Bacterial cell wall synthesis and structure in relation to the mechanism of action of penicillins and other antibacterial agents

Bacterial Cell Wall Synthesis and Structurein Relation to the Mechanism of Action ofPenicillins and Other Antibacterial Agents*

JACK L . STROMINGER, M.D . and DONALD .I. TIPPER, PH .D .

Madison, Wisconsin

I T is thirty-six years since penicillin was dis-covered and twenty-five years since the

modern era of chemotherapy began with therealization that this small organic molecule,isolated from a fungus, could kill a large numberof bacterial species and was of incredibly lowtoxicity when given as injections to experi-mental animals [1] . This latter observationfollowed on the heels of the discovery that asynthetic azo dye, prontosil, also possessed theselective toxicity required of an agent whichmight be used for the therapy of infections inman. It was soon found that prontosil itself wasnot an antibacterial agent but was reductivelycleaved by animal enzymes to sulfanilamide .The following two decades have witnessed anintensive chemical investigation of these com-pounds and their modification in many usefulways. A dozen or more additional useful anti-bacterial agents, such as streptomycin, chloram-phenicol and tetracycline, have since been in-troduced. The impact of these discoveries onhuman disease can hardly be comprehended bythose of us whose experience in medicine islimited to the period after World War it .

Still another important consequence of theisolation of these new antibacterial agents hasbeen their use as tools with which to explore newareas of biology . The fact that antibacterialagents are useful in chemotherapy implies thatin various ways the metabolic processes of bac-teria are different from those of their humanhosts, and that advantage of these differencescan be taken in the treatment of disease. Anti-bacterial agents have therefore been exceed-ingly useful tools with which to probe at thosecell processes which distinguish one cell type

from another and ultimately determine theuniqueness of every organism. Perhaps the moststriking example of this utility was the discoverythat yeast extract contained a competitive in-hibitor of the action of sulfonamides. This sub-stance was isolated and identified in 1940 . Itwas, of course, para-aminobenzoic acid, anessential metabolite in the growth of most bac-teria but not utilized by animal cells . It wassoon found that this substance was a precursorof folic acid and that sulfonamides were competi-tive antagonists of the utilization of para-amino-benzoic acid for folic acid synthesis [2] . It tooktwenty years more to elucidate the mechanismof folic acid synthesis and to find the preciseenzymatic reaction which is inhibited by sul-fonamides. The selective toxicity of sulfonamidesis due to the fact that animal cells require andutilize pre-formed dietary folic acid, originallysynthesized by bacteria and plants .

It is remarkable that this early and elegantstudy of the mechanism of action of an antibac-terial agent took place even before the isolationof penicillin and its introduction into chemo-therapy. From the early studies of penicillinaction, three important facts stand out in retro-spect. Penicillin G kills gram-positive bacteriafar more readily than gram-negative bacteria[3] . It kills growing cells but not resting cells[4], and it results in the formation of large orfilamentous forms of bacteria [5,6] . As we shallsee, an explanation of these last two facts isavailable, but the basis of the relative resist-ance of gram-negative bacteria is not yet en-tirely clear .

Further progress took another ten years .During this time an important area of micro-

- From the Department of Pharmacology, University of Wisconsin School of Medicine, Madison, Wisconsin .This study was supported by U. S. Public Health Service Grant AI-06247, National Institutes of Health; GrantG&1823, National Science Foundation ; and by a grant from Eli Lilly & Co., Indianapolis, Indiana .

708

AMERICAN JOURNAL OP MEDICINE

Bacterial Cell Wall Synthesis and Structure-Streminger, Tipper

709

biology began to develop . In the early 50's theunique features of the surface of bacterial cellsbegan to be recognized and procedures weredeveloped for the isolation of bacterial cell walls .Examination of the components of acid hy-drolysates of the walls of gram-positive bacteriarevealed that they had a remarkably simplecomposition, containing four or five aminoacids and two amino sugars, one of which couldnot be identified [7] . The peptide released dur-ing germination of bacterial spores had a similarcomposition, The walls of gram-negative bac-teria were far more complex, contained a widervariety of amino acids and sugars, and charac-teristically lipid, absent from the walls ofgram-positive bacteria . The components ofthe cell walls of gram-positive bacteria, includ-ing the novel amino sugar, were also present.Moreover, it was recognized that n-alanine andn-glutatnic acid were both present [8) . Theunusual D-forms of amino acids had at that timebeen found in very few other natural products .

EFFECrs OF LYSOZYME AND PENICILLINON BACTERIAL MORPHOLOGY

Bacterial cell walls have been identifiedmorphologically by staining methods, and werelater seen also as a distinct layer in electronmicrographs of thin sections of bacteria . Thentwo exceedingly important observations weremade. The enzyme, lysozyme, had been knownto lyse a variety of bacterial species, since Flem-ing discovered it in the early 20's . This lysis wasfound to be the consequence of hydrolysis of aglycosidic linkage in the bacterial cell wall [9] .Moreover, if a sensitive Bacillus was treated withlysozyme in hypertonic broths, the rod-shapedorganisms were converted to spherical forms,called protoplasts [10] . When the cell wall issolu-bilized by lysozyme, the cell membrane can-not withstand the high internal osmotic pressureof bacteria. As the result, the cell bursts anddies. If hypertonic sucrose or sodium chloride ispresent in the medium, the internal osmoticpressure is counterbalanced and the cell isstabilized . However, it assumes a sphericalshape because it has lost the wall which has theshape and rigidity characteristic of the bacterialcell .

