JOURNAL OF BACTERIOLOGY, JUIY 1985, p. 69-74 Vol. 163, No. 10021-9193/85/070069-06$02.00/0Copyright © 1985, American Society for Microbiology

Analysis of Neisseria gonorrhoeae Peptidoglycan by Reverse-Phase,High-Pressure Liquid Chromatography

THOMAS J. DOUGHERTYLaboratory of Microbiology, The Rockefeller University, New York, New York 10021

Received 13 February 1985/Accepted 2 April 1985

The muramidase digest of peptidoglycan from Neisseria gonorrhoeae was isolated and analyzed by the use ofa reverse-phase, high-pressure liquid chromatography system. As was found previously in the case ofEscherichia coli, gonococci peptidoglycan is also composed of a greater number of muropeptides than can beresolved with thin-layer chromatography systems. Preliminary classification of the muropeptide componentsinto subclasses based on O-acetyl modification and degree of cross-linkage was achieved. Examination of apenicillin-susceptible strain and a highly resistant strain with two penicillin-binding protein alterationssynthesized distinctly different peptidoglycan structures, as revealed by this technique.

All gram-negative bacteria studied possess one chemicaltype of peptidoglycan, Aly, and variations have been be-lieved to be limited to degree of muropeptide cross-linkage,degree of amidation of the carboxyl groups of the peptidesubunit, and O-acetylation of the disaccharide (15, 21).Recent investigations by Glauner and Schwarz, who usedthe extreme resolving power of reverse-phase, high-pressureliquid chromatography (HPLC), established that Escherichiacoli peptidoglycan has a much greater heterogeneity inmuropeptide structure than previously thought (5). Varia-tions include substitution of amino acids, such as glycine foralanine, and direct cross-links between diaminopimelic acidresidues at the site of lipoprotein attachment. These findingsraise the possibility of specialized domains within the pepti-doglycan structure and roles for the multiple penicillin-bind-ing proteins (PBPs) in assembly of these regions. In anotherrecent study, this technology was used to characterizeseveral modifications that the peptidoglycan of E. coli under-goes during transitions in growth states (11).

In the present study, the reverse-phase HPLC system wasused for examination of the peptidoglycan structure ofNeisseria gonorrhoeae. A complex, reproducible pattern ofmuramidase-digested peptidoglycan fragments was ob-tained. The fragments were classified into groups by thedegree of peptide cross-linkage and O-acetylation, and pre-liminary identification of some species was assigned. Pepti-doglycan from a highly penicillin-resistant strain with re-duced binding affinity in two of its PBPs was examined.Several differences in the peptidoglycan digest from penicil-lin-susceptible and -resistant strains of gonococci were evi-dent.

MATERIALS AND METHODS

Strains and growth conditions. N. gonorrhoeae FA19 wasobtained from P. F. Sparling, University of North Carolina,Chapel Hill, N.C., and strain CDC 77-124615 was from theCenters for Disease Control, Atlanta, Ga. These strains havebeen extensively characterized in previous studies (2, 3). Aseries of non-beta-lactamase-producing, high-level penicil-lin-resistant strains was from J. Biddle, Centers for DiseaseControl. Several penicillin-susceptible isolates were ob-tained from the clinical laboratory at the New York Hospital-Cornell Medical Center, New York, N.Y. Strains were keptfrozen in gonococcal broth with 15% glycerol at -70°C.

Stocks were thawed, and cultures were maintained by dailypassage on GCBA agar plates with 1% IsoVitaleX supple-ment (BBL Microbiology Systems, Cockeysville, Md.). In-cubation was at 37°C in 5% CO2. Colonies were p-, transpar-ent by the criteria of Swanson (19).

Peptidoglycan preparation. Gonococci were inoculatedinto 500-ml volumes of gonococcal broth with 1% IsoVitaleXand 420 ,ug of NaHCO3 per ml. These cultures, in 2-literflasks, were grown at 37°C with agitation (140 rpm) in a NewBrunswick G-25 shaker (New Brunswick Scientific Co.,Inc., Edison, N.J.). Growth was monitored at frequentintervals with a Sequoia-Turner model 340 spectro-photometer at 610 nm. At absorbance values of 0.6 to 0.7(ca. 246 mg/liter [dry weight], 5 x 108 CFU/ml), culturevessels were swirled in ice-alcohol baths with temperaturemonitoring. Culture temperatures dropped from 37 to 2°C inless than 5 min. This was essential to avoid autolysisartifacts.

