechanism as nitrite. One is the S-nitrosothiols,with a general structure of RSN=O (9-13). These compounds are analogs of nitrous acid (HON=O), in which the hydroxyl group has been replaced by an RS group. By observing Snitrosothiols effects on outgrowing Bacillus cereus spores, we have established that inhibition is a consequence of interfering with sulfhydryl groups on the exterior of freshly germinatedspores (14-16), probably to give a symmetrical and reversible intermediate such as RSN(0H)-SX, where -SX is derived from a n unusually reactive spore sulfhydryl group, which may be in the membrane(14, 15). In this report,we more firmly establish that thesulfhydryl groups are located in the membrane, characterize the sulfhydryl group-containing membrane components, and study the reactivity of the membrane sulfhydrylgroupswithseveral sulfhydryl agents and outgrowth inhibitors. We also find that nisin at very low concentrations (0.1 p M amounts) inactivates the sulfhydryl sitestoward sulfhydrylagents. Thisresult suggests that these membranesulfhydryl groups are the natural target of nisin bacteriostatic action. MATERIALS AND METHODS AND RESULTS’ DISCUSSION
The results presented here along with our previous work (14-16) provide evidence that the membranesof freshly germinated B. cereus spores contain sulfhydryl groups whichcan be covalently modified to cause profound inhibition of subsequent spore outgrowth. The information we have obtained allows us to describe the apparent natureof these sulfhydryl sites. They are quite reactive and are irreversibly modified by iodoacetate and reversibly modified by S-nitrosothiols (1416).Themechanism of modification iscovalent,andthe reactivity of nitrosothiol(RSN=O)isdetermined by and Portions of this paper (including “Materials and Methods, “Results,” Figs. 1-6, and Tables I and 11) are presented in miniprint at the end of this paper. The abbreviations and trivial name used are: SDS, sodium dodecylsulfate; DEPC, diethylpyrocarbonate or ethoxyformic anhydride; diamide, 1,l’-azobis(N,N-dimethylformamide; PPO, 2,5-diphenyloxazole;POPOP, p-bis[2-(5-phenyloxazolyl)]benzene; TCA, trichloroacetic acid; o-NPG, o-nitrophenylgalactopyranoside. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, MD 20814. Request Document No. 84M-1250, cite the authors, and include a check or money order for $6.80 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press.
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Membrane Sulfhydryl Groups Sensitive directly related to thepolar or electron-withdrawing effect of the R group (15). The effectiveness of S-nitrosothiols is not a function of their hydrophobicities, so transport to an intracellular site is unlikely. Also, nontransportable S-nitrosothiols are effective (15). The sensitive sulfhydryl sites are not available in the ungerminated spore; and, upon germination, the appearance of sensitivity toward iodoacetate inhibition appears simultaneouslywith the uptakeof iodoacetate label into the germinated spores (15). We show here that the label is incorporated into themembrane and thatonly a small number of membrane components,presumably proteins, are involved. The highly reactive sulfhydryl groups appear to be in communication with one or more histidines, and covalent modification of either sulfhydryl group or histidine has a profound reciprocal effect on the reactivity of the other. Finally, the sulfhydryl groups exist in a highly cooperative environment, there may be more than one type of sulfhydryl site, and there may be closely neighboring sulfhydryl groups which are more effectively inhibited by a bifunctional sulfhydryl agent than a monofunctional one. These results allow us tobegin to devise rules for identifying or designing bacteriostatic agentsthat arespecifically directed toward these membranesulfhydryl sites. The Taftplots which relate S-nitrosothiol structure with bacteriostatic effectiveness (15) establishthat such thingsas size, shape, charge, and hydrophobicity are not significant factors in inhibitor effectiveness. One can conclude that the sulfhydryl groups are in a sterically unhindered environment that is readily accessible by agents which cannot traverse the membrane. A good agent would accordingly be one which is a multifunctional sulfhyof moderate reactivity in a relatively bulky, drylagent charged, and nontransportable molecule. These properties would not interfere with interaction at the target site, but would greatly reduce the likelihood of toxic side effects by attack at other sites, such as sulfhydryl groups of critical cytoplasmic enzymes. Antibiotic agents which interfere with specific sulfhydryl groups appear to be common. Showdomycin contains a maleimide moiety that inhibits UMP kinase and uridine phosphorylase (39). Fuscin contains a quinoid moiety which inhibits mitochondrial and bacterial transport by irreversibly modifying membrane cysteine residues (8). Nisin is particularly interesting in thatall the above design criteria for a specific inhibitor of membrane sulfhydryl groups seem to be met. It is a 34-residue peptide antibiotic (40) that is rather bulky and should be nontransportable, unless there is some specific transport mechanism for it. The activity of nisin is associated with dehydroalanine residues which could react with sulfhydryl groups (7, 40). There are two of these dehydroalanines and one methyldehydroalanine in the nisin molecule, so each molecule might react with more than one neighboring sulfhydryl group. Considering thenature of dehydroalanine, it should behave as a Michael acceptor of moderate reactivity, although its environment in the peptide makes this expectation tenuous without experimentalevidence. Our results show that nisin is remarkably effective, both as an inhibitor of spore outgrowth andasan inactivator of the membrane sulfhydryl groups of germianted B. cereus spores. In view of
to Bacteriostatic Agents
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the high specificity of action typically displayed by natural antibiotics, it seems likely that nisin has evolved the specific capability to inactivate membrane sulfhydryl groups of this type. The fact that a naturally evolved antibiotic interacts at these sulfhydryl groups creates the expectation that these groups will prove to be of common occurrence in bacteria, and hence be important target sites toward which a wide variety of bacteriostatic agents could be directed. A thorough understanding of the topology and chemistry of these sulfhydryl groups and the mechanism by which they are inactivated should permit new bacteriostatic agentsto be made by rational design or selected from known chemical agents and natural products. REFERENCES 1. Jocelvn. P. C. (1942)
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