These chemical and morphologic studies setthe stage for the hypothesis that penicillin is aselective inhibitor of bacterial cell wall syn-thesis. When Escherichia coli was treated withpenicillin in hypertonic broth, spherical organ-

vol . . 39, NOVEMBER 1965

Fm. 1 . Formation of spheroplasts of Esch . coli underthe influence of penicillin G in hypertonic sucrose solu-tion. From HAHN, R . E . and CIAS, ,1 .,. Science, 125 :119,1957 [121 .

isms were produced [171 . (Fig . 1 .) If the peni-cillin was washed out, they reverted to bacilli .These observations indicated that penicillin hadno lethal effect on the protoplasts, which con-tinued to enlarge in its presence and reverted tobacilli in its absence . The various intermediateforms are the large and filamentous forms in-duced by penicillin and observed by a numberof early investigators . The continued enlarge-ment of the protoplasts explains the require-ment of growth for penicillin action. The grow-ing protoplast emerges, apparently through a"crack" or hole in the wall . Unless the proto-plast grows and emerges, it remains a stableform within the wall, despite the fact that wallsynthesis has been blocked by penicillin . Thatis, the inhibition by penicillin of wall synthesis isnot itself lethal, but it permits the outgrowth ofa form of the cell which is unstable and bursts .The mechanism of antagonism of penicillinaction by agents such as chloramphenicol, whichinterfere with the growth of the protoplast, isapparent. Similarly, bacteria might continue tosurvive as viable protoplasts during therapy ofinfections in loci in which the medium is hyper-tonic, such as in the renal medulla or even possi-bly in purulent accumulations. L forms or pleuro-pneumonia-like organisms, bacteria which existas stable forms despite the presence of a defec-tive cell wall or the absence of a cell wall, are

710

Bacterial Cell Wall Synthesis and Structure-Strorninger, Tipper

Senlia. Vi apt

UOP-SNAC-Iactyi L alay

uov-GNAC-lact ylL-am- o- q w

~CLyei a oemi .man

UDP-GNAC-Icclyi- L-ala- 0-qlu' L-iysD-11111 1e

0 -cycloudne-O-al. e-ola+-,~-D-ala - - I,7 .

UOP-GNAC-lacty I L-ala' 0-glu' Dlyc Dtia DoleUOP-GNAC

PeniciNn ; Bac,lracln • N

s cm,vancomvcm or Rslacetin

1 ~ATPUOPj

•\4Aaceplar

r6GCferial

/

Cell wall F

pi, rlmtdine

ribonucleic

nauemme

acid

wecursors

FIG. 2 . Reaction cycle which leads to synthesis of uridine nucleotide precursorsof the cell wall of Staph, aureus. From STROMINGER, J . L . In : The Bacteria, vol .3, pp. 413-470 . Edited by Gunsalus, L . C . and Starrier, R . Y. New York, 1962 .Academic Press, Inc . [30] .

ODe-GNAc-lactic acid ether4

not sensitive to penicillin or other inhibitors ofcell wall synthesis .

ACCUMULATION OF URIDINE NUCLEOTIDESIN ANTIBIOTIC-TREATED BACTERIA

Simultaneously with the observation of thesemorphologic effects of penicillin, the study ofthe structure of a uridine nucleotide whichaccumulated in penicillin-inhibited Staphylo-coccus aureus [13] and of the role of uridinenucleotides in general in cellular metabolism[14] led to the same hypothesis . The nucleotidecontained the same constituents as the cellwall including the novel acetamido sugar, bynow identified as a 3-0-D-lactic acid ether ofN-acetyl-n-glucosamine (acetylmuramic acid)and the n-amino acids. It was therefore hy-pothesized that the uridine diphospho-acetyl-muramyl-pentapeptide (the major accumulatednucleotide) was a precursor of the cell wall andaccumulated as the consequence of a block insynthesis of the cell wall, induced by penicillin[15,161 . That hypothesis is now amply confirmedby direct isotopic measurement of wall synthesisin both gram-positive and gram-negative bac-teria .

It is remarkable that Fleming discovered twoimportant classes of antibacterial agents whichact through similar mechanisms : one, anenzyme which catalyzes the hydrolysis of thecell wall, and the other, a small organic com-pound which inhibits cell wall synthesis. Themechanism of the greater resistance of gram-negative bacteria to penicillin G has neverbeen explained . At the present time the most

likely hypotheses arc failure of this agent topenetrate through the wall to the site of its ac-tion, or a decreased affinity of the "penicillin-binding component" for the drug, since it seemsclear that, despite their relatively greater re-sistance, penicillin kills gram-negative bacteriain the same manner as gram-positive cells[ 17,18 j .

By this time a method had been developedwhich permitted relatively simple measurementof uridine nucleotide accumulation [19J, andit was therefore possible to search for otheragents which might induce uridine nucleotide ac-cumulation. All the penicillin and cephalospor-ins induce this accumulation in Staph . aureus,and it has been found to occur with bacitracin,D-cycloserine, vancomycin, ristocetins, novo-biocin and gentian violet [ 13,19-29] .