Cells were collected by centrifugation (Sorvall RC-2B,DuPont-Sorvall, Wilmington, Del.) at 8,000 x g for 10 min at2°C. The cells were washed once with ice-cold 20 mMsodium acetate (pH 5.0) and then were resuspended in 15 mlof the same buffer on ice. The cells were added dropwise to15 ml of 10% sodium dodecyl sulfate (SDS) buffered with 20mM acetate (pH 5.0) at 100°C. After all of the cells wereadded (ca. 5 min), the SDS extraction at 100°C was contin-ued for 20 min.

After cooling, the SDS-insoluble material was collected bycentrifugation (Beckman L5-50, Beckman Instruments, Inc.,Palo Alto, Calif.) at 80,000 x g for 30 min. The material wasthen washed by centrifugation three times with glass-distilled water. The SDS-insoluble material was treated for 1h with 200 ,ug of a-amylase (Sigma Chemical Co., St. Louis,Mo.) and then with 200 ,ug of pronase (Pronase-CB, Calbio-chem-Behring, La Jolla, Calif., preheated for 1 h at 60°C).Both incubations were in 20 mM Tris-chloride (pH 7.4) and10 mM NaCl at 24°C. After 2 h of pronase treatment, theSDS-insoluble fraction was reextracted with 0.1% SDS at100°C for 5 min. The material was collected and washed bycentrifugation, as described above, four times. After allwashes, residual fluid was carefully removed with a Pasteurpipette, leaving a clear, gelatinous pellet. The peptidoglycanpellet was resuspended in distilled water by brief (5-s)treatment with a low-power sonicator (Kontes Micro Dis-ruptor, Kontes Co., Vineland, N.J.). The final pellet was

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MINFIG. 1. (A) Separation of radiolabeled gonococcal peptidoglycan digest by thin-layer chromatography in isobutyric acid-ammonia system

(2). Detection was by fluorography. Components: 1, mono-O-acetylated disaccharide monomer; 2, disaccharide monomer; 3, di-O-acetylatedbis-disaccharide dimer; 4, mono-O-acetylated bis-disaccharide dimer; 5, bis-disaccharide dimer; 6, oligomers. (B) Reverse-phase HPLCpattern of strain FA19 gonococcal peptidoglycan. The salt peak contained both the borate buffer and the muramidase used in the digestion.

suspended in 200 ,ul of 25 mM sodium phosphate buffer (pH6.5) with 0.1 mM MgCl2. Streptomyces globisporus muram-idase (Miles Scientific, Naperville, Ill.) was added to aconcentration of 25 ,ug/200 RI1 (ca. 55 U). Sodium azide wasadded (0.05%), and the material was incubated at 37°Cfor 18 h.

Before analysis, the digested peptidoglycan was reducedwith sodium borohydride (5) to prevent anomerization at theCl position. The digested peptidoglycan was mixed with anequal volume of 0.5 M borate buffer (pH 8.0) in whichNaBH4 (10 mg/ml) had been freshly dissolved. After exactly20 min, the reaction was terminated by the addition ofphosphoric acid, and the reaction mixture was brought to afinal pH of ca. 4.0 and filtered (0.2-,um pore size filter).Because of the effect of high pH on O-acetyl groups, theO-acetyl data were not considered quantitative.Reverse-phase HPLC analysis. The HPLC system con-

sisted of two Waters 510 pumps (Waters Associates, Inc.,Milford, Mass.), a Waters 721 system controller, a WatersU6K injector, and a Waters 730 Data Module that integratedthe areas under the curves and performed other documenta-tion functions. Detection of the peptide side chains on thepeptidoglycan was at 205 nm in an Isco V4 detector (Isco,Lincoln, Nebr.) equipped with a heat exchanger flow cellwith a 10-mm path length and 7-,u volume.The column was a Shandon ODS-Hypersil (4.6 by 250

mm; 5 p.m particles; Shandon Southern Instruments Inc.,Sewickley, Pa.). The analysis consisted of a linear gradientfrom 50 mM phosphate (pH 4.33) to 50 mM phosphate buffer(pH 5.10) with 15% methanol over 120 min at a flow rate of0.5 ml/min. Final conditions were held for an additional Smin. HPLC grade reagents were from Burdick & JacksonLaboratories Inc. (Muskegon, Mich.). Sodium hydroxide(solid) and orthophosphoric acid were analar grade fromBDH Chemicals, Ltd. (Poole, England). Determinations ofbuffer pH values, which were critical, were made on anOrion Research model 701 A digital ionalyzer with an Orion

research grade electrode (Orion Research Inc., Cambridge,Mass.). Buffers for pH standardization were from Corning(Corning Science Products, Medfield, Mass.) and were ref-erenced to National Bureau of Standards standards at + 0.01pH unit.

