Mechanism of Action - Antimicrobial Agents and Chemotherapy

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Sept. 1980, p. 416-423 0066-4804/80/09-0416/08$02.00/0

Vol. 18, No. 3

Pyocin Inhibition of Neisseria gonorrhoeae: Mechanism of Action STEPHEN A. MORSE,* BENJAMIN V. JONES, AND PAUL G. LYSKO Department ofMicrobiology and Immunology, University of Oregon Health Sciences Center, Portland, Oregon 97201

Purified R-type pyocins (611 131) from Pseudomonas aeruginosa PA103 exhibited bactericidal activity against Neisseriagonorrhoeae. Killing of gonococci was a single-hit process requiring as few as 1 pyocin per colony-forming unit. Deoxyribonucleic acid, ribonucleic acid, protein, and lipid syntheses were rapidly and completely inhibited. Oxygen uptake was also inhibited, but occurred after the inhibition of macromolecular synthesis. The cell lysis which occurred after pyocin inhibition of gonococcal growth was the result of endogenous gonococcal autolysin activity.

Bacteriocins are antibacterial substances produced by many species of bacteria, usually against strains of the same or closely related species (29). Bacteriocins are protein in nature and range from simple proteins to particles resembling bacteriophage components (19). Bacteriocinogenic strains of Pseudomonas aeruginosa produce several types of bacteriocins (pyocins) (11): the S-type pyocins (2), which are characterized by sensitivity to proteolytic enzymes and a lack of structure in electron microscopy, and the R-type (2) and flexuous (20) pyocins, which are not sensitive to proteolytic enzymes and resemble bacteriophage components. Geizer (7) reported the inhibition of gonococcal growth by unidentified substance(s) produced by P. aeruginosa. Subsequently, we have shown that the R-type pyocin, 611 131, was responsible for growth inhibition of Neisseria gonorrhoeae by culture supernatants of P. aeruginosa PA-103 (27). Similar inhibition of gonococcal growth by R-type pyocins was reported by Sidberry and Sadoff (33) and by Blackwell et al. (1). Kageyama et al. (16) identified a muramidaselike enzyme as a component of R-type pyocins. This enzyme could account for the rapid lysis of gonococcal cultures that were exposed to high concentrations of pyocin (27). However, other effects of pyocins, such as inhibition of macromolecular synthesis or other cell functions, may lead indirectly to cell lysis. This study examines the mechanism of pyocin-induced killing of N. gonorrhoeae. MATERIALS AND METHODS

aeruginosa strain PA 103 (ATCC 29260) was obtained from P. V. Liu (University of Louisville, Louisville, Ky.). Medium. Gonococci were grown in a chemically defined medium as previously described (24). Viable counts of N. gonorrhoeae (colony-forming units [CFU] per milliliter) were determined on GC agar (Difco) plates containing glucose (5 g/liter) and a growth factor supplement (22). Growth conditions. Plates of GC agar were inoculated with N. gonorrhoeae and incubated for 16 to 18 h in a C02 incubator (4% C02) at 37°C. Cells were suspended, washed once, and resuspended to ca. 5 x 109 CFU/ml in a 20 mM potassium phosphate buffer, pH 7.4. Nepheloflasks (300-ml flasks containing 50 ml of defined medium) were inoculated with a sufficient number of bacteria to achieve an initial turbidity of ca. 15 Klett units (3.5 x 107 CFU/ml) as measured with a Klett-Summerson colorimeter at 540 nm (no. 54 filter). All liquid cultures were incubated at 37°C in a gyratory shaker (New Brunswick Scientific Co., New Brunswick, N.J.). Induction and purification of pyocin 611 131. Pyocin 611 131 was induced in cultures of P. aeruginosa 103 with mitomycin C and purified by (NH4)2SO4 precipitation and diethylaminoethyl (DEAE)-cellulose column chromatography as previously described

(27).

Assay of pyocin activity. Organisms to be tested for susceptibility to pyocin 611 131 were grown overnight on GC agar plates. A suspension of these organisms was prepared in a diluent consisting of 0.85% NaCl and 0.1% cysteine-hydrochloride (pH 6.4) and adjusted to a Klett reading of 50 to 60. GC agar plates were inoculated by means of a swab dipped into the cell suspension. Undiluted or serially diluted pyocin preparations (5 ,ul) were applied to the surface of the agar plate. All plates were incubated overnight at 37°C with increased CO2 (4% C02) before being examined for pyocin inhibition of growth. Pyocin titers were expressed as 200 times the highest dilution that showed complete inhibition. The procedure of Kageyama et al. (16) was used to calculate the number of active pyocin particles. The values thus obtained were

Organisms. N. gonorrhoeae strains JW-31 (Arg-), JW-31R (Arg-), (a pyocin 611 131-resistant mutant), and CS-7 (Glu-) were used in these studies. Strains were maintained as previously described (23). P. 416

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PYOCIN INHIBITION OF N. GONORRHOEAE

used as a basis for calculating the multiplicity of infection (MOI). Cell fractionation, incorporation, and release of labeled precursors. Cell constituents were fractionated chemically by a modification (26) of the procedure described by Roberts et al. (30). The total incorporation of labeled precursors into the trichloroacetic acid-insoluble cell fraction was determined as previously described (21). Pyocins (MOI = 10/CFU) were added to mid-log phase cultures 2 to 3 min after the addition of the labeled precursor. Samples (1.0 ml) were removed, added to an equal volume of 10% (wt/vol) trichloroacetic acid, kept in ice for several hours, and filtered onto 0.45-,tm filters (Millipore Corp.). The release of incorporated hypoxanthine (nucleic acid) was measured after the growth of gonococci for two generations (generation time, 75 min) in defined medium containing 0.4 yCi of ['4C]hypoxanthine per ml. Cells were centrifuged, washed, diluted 1:4 in fresh defined medium, and placed into two flasks. When the turbidity reached 80 Klett units, pyocins (MOI = 10/ CFU) were added to one flask. Samples (1 ml) were removed at various intervals before and after pyocin addition and centrifuged for 5 min (Microfuge B, Beckman Instruments). The supernatant (0.5 ml) was added to an equal volume of 10% (wt/vol) trichloroacetic acid and kept on ice for several hours. The trichloroacetic acid-insoluble material was collected by filtration as previously described. Oxygen uptake. Oxygen uptake was measured with a Yellow Springs Instrument Co. model 53 oxygen monitor attached to a circulating water bath adjusted to 37°C. Exponential-phase cultures of gonococci were diluted with fresh defined medium, sparged, and added to the electrode chambers at a cell density of ca. 2.0 x 108 cells in a volume of 4 ml. The number of cells was estimated with a Petroff-Hauser counting chamber. Diplococci were counted as two cells. The defined medium used for dilution contained 5 mM glucose. When the rate of 02 uptake was linear, pyocins (MOI = 10) were added to one of the chambers. Pyocin inhibition of oxygen uptake was the ratio (in percent) of the