A study of the uridine nucleotides whichaccumulated in each case indicated the exist-ence of a reaction cycle which led to formationof UDP-acetylmuranryl-pentapeptide and whichwas blocked at different points by these agents .One other kind of block, a nutritional one, wasimportant . Deprivation of lysine led to accumu-lation of a nucleotide which was not observed toaccumulate with any of the antibacterial agents .(Fig . 2 .)

ENZYMATIC SYNTHESIS OF URIDINENUCLEOTIDES AND THE MECHANISM

OF ACTION OF D-CYCLOSERINEWith these intermediates in hand, it was then

possible to work out the reaction mechanismwhich led to synthesis of the nucleotides . Fifteen

AMERICAN JOURNAL OF MEDICINE

Bacterial Cell \Vall Synthesis and StructureStrmnitn.?er . TipperUMP

UDP2 1UTP

3 ~GIcNAc-1-P 211 GIcNAc-6-P i t Glc-6-P to fructose-6-P

LID -GIcNAe4 j phosphornolpyrucatr5 1UDP-MurNAc6 j c-alanine7 j D-glu .1-'; c-glu8 j L-lysine9 j D-ala .n-ala I D-ala .t-l t-alaUDP-MurNAc-pentapeptide16 1 phospholipidMurNAc(-pentapeptide)-P-phospholipid17 1 UDP-GIcNAcDisaccharide(-pentapeptide)-P-phospholipid18 4 gly-sRNA :-z gly + ATP + sRNA

Phospholipid + Pi

Disaccharide(-pentapeptide-pentaglycine)-P-phospholipidk21

19-4 Acceptor linear glycopeptidePhospholi pid-P201

Cross-linked glycopeptidc + 1)-ala

Fle. 3 . Reactions involved in cell wall synthesis .

enzymes are required for the synthesis of thisnucleotide from simple nucleotides, hexosephosphate and amino acids, and at least eightare involved in its utilization for cell wall syn-thesis (dotted lines, Fig . 2) .

Nine enzymes are directly concerned with thereaction cycle shown . (Fig. 2.) Two are phos-phorylation reactions (reactions I and 2, Fig .3), three are involved in the activation of thesugar, acetylglucosamine, and its modificationto form acetylmuramic acid (reactions 3, 4 and5), and four participate in a sequential additionof three amino acids and a dipeptide to form thepentapeptide (reactions 6, 7, 8 and 9) . Thespecificities of these-various enzymes have beendescribed repeatedly [.301 and need not bebelabored here . Six other enzymes are requiredfor the synthesis of substrates for these ninereactions, viz : the synthesis of acetylglucosamine-I-phosphate from fructose-6-phosphate (reac-tions 10, 11 and 12), the formation of D-glutamicacid catalyzed either by L-glutamic acidracemase or by a stereospecific o-alanine-a-ketoglutarate transaminase (reaction 13), andthe formation of the dipeptide, n-alanyl-n-ala-nine from L-alanine (reactions 14 and 15, equa-tions 1 and 2, catalyzed by alanine racemaseand o-alanyl-D-alanine synthetase)

t.ala,=n-ala

(1)

2 D-Ma + ATP

n-ala D-ala + ADP + Pi (2)

VO1., 39, NOVEMBER 1965

The last sequence is of particular interest toour theme because the antibiotic, n-cycloserine,is a true competitive antagonist of the two en-zymes involved in o-alanyl-n-alanine synthesis[23,30-32]_ It is a structural analogue ofo-alanine (Fig . 4) and is, in fact, the onlyone of the inhibitors of cell wall synthesis forwhich the precise mechanism is known . Oneinteresting feature of the competition has re-cently been elucidated [33] . Both alanineracemase and n-alanyl-n-alanine synthetase arecompetitively inhibited by n-cycloserine . Infact, alaninc racemase is inhibited by D-cyclo-serine whether the reaction is measured in thedirection, L alanine -* D-alanine, or in thedirection, D-alanine - L-alanine . However,L-cycloserine does not inhibit the reaction ineither direction. This apparent anomaly can beexplained by considering the conformation ofthe antibiotic and of the substrates for alanineracemase . (Fig. 4.) The closed ring severelyrestricts the possible conformations of thecycloserines ; and, in fact, except for minorvariations, the one photographed is the onlyconformation possible. On the other hand,n-alanine can have a number of conformations,one of which on one surface is identical with thatof the antibiotic, o-cycloserine In this con-formation, the relative positions of the carbonylgroup, the asymmetric carbon and the aminogroup are the same as in the antibiotic . L-Alauinesimilarly can have this same conformation, dif-fering only in that the relative positions of the

712

Bacterial Cell Wall Synthesis and Structure-Slromin,ger . Tipper

4A

4B

D-alanine

HH

D-cycloserine

L-alanine

0

0 -

N

H

L-cycloserine

Fin . 4 . Structures and molecular models (Stuart-Briegleb) of D- and L-alanine and of D- and L-cycloserine .From ROZE, U . and STROMINGER, J . L . Molec . Pharmacnl ., in press [33' . A, these models are photographedlooking down on the hydrogen atom on the asymmetric carbon atom . The closed ring in the cycloserinescan be seen . The formulas were drawn from a projection of this photograph, and therefore positions of theatoms in the formulas and in the photograph correspond . B, these models are photographed looking down onthe positively charged -NII 5 ~ and negatively charged --C-O- . Here the dihedral angles involving thecharged groups are opposite in sign in then- and L-series . C, the model of L-alanine has been rotated so thatthe dihedral angle has the same sign as in o-alanine and o-cvcloserine . L-Cycloserine cannot be rotated intothis conformation . In these atomic models black is carbon, white is hydrogen, blue is nitrogen and red isoxygen .