Peptidoglycan fractionation. Peptidoglycan prepared froma 500-ml culture of FA19S was mixed with 3H-labeledpeptidoglycan (300,000 cpm) and digested with muramidaseas described above. The material was loaded onto coupledSephadex G-50-G-25 columns (1.8 by 70 cm each;Pharmacia Fine Chemicals, Inc., Piscataway, N.J.) andeluted with 0.1 M LiCl (4). Fractions of 1.2 ml volume werecollected, and 100-,ul samples were counted in Ultrafluorscintillation fluid (National Diagnostics, Inc., Somerville,N.J.). Each set of peak fractions was pooled, taken todryness on a rotary evaporator, and suspended in 1 ml ofwater. The individual peaks were desalted on a SephadexG-10 column (1.8 by 100 cm) eluted with distilled water. Thematerial was concentrated again, reduced with borohydrideas described above, and injected into the HPLC system.The 1-6 anhydro muramyl peptide was prepared by R.

Rosenthal, Indiana University School of Medicine, Indian-apolis, Ind. An E. coli anhydromuramidase preparation wasused to digest the gonococcal peptidoglycan, and theanhydro muramyl monomer was isolated.Amino acid analysis. Peptidoglycan was hydrolyzed for 4

and 18 h in 5.8 N HCl at 110°C in vacuo. The use of twohydrolysis times allowed estimation of the degree ofmuramic acid and glucosamine breakdown under these con-ditions. Samples were analyzed on a Durrum analyzer,model D-500.

RESULTSBasic characteristics of gonococcal peptidoglycan. Gono-

coccal peptidoglycan from strain FA19 was prepared asdescribed above, and a portion was subjected to amino acidanalysis after acid hydrolysis. A composition identical to

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HPLC ANALYSIS OF GONOCOCCAL PEPTIDOGLYCAN 71

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FRACT ION (1.2 ml)FIG. 2. Separation of muramidase-digested gonococcal peptidoglycan on coupled Sephadex G-50 and G-25 columns. Peak fractions: 1,

tetramer; 2, trimer; 3, dimer; and 4, monomer. For the subsequent analysis by HPLC, tetramer and trimer peaks were combined beforedesalting on Sephadex G-10. Peak identities were confirmed by thin-layer chromatography with known standards. Void and total columnvolumes for the coupled columns are indicated.

that previously reported by others was obtained, includingsmall amounts of glycine and aspartic acid present (9, 14,22). The remaining portion was digested with S. globisporusmuramidase. After reduction with borohydride, the materialwas adjusted to pH 4.0, and 50 to 100 ,ug of peptidoglycanfragments was injected into the reverse-phase HPLC sys-tem. The resulting chromatograph, and an analysis of pepti-doglycan by thin-layer chromatography (2), are shown inFig. 1. Several notable features are evident. The thin-layersystem sorts components solely on the basis of cross-linkageand O-acetylation. There are by reverse-phase HPLC analy-sis ca. 13 major species of muropeptide that comprise about70% of the total peptidoglycan. The 40 to 45 minor peaksmake up the remaining 30% of the material.During the early stages of this work, several peaks were

noted to be metastable. This instability could be attributed totwo sources, namely, the borohydride reduction at high pH,at which the O-acetyl groups can be removed from thedisaccharide units (1), and the highly active system ofpeptidoglycan hydrolases present in the gonococcus (6, 12,16). Standardization of the reduction time to exactly 20 minfor each sample greatly improved the reproducibility of thesystem. Care must also be exercised during the initial cellharvesting, when rapid cooling is essential.For a preliminary assessment of the structural features of

the muropeptides, a preparation of strain FA19 peptidogly-can was mixed with 0.2 ,uCi of [3HJglucosamine-labeledFA19 peptidoglycan (2) and digested with muramidase. Thedigest was chromatographed on coupled Sephadex G-50-G-25 columns in 0.1 M LiCl. The peaks, detected by radio-activity, were identified as disaccharide peptide monomers,bis-disaccharide peptide dimers, and higher oligomers (Fig.2). After concentration and desalting, these peaks were