4C

.AMERIC .AN JOURNAL OF MEDICINE

VOL . 39, NOVEMBER 1965

Bacterial Cell Wall Synthesis and Structure-Serominger, Tipper

hydrogen and methyl groups on the asymmetriccarbon atom are interchanged . L-Cycloserine,because of the closed ring, cannot be rotated intothis conformation . If the substrate binding site onalanine racemase prefers this conformation, thisconcept explains why D-cycloserine, which isfixed in this preferred conformation, is bound tothe enzyme 100 times as effectively as the en-zyme binds its natural substrate . These factsprobably account for the effectiveness of thissubstance as an antibacterial agent .

The mechanisms of action of four other sub-stances, the penicillins, bacitracin, vancornycinand ristocetins, remain to be elucidated in com-parable detail. Two others, novobiocin andgentian violet, can be excluded from furtherdiscussion because they are not specific inhibitorsof cell wall synthesis and a large number of othermacromolecular syntheses are simultaneouslyinhibited by these substances .

At this stage it became apparent that if fur-ther progress was to be made, a great deal ofinformation on the structure of the cell wallwould be required, and such studies were under-taken as an accompaniment of further biosyn-thetic work .

CHEMICAL STRUCTURE OF THE CELL WALL

The structure of the cell wall of Staph . aureuswhich has emerged from those studies is shownin Figure 5 . The structure illustrated is almostcertainly not correct in all its details, but in ageneral way it certainly represents the outline ofthe structure of the cell wall of this organism[341. The cell walls of other bacteria have notyet been investigated in comparable detail, butsuch information as is presently available sug-gests that the structures of other cell walls followthe same general pattern although they differ inimportant details. The polysaccharide backboneof the cell wall consists of two alternating sugars,acetylglucosamine and acetylmuramic acid .Four chains of these sugars are represented inthe illustration, but to account for the dimen-sions of the wall at least twelve chains wouldprobably he required . The two sugars are linkedto each other by #-1 ,4 linkages and therefore thepolysaccharide is a substituted chitin, differingfrom the substance found in the exoskeleton ofinsects and crustacea only in that every otheracetylglucosamine residue is substituted at its3-position by lactic acid, linked as an ether . Tothe carboxyl group of the lactic acid is linked atetrapeptide, the sequence of which is L-ala-D-

2

2

7

X=N-ACETYLGLUCCLSAMINEY=N-ACETYLMURAMC AGD OR N,O-fACEnJ,IURAMKACIDO=AN MAINOOLD OFT€ TETRAPEP TIDE OR PENTAPEP rDE•M .-A PENrAGLYCINE CRO55-BRIDGE

----=BONDS CLEAVED BY ACETYLMURAMIOASE-=RANDOM BREMCS IN THE STHOCTURE

713

FIG . 5. A representation of the proposed structure of thecell wall ofStaph. aureus. From GItUYSEN, J.-M ., TIPPER,D. J . and STROMINGER, J . L . Biochemistry, 4 : 474, 1965[341 .

glu-L-lys-D-ala. As already mentioned, the pre-cursor of this structure is a uridine nucleotidewhich contains a pentapeptide. A mechanism bywhich the terminal D-alanine of the pentapep-tide may be lost in the course of cell wall syn-thesis will be discussed later .

The tctrapeptide units are in turn linked toeach other through peptide cross bridges . In thecase of Staph. aureus these bridges are composedof polyglycine units averaging five glycine resi-dues in length, linked between the E-aminogroups of lysine and the carboxyl group of theterminal D-alanine in the tetrapeptide [35,36 .This type of bridge is, as far as known, founduniquely in the genus Staph. aureus, and thenature of the bridge is one feature of cell wallstructure which appears to differ considerablyamong different bacteria . In this manner a two-dimensional structure can be built up . However,the wall is a three-dimensional structure . Physi-cal methods presently available do not appearto he adequate to elucidate the three-dimen-sional structure of a noncrystalline network suchas the cell wall, and at the present time it istherefore possible only to speculate that a three-dimensional structure could be built up bypolyglycine bridges extending to a plane infront of or in back of the illustration, rather thanwithin it .

Most if not all bacterial cell walls containadditional structures which may be protein,carbohydrate or lipid, and which are antigenic

714

0 a

160TIME, HOURS

FIG . 6 . Degradation sequence leading to isolation of thedisaccharide-tetrapeptide from the cell wall of Staph .aureus. From GHUY5EN, J.-M., TIPPER, D. J., BIRGEC. H . and STROMINGER, J . L. Biochemistry, in press [35] .In the first section, about 15 per cent of the glycinebridges are opened with glycine bridge-splitting enzyme1 . In the second section, the actions of glycine bridge-splitting enzyme 2 and glycinee aminopeptidase result inopening of additional bridges, liberation of free glycineand eventually of c-amino groups of lysine. In the lastsection a C-terminal glycine residue is removed with aglycine carboxypeptidase . Treatment of the product ofthis sequence with (i-acetylglucosaminidase yieldedacetyhnuramyl-tetrapeptide.

Bacterial Cell Wall Synthesis and Structure-Strominger, Tipper

isolate many different types of small fragmentswhose structures could be elucidated . For ex-ample, elucidation of the structure of thepolysaccharide required the isolation of thetwo isomeric disaccharides, acetylglucosaminyl-acetylmuramic acid and acetylmuramyl-acetyl-glucosamine, as well as the intact polysac-charide, free of peptide. Just one example willserve to illustrate the usefulness of these en-zymes in studies of the bacterial cell wall . Inthis example (Fig . 6), the tetrapeptide was ob-tained by employing the specificities of twoglycine-bridge-splitting . enzymes, a glycineaminopeptidase and a glycine carboxypeptidase .

From the point of view of the present dis-cussion, another glycine-bridge-splitting en-zyme, termed lysostaphin, is especially inter-esting [38,39] . This protein, with a molecularweight of about 30,000, kills bacteria by hydro-lyzing the bacterial cell wall . Because it cleavesthe pentaglycine bridge, it is specific for thegenus Staph. aureus, and indeed its useprovides a simple tool for identificationof this organism. It is able to cure someexperimental staphylococcal infections inanimals. It has a potency on a weight basisseveral times that of penicillin G, and on amole basis several hundred times that of peni-cillin G. It is thus one of the most powerful anti-biotic substances to have been discovered .It can kill resting as well as growing cells,and no resistant strains of Staph . aureus werefound among 250 clinical isolates examined[40,41]. Its isolation could suggest a new ap-proach in antibacterial chemotherapy .

in higher animals . In the case of Staph. aureus,the antigen is a teichoic acid, a substitutedribitol phosphate polymer linked through itsphosphomonester end to one of the sugars in thepolysaccharide . These molecules are shown inFigure 5 as linked at the surface but it should bepointed out that their distribution within thecell wall is unknown . Indeed, it is not knownwhich side of the structure illustrated representsthe outside or the inside of the cell wall ; theproblem of orientation of molecules within thewall is exceedingly difficult to approach .

The analysis of this structure has been carriedout with the aid of more than twenty bacterio-lytic enzymes which catalyze the selective hy-drolysis of various linkages within the wall [37] .With the aid of these enzymes it was possible to

ENZYMATIC SYNTHESIS OF THE CELLWALL AND THE MECHANISM OF ACTION

OF VANCOMYCIN AND RISTOCETIN

The enzymes described earlier, which cata-lyze the synthesis of the nucleotide intermediatesin bacterial cell wall synthesis, are soluble en-zymes. These nucleotides are utilized for cellwall synthesis by particulate enzymes presum-ably bound in the cell membrane . The first par-ticulate enzymes studied were two required forthe synthesis of a teichoic acid, the antigen in thecell wall of Staph. aureus . One of these enzymesutilizes CDP-ribitol for the synthesis of poly-ribitol phosphate . The second utilizes UDP-acetylglucosamine to glycosylate the polyribitolphosphate polymer [30] . Actually, two ace-tylglucosamine transferases are found in strainsof Staph. aureus, one of which catalyzes the


Bacterial Cell Wall Synthesis and Structure Strominger, Tipper

715

tJD P-MurNAc

UMPaL-alaD- glu

~

~MurNAc-P-lipidL-IYs

L

\IIalalipid

D-ala

D ID-ala

L-h•sD IalaD

Iala

UDP


(P-hpid)

,GlcNAc-MurNAc-P-lipid

GIcNAc-MurNAc-accepto ~-

L alaL-ala

D-gluAcceptor

D-glu

L-lys

D lalaD

Iala

L-lys

D Lla

D -als

Fm, 7 . The lipid cycle in wall biosynthesis in Staph . aureus . From ANDERSON,J. S., MATSUHASHI, M., HASKIN, M . A . and SIROMINGER . J . L . Proc. Nat. Acad.Sc ., 53 : 881, 1965 [451 .

transfer of the sugar residue in the a-configura-tion and the other catalyzes the transfer of thesugar residue in the $-configuration . These twoenzymes determine an important immunologicspecificity in different strains of Staph . aureus .Polyribitol phosphate formation and its gly-cosylation proceed in the intact cell as a coupledreaction, so that every ribitol unit is glycosy-lated. Similar reactions involved in the synthesisof the teichoic acid antigen in Lactobacillusplantarum and Bacillus subtilis have also beeninvestigated [42] .

Recently most attention has been paid to theenzymes which utilize UDP-acetylmuramyl-pentapeptide and UDP-acetylglucosamine toform an incomplete glucopeptide, incomplete inthe sense that the polyglycine bridges have notbeen added [43-47] . In the course of the studyof this reaction (equation 3) it was observed thatone of the substrates, IJDP-acetylglucosamine,yielded UDP as the nucleotide reaction product,as had been observed in several dozen reactionsof this kind which had been studied previously .

UDP-MurNAc-pentapeptide + UDP-GIcNAAc--> (GlcNAc-MurNAc-pentapeptide)„ +

UDP + UMP + Pi (3)

However, surprisingly, UDP-acetylmuramyl-pentapeptide yielded UMP and inorganic phos-phate as the nucleotide reaction products [45] .