injected separately into the reverse-phase HPLC system(Fig. 3). The elution order was disaccharide monomers (15 to65 min), bis-disaccharide dimers (55 to 95 min), and thehigher oligomers (80 to 120 min). Two of the disaccharidemonomers had retention times identical to those of E. colidisaccharide tripeptide (22 min) and disaccharide tet-rapeptide (35 min). Interestingly, the distribution of thesetwo components was ca. 25% tripeptide and 75% tet-rapeptide, which agrees well with previously reported valuesfor gonococci (14). In the bis-disaccharide class, only E. colibis-disaccharide connected by two tetrapeptide side chainswas unambiguously identified with a gonococcal component(82 min) of identical retention time. In the oligomers, theTris-disaccharide with three tetrapeptides was tentativelyfound at 101 min.Treatment of O-acetylated peptidoglycan under basic con-

ditions (NH40H, pH 10 for 6 h) removes the majority of theO-acetyl groups from the disaccharide residues (1). Thisallowed assignment of the O-acetylated residues in themonomer, dimer, and oligomer classes (Fig. 3). A smallnumber of additional minor peaks that eluted after the saltpeak were noted after the treatment with base; these are thebeta-elimination products (20). Gonococcal peptidoglycanwas treated with 1-6 anhydro muramidase isolated from E.coli and fractionated to obtain the 1-6 anhydro peptidoglycanmonomer. The main product peak (Fig. 3) eluted much laterthan the other monomeric products.

Analysis of peptidoglycan from a gonococcal strain withhigh-level penicillin resistance. Peptidoglycan was preparedfrom FA19, a penicillin-susceptible strain (MIC, 0.01 ,ug/ml),and CDC 77-124615, a highly resistant (MIC, 2.0 ,ug/ml)gonococcus. These two strains differ in that two of the threePBPs in the resistant strain have a much-reduced affinity for

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30 60 90 120MIN

FIG. 3. Analysis of peptidoglycan digest fractions from Sephadex columns by HPLC. (A) HPLC pattern of disaccharide monomers; (B)HPLC pattern of bis-disaccharide dimers; (C) HPLC pattern of combined trimers and tetramers. Tentative identifications of the O-acetylatedmuropeptides, made in a separate experiment by treatment with base, are indicated by solid arrows. Open arrow indicates the position of the1-6 anhydro monomer generated by treatment of gonococcal peptidoglycan with the E. coli anhydromuramidase. Tentative identifications ofdisaccharide tripeptide (a), disaccharide tetrapeptide (b), bis-disaccharide tetrapeptide, tetrapeptide (c), and Tris-disaccharide tetrapeptide,tetrapeptide, tetrapeptide (d) are indicated.

penicillin (3). The PBPs are involved in the terminal stages ofpeptidoglycan assembly, so it was of interest to examine thestructural details of peptidoglycan from these organisms.Figure 4 shows the peptidoglycan digest reverse-phaseHPLC patterns of these two gonococcal strains. A numberof differences, indicated in the figure by arrows, are evident.

Most of these changes appear to be either amplification ordiminution of preexisting muropeptides.

DISCUSSIONAnalysis of the peptidoglycan structure of N. gonorrhoeae

indicated that, similar to the situation in E. coli (5), the

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HPLC ANALYSIS OF GONOCOCCAL PEPTIDOGLYCAN 73

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MINFIG. 4. HPLC pattern of peptidoglycan digest from a penicillin-susceptible strain and highly penicillin-resistant strains of N. gonorrhoeae.

Peptidoglycan was prepared and muramidase digested from (A) FA19 (MIC, 0.01 ,ug of penicillin per ml) and (B) CDC 77-124615 (MIC, 2.0,ug of penicillin per ml). Areas of major difference between the strains are indicated by arrows.

muramidase digestion products present in gonococci aremore numerous than previously believed (Fig. 1). In thepresent study, several techniques were used to obtain apreliminary classification of the muramidase-treated pepti-doglycan products into subgroups. In general terms, disac-charide peptide monomers have shorter retention times thanbis-disaccharide peptide dimers, which in turn are lessretarded than the oligomers. There is considerable overlapamong these classes (Fig. 3). Another point evident in Fig. 3is that the number of major monomeric species is very highcompared with that of the dimer and oligomeric digestionproducts. One might expect that if bis-disaccharide peptidedimers were composed of any possible combination ofdisaccharide monomers, then there should be a much largerrepertoire of the dimer form. The fact that this is not evident(nor is it found in the trimer case) and only a few majorspecies predominate may indicate that the transpeptidasesystem responsible for cross-linking may have tight re-

straints on donor-acceptor structure. It remains to be seenwhether the large number of minor peaks in Fig. 3B mayrepresent rare cases of these possible combinations.