UDP-GIcNAc

It could be shown that these were the primaryproducts of this reaction and that UMP and Piwere not formed by degradation of UDP . Theassay employed for this reaction involved paperchromatography of incubation mixtures. Inaddition to the glycopeptide product whichremained at the origin of the chromatogram, anadditional fast moving product was formedwhich is now known to contain several lipidintermediates in the reaction sequence [4.5] . Infact, careful kinetic studies indicated that a lagconsistently observed in the formation of gly-copeptide is accounted for by the time requiredto form the lipid intermediates . These clues ledto elucidation of the reaction cycle shown inFigure 7. As can be seen, a membrane-boundphospholipid serves as an initial acceptor of phos-phoacetylmuramyl pentapeptide from the nu-cleotide. Acetylglucosamine is then transferredto this lipid intermediate with formation ofUDP and disaccharide(-pentapeptide)-P-phos-pholipid . Finally, the disaccharide-pentapeptidemoiety is transferred to an acceptor, with even-tual formation of inorganic phosphate andliberation of the phospholipid . By repeatedcycles of this kind a linear glycopeptide isformed. Each of the lipid intermediates hasbeen isolated, but for the purpose of the presentdiscussion these studies are of interest becausetwo of the antibiotics mentioned earlier, vanco-mycin and ristocetin, are specific inhibitors of

7 1 6

(Lipid-P)


UDP-MurNAc-pentapeptidet1MP

Lipid

M

P-lipidpentcpepnapeptide UDP-GICNAc

GicNAc-MurNAC-P-lipid

GIeNAC-MurNAc-Acceptor

pentagf~nepentapeptide

Acceptorpentoglycine

FIG. 8 . The lipid cycle in cell wall biosynthesis inStaph. aureus, including addition of glycine chains .From MATSUHASHI, M., DIETRICH, C, P . and STROMINGER,J. I.. Proc . Nat . Acad. Sc., 54 : 587, 1965 [477 .

the utilization of disaccharide(-pentapeptide)-P-phospholipid [45] . The inhibitory concentra-tions (10 to 20 µg . per ml.) are the same as arerequired to inhibit growth of the organism . Atthese concentrations the antibiotics do not inter-fere with formation of the lipid intermediate butat twenty times higher concentrations the for-mation of the lipid intermediates is also in-hibited . Penicillin does not inhibit this reactionsequence even at exceedingly high concentra-tions . This is true whether penicillin is added tothe enzyme system in vitro or whether the cellsare pretreated with penicillin prior to prepara-tion of the particles from them . Bacitracin hassome inhibitory action too, but for reasons thatwill not be elaborated here, its basis of action isnot yet certain . Vancomycin and ristocetin arestructurally related antibiotics. They both con-tain amino acids and sugars . That is, they arethemselves glycopeptides . The precise mannerin which they inhibit the utilization of the lipidintermediates remains to be elucidated .

One other important point about this reactioncycle should be mentioned . Careful investigationby means of double labeling experiments hasshown that the glycopeptide product of thereaction still contains both of the D-alanineresidues which were present in the uridinenucleotide substrate [45] . As indicated earlier,the terminal D-alanine residue must be lost insome subsequent reaction .

The mechanism of addition of the glycinechains to the glycopeptide has proved to beexceedingly interesting . The lipid cycle shown in

Figure 7 is in fact a simplified form of the cyclewhich occurs in the cell, In the course of cellwall synthesis virtually every lysine residuebecomes substituted on its e-amino group by thecarboxyl end of the glycine bridges . These gly-cine chains are synthesized from glyeyl-sRNAas the activated intermediate . This is the onlyexample so far known of the involvement of anaminoacyl-sRNA in a reaction other than pro-tein synthesis. Glycine is transferred to bothlipid intermediates in vitro but neither to theuridine nucleotide nor to the glycopeptide .(Fig. 8.) Disaccharide(-pcntapeptide)-P-phos-pholipid is a better acceptor than acetyl-muramyl(-pentapeptide)-P-phospholipid . TheN-terminal ends of the glycine chains in thelinear glycopeptide product of this reactioncycle are unsubstituted ; therefore the bridgesare still open . Although these reactions aremechanistically very interesting, they will not beelaborated further here because none of theantibiotics which inhibit cell wall synthesis hasany effect on any of the reactions which lead tothe introduction of the glycine chains .

The lipid intermediates are presumably in-volved in membrane transport . The cell wall isessentially an extracellular product, i .e ., it is onthe outside of the permeability barrier of thecell, the cell membrane . On the other hand, thenucleotide intermediates which are utilized forits synthesis are synthesized within the cell . Thecell membrane is not permeable to these sub-stances. It can therefore be presumed that thelipid cycle illustrated in Figures 7 and 8 rep-resents a process by which intracellular pre-cursors are transported through the membranefor the synthesis of an extracellular product . Itseems possible that this process represents amodel for synthesis of other extracellular prod-ucts, but the extent to which this mechanismcan be generalized remains to be determined,as do many of the details of the reactions whichhave been described .