Isolation of the component peaks in quantities sufficientfor further chemical characterization is under way. It isanticipated from data in the literature that some of the peaksmay be due to variations in the amino acid composition ofthe peptide side chains. There are several reports fromdifferent groups of the presence of glycine and aspartic acidin gonococcal peptidoglycan (9, 14, 22). In addition, in thebetter-characterized E. coli system, amino acid substitutionsin the side chain account for a proportion of the speciesobserved (5). In E. coli, the diaminopimelyl-diaminopimelicacid cross-links serve as sites for lipoprotein attachment (5).No protein with a structure analogous to that of lipoproteinhas been reported in gonococci; however, growth of severalgonococcal strains at low pH causes increased amounts of aprotein that is apparently covalently attached to the pepti-

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doglycan (7). There is a strain, CS-7, that produces largeamounts of this peptidoglycan-associated protein (8). Stud-ies on this strain are currently in progress.The observations with the penicillin-susceptible and

-resistant strains of N. gonorrhoeae are of great interest.These two strains have been extensively characterized, andthe decreased penicillin-binding affinity of two of the threePBPs in the resistant strain has been documented (3).Penicillin binds to the active site of the PBPs, and the changein penicillin affinity presumably reflects a mutational modi-fication of these enzymes (17, 18). The notable differences inthe peptidoglycan synthesized, particularly in the cross-linked muropeptide species, suggest that the PBP alterationsaffect the assembly of this macromolecule. Most of thechanges appear to be in the relative concentrations ofpreexisting muropeptide peaks, and this may reflect a shift inprecursor utilization by the modified PBPs. It should beemphasized that this peptidoglycan is the product of loga-rithmically growing cultures of these two strains, neither ofwhich had penicillin present during growth. An additionalpair of susceptible and resistant strains with virtually identi-cal penicillin MICs yielded the same reverse-phase HPLCpatterns (not shown).With the high-resolution system developed by Glauner

and Schwarz (5) in E. coli, gonococci have been shown topossess remarkable heterogeneity in their peptidoglycanconstituents. There are several points of significance to thesefindings. The change in peptidoglycan structure observed inhighly penicillin-resistant strains indicates that a change inthe penicillin target enzymes is not without consequence tothe structure of the gonococcal cell envelope. Anotheraspect of gonococcal biology in which the diverse peptido-glycan structures may be of importance is host interactions.Gonococci exhibit substantial cell wall turnover, and releaseof gonococcal peptidoglycan fragments has been implicatedin several pathological host responses (10, 13). The possibil-ity of variations in host effects by different peptidoglycansubunits must be entertained.

In addition to chemical characterization of the peptidogly-can subunits, studies of the effects of beta-lactam antibioticson the sacculus structure are planned. These investigationsare expected to further delineate the process of cell surfaceassembly in gonococci.

ACKNOWLEDGMENTSI thank Bernd Glauner, Joachim-V. Holtje, and Uli Schwarz for

their generosity and hospitality during my visit to the Max-Planck-Institut fur Virusforschung, Tubingen, Federal Republic of Ger-many. I also thank R. Rosenthal for the anhydro muramyl peptidemonomer compound, and Margaret Geller for help in manuscriptpreparation.

This work was supported by Public Health Service grants Al20020 from the National Institute of Allergy and Infectious Diseasesand BSRG S07 RR 07065 from the National Institutes of Health.

LITERATURE CITEI)1. Blundell, J. K., G. J. Smith, and R. H. Perkins. 1980. The

peptidoglycan of Neisseria gonorrhoeae: O-acetyl groups andlysozyme sensitivity. FEMS Microbiol. Lett. 9:259-261.

2. Dougherty, T. J. 1983. Peptidoglycan biosynthesis in Neisseriagonorrhoeae strains sensitive and intrinsically resistant to 13-lactam antibiotics. J. Bacteriol. 153:429-435.

3. Dougherty, T. J., A. E. Koller, and A. Tomasz. 1980. Penicillin-binding proteins of penicillin-susceptible and intrinsically resist-ant Neisseria gonorrhoeae. Antimicrob. Agents Chemother.

18:730-737.4. Ghuysen, J. M., E. Bricas, M. Lache, and M. Leyh-Bouille. 1968.