MECHANISM OF ACTION OF PENICILLIN

Several additional reactions are required tocomplete the cell wall, including, in Staph .aureus, amidation of the a-carboxyl group ofglutamic acid [36, 53] and closure of the glycinebridges. The bridge closing reaction is of specialinterest because it is probably the site of actionof penicillin . A number of observations havesuggested that this reaction, which may be thelast reaction in cell wall assembly, involves a


Bacterial Cell Wall Synthesis and Structure--Strnminger, Tipper

transpeptidation involving a glycine chain inone glycopeptide molecule and the end ofthe acetylmuramyl-pentapeptide fragment inanother, thus cross linking the two glycopeptides :

GlycopeptideMurNAc

The idea that the mechanical rigidity of the cellwall was in part accounted for by cross linkingwas first deduced from the paucity of free aminogroups of lysine or glycine in cell wall [48-50] .The absence of free s-amino groups of lysine inparticular indicated that the cell wall glyco-peptide was a branched peptide . Recently, moreextensive analyses with the aid of the enzymesreferred to previously have shown conclusivelythat the peptide unit attached to acetylmuramicacid is a tetrapeptide, rather than a pentapep-tide as is found in the uridine nucleotide pre-cursors [.3,51 . Moreover, the glycine chains havebeen shown to bridge between the terminalcarboxyl group of this tetrapeptide and thes-amino group of lysine [36] . Thus the closureof the bridge must be coupled in some manner tothe loss of the terminal D-alanine residue . It ispostulated that these two phenomena are in factdirectly coupled, and that the energy of then-ala-D-ala bond is utilized in the transpeptida-tion in which the bridge is closed . This hy-pothesis is attractive because bridge closure mustoccur at the outside of the cell membrane on thepre-existing cell wall to which new fragmentsbecome attached, and at this extracellular siteadenosine triphosphate is almost certainly notavailable as an energy source for synthetic reac-tions. Cross linking with loss of a terminal


71 7

D-alanine residue appears to be a general featureof wall structures in bacteria .

The concept of transpeptidation has made itpossible to formulate and to test a detailed hy-

GlycopeptideMurNAc

---,gly-D-ala-L-lys-gly-gly-glY-gly-gly-D-ala-Ljys-gly-gly`gly-gly-gly--- + 2 b-ala

pothesis regarding the mechanism of action ofpenicillin . From a structural standpoint, peni-cillin can be viewed as an analog of D-alanyl-D-alanine . (Fig . 9 .) Penicillin is a cyclic dipeptideof L-cysteine and D-valine . Ring closure fixes the"dipeptide," penicillin, in a single conforma-tion, just as has been described for n-cycloserine .One of the many possible conformations ofn-alanyl-n-alanine is nearly identical with thatof penicillin (even though one of the carbonatoms in penicillin has the L-configuration) .This situation is also analogous to the relation-ship between L-alanine and n-cycloserine al-ready described .

It is therefore postulated that penicillin hasthe conformation in which the D-alanyl-n-alanine end of the acetvlmuramylpentapeptidefragment is fixed to the substrate binding site ofthe transpeptidase. Because it is fixed in thisconformation, it would have a very high affinityfor the substrate binding site . The transpeptidasenormally catalyzes a reaction in which theD-alanyl-D-alanine bond in the substrate iscleaved, presumably with preservation of thebond energy as a substituted D-alanyl-enzymeintermediate . Then, the bridge would be closedby transfer of the substituted D-alanyl residueto the free amino end of the glycine chain .(Fig . 10 .)

L-alaI

o-glu

L-ala

D-glu

L-lYI s-gly-gly-gly - gly-gly L-lys-gly-gly - gly-gly-gly

D-ala D-ala

D-ala D-ala

Glycopeptide Glycopeptide

MurNAc MurNAcI I

L-ala L-alaI

o-glu D-glu

7 1 8 Bacterial Cell Wall Synthesis and Structure -Slrominger, Tipper

N'

H

N

H

CH3

N

CH3

Fm. 9. Photograph of Dreiding stereomodels of asubstituted 6-aminopenicillanic acid (penicillin) and asubstituted D-alanyl-n-alanine . From TIPPER, D . J . andSTROmNOER, J. L., in press [53] . The distances N' toN" (33 A) and N" to C' (2 .5 A) in the two modelsare identical, while the distances N' to C' (5 .4 A, peni-cillin and 5 .7 A, n-alanyl-D-alanine) are quite similar .The carbon atom common to the two rings and thesulfur atom of the penicillin nucleus have no analog inthe n-alanyl-o-alanine structure. They serve to fix theconformation, and presumably they would not affectthe approach of an enzyme molecule to the top (asphotographed) of the molecules. The major differencebetween the models depends on the configuration aboutN", determined in penicillin by the ring structures, andin D-alanyl-D-alanine by the double-bond character ofthe CO-N bond . It seems likely that a transition stateduring the scission of this bond would have approxi-mately single-bonded character with a non-planar con-figuration about N". Such a model (not illustrated)can be made to fit the appropriate parts of the penicillinskeleton very closely indeed, and the N' to C' distancesare then identical .

0-ow +

R-CONHCCMI-O-ala-C-NH-O-ala

6

COOH -N/f NHCO-gIy-NHZ O'

CCOON

PENICILLIN

R-CON

/CONH-D -C

,/NHCO-gIy-NHZCOOH

PENICILLOYL ENZYME

ACCNH-D-ola--0/

/NHCO-gy-NH

CROSS-LINKED GLYCC EPTIOE

Fm . 10 . Proposed transpeptidation sequence and itsinhibition by penicillin .

The highly reactive amide bond of the(3-lactam ring of penicillin is the equivalent ofthe peptide bond in D-alanyl-D-alanine. Whenfixed to the transpeptidase, a facile acylation ofthe transfer site would occur, with opening ofthe Q-lactam ring, forming a penicilloyl-enzyme,thus inactivating the transpeptidase . Penicillinis in fact irreversibly bound to a "penicillin-binding component" of bacterial cells, which isprobably the protein component of a lipoproteincomplex located at the outside of the cell mem-brane [51,52] . The extent of binding is consist-ent with the hypothesis that this binding com-ponent is a membrane-bound enzyme, present inrelatively small amount, presumably the trans-peptidase. An interesting extension of the hy-pothesis is the possibility that penicillinase is amodified transpeptidase, no longer bound to thelipid membrane, and in which the penicilloyl-enzyme is readily hydrolyzed by water . It isbelieved that the sites of binding of penicillin forkilling and for induction of penicillinase areclosely related. Induction of penicillinase couldbe the consequence of a defect in regulation oftranspeptidase synthesis .

In order to test the hypothesis, the nature ofthe glycopeptide formed by cells growing innormal culture medium in the presence of peni-cillin was examined [53] . Untreated cells con-tained a small amount of a low molecularweight glycopeptide fragment . The amount ofthis fragment strikingly increased in the pres-ence of penicillin (Fig . 11), and the amount offully cross-linked glycopeptide synthesized de-creased. In fact, 51 per cent of the new glyco-peptide units formed in the presence of penicillinwere the small fragments, whereas in untreated


Bacterial Cell Wall Synthesis and Structure-Slrm

cells only 7 per cent were in this form . Analysesindicate that the material found in small amountin normal cells is identical with that which in-creases in amount in the penicillin-treated cells.Both appear to be acetylglucosaminyl-acetyl-auuramyl-pentapeptide-pcntaglycinc, i .e ., thenascent uncross-linked glycopeptide unit . Ex-periments similar to these had been carried outby Martin [58,55] who observed that the gly-copeptide synthesized by the pennicillin-inducedspheroplasts of Proteus mirabilis was composedlargely of low cross-linked glycopeptide, incontrast to the polymeric glycopeptide of un-treated cells . Based on these observations,Martin had suggested that penicillin was insome manner interfering with an unknown cross-linking reaction . A similar hypothesis has beenproposed recently by Wise and Park [56] but itdiffers in important details from that here de-scribed .

The hypothesis explains the requirement of afree carboxyl group and a substituted 6-aminogroup for penicillin action, as well as the fact thatalteration of the thiazolidine ring (as in cephalo-sperins) has no effect on activity as long as theposition of the free carboxyl group is retained .However, the presence of the 0-lactam ringappears to he essential for activity . The presentstudies suggest additional modifications of peni-cillin which might result in even more effectiveantibacterial agents . In this hypothesis acylsubstitutents (phenylacetic acid in penicillin G)on 6-aminopenicillanic acid and 7-amino-ceph-alosporamic acid may be regarded as analogsof the amino acids preceding n-alanyl-n-alanine in the acetylmuramyl-pentapeptide.Thus, addition of lysine, substituted lysines,diaminopimelic acid, derivatives of diatnino-pimelic acid or other dibasic amino acids orsmall peptides related to the cell wall tetra-peptides (as, for example, L-alanyl-n-isoglut-aminyl-L-Iysyl-, or D-lactyl-L-alanyl-o-isoglut-aminyl-L-lysyl-, or phenylacetyl-L-alanyl-n-iso-glutaminyl-L-lysyl-, or L-alanyl-n-isoglutaminyl-(N`-pentaglycyl)L lysyl- or other N-acyl de-rivatives of these) should result in an evencloser resemblance to the glycopeptide sub-strate. Similarly a 6-methyl penicillin or 7-methyl cephalosporin would bear a methylgroup in the same position as is found in the n-alanyl residue and this might enhance itseffectiveness as an antibacterial agent .

Verification of the hypothesis and detailedstudies of the mechanisms depend upon the

VOL . 39, NovEMnsn 1965

ger, Ttt'per

UNTREATED CELLS

-y-~-- PENICILLIN TREATED CELLS

719

Fta. 11. Accumulation of low cross-linked fragments inthe cell wall of Staph . aureus on treatment with peni-cillin [53] . After logarithrnic growth was established,penicillin (0 .15 pg . per ml.) was added to one culture,C'4 -glycine was added to this and the control 30 minuteslater. The bacteria were harvested after an additional20 minutes . The cultures still had parallel growth curvesat this time. The cell walls were isolated and solubilizedby treatment with the Chalaropsis B enzyme . After re-moval of the teichoic acid-glycopeptide complex by ad-sorption on a column of ECTEOLA-cellulose, the glyco-peptide was fractionated on a column of Sephadex G-25 .The highly cross-linked glycopeptide is eluted at theleft (void volume of column, 21 nd .) and the low cross-linked fragment at the right (salts are eluted at 52 ml .) .The distribution of glycopeptide components (hexos-amines and amino acids) was also measured and wasvirtually identical to counts per minute incorporated .

isolationn of an active transpeptidase . This goalis certainly now within reach, and it may behoped that the twenty-year search for themechanism of action of penicillin may be near-ing its conclusion .

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~,

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Documents

Bacterial cell wall synthesis and structure in relation to the mechanism of action of penicillins and other antibacterial agents