Structure of the cell walls of Micrococcus lysodeikticus. III.Isolation of a new peptide dimer, N-(L-alanyl-y-(cx-D-glutamyl-glycine)) - L - lysyl - D - alanyl - N(L - alanyl - 'y - (a - D - glutamyl-glycine))L-jysyl-D-alanine. Biochemistry 7:1450-1460.

5. Glauner, B., and U. Schwarz. 1983. The analysis of mureincomposition with high pressure liquid chromatography, p.29-34. In R. Hakenbeck, J.-V. Holtje, and H. Labischinski(ed.), The target of penicillin. W. de Gruyter, Berlin.

6. Goodell, E. W., M. Fazio, and A. Tomasz. 1978. Effect ofbenzylpenicillin on the synthesis and structure of the cellenvelope of Neisseria gonorrhoeae. Antimicrob. Agents Che-mother. 13:514-526.

7. Hebeler, B. H., S. A. Morse, W. Wong, and F. E. Young. 1978.Effect of pH on the chemical and biological properties of the cellenvelope of Neisseria gonorrhoeae: isolation of a protein-peptidoglycan complex, p. 18-25. In G. F. Brooks, E. C.Gotschlich, K. K. Holmes, W. D. Sawyer, and F. E. Young(ed.), Immunobiology of Neisseria gonorrhoeae. American So-ciety for Microbiology, Washington, D.C.

8. Hebeler, B. H., W. Wong, S. A. Morse, and F. E. Young. 1979.Cell envelope of Neisseria gonorrhoeae CS7: peptidoglycan-protein complex. Infect. Immun. 23:353-359.

9. Hebeler, B. H., and F. E. Young. 1976. Chemical compositionand turnover of peptidoglycan in Neisseria gonorrhoeae. J.Bacteriol. 126:1180-1185.

10. Petersen, B. H., and R. S. Rosenthal. 1982. Complement con-sumption by gonococcal peptidoglycan. Infect. Immun. 35:442-448.

11. Pisabarro, A. G., M. A. de Pedro, and D. Vazque'z. 1985.Structural modifications in the peptidoglycan of Escherichia coliassociated with changes in the state of growth of the culture. J.Bacteriol. 161:238-242.

12. Rosenthal, R. S. 1979. Release of soluble peptidoglycan fromgrowing gonococci: hexaminidase and amidase activities. In-fect. Immun. 24:869-878.

13. Rosenthal, R. S., W. J. Folkening, D. R. Miller, and S. C. Swim.1983. Resistance of O-acetylated gonococcal peptidoglycan tohuman peptidoglycan-degrading enzymes. Infect. Immun.40:903-911.

14. Rosenthal, R. S., R. M. Wright, and R. K. Sinha. 1980. Extentof peptide cross-linking in the peptidoglycan of Neisseria gonor-rhoeae. Infect. Immun. 28:867-875.

15. Schleifer, K. H., and 0. Kandler. 1972. Peptidoglycan types ofbacterial cell walls and their taxonomic implications. Bacteriol.Rev. 36:407-477.

16. Sinha, R. K., and R. S. Rosenthal. 1980. Release of solublepeptidoglycan from growing gonococci: demonstration of an-

hydro-muramyl-containing fragments. Infect. Immun. 29:914-925.

17. Spratt, B. G. 1978. Escherichia coli resistance to beta-lactamantibiotics through a decrease in the affinity of a target forlethality. Nature (London) 274:713-715.

18. Spratt, B. G. 1983. Penicillin binding proteins and the future ofbeta-lactam antibiotics. J. Gen. Microbiol. 129:1247-1260.

19. SWanson, J. 1978. Studies on gonococcus infection. XII. Colonycolor and opacity variants of gonococci. Infect. Immun.19:320-331.

20. Tipper, D. J. 1968. Alkali-catalyzed elimination of D-lactic acidfrom muramic acid and its derivatives and the determination ofmuramic acid. Biochemistry 7:1441-1449.

21. Tipper, D. J., and A. Wright. 1979. The structure and biosyn-thesis of bacterial cell walls, p. 291-426. In J. R. Sokatch andL. N. Ornston (ed.), The bacteria, vol. 7. Academic Press, Inc.,New York.

22. Wolf-Watz, H., T. Elmros, S. Normark, and G. D. Bloom. 1975.Cell envelope of Neisseria gonorrhoeae: outer membrane andpeptidoglycan composition of penicillin-sensitive and -resistantstrains. Infect. Immun. 11:1332-1341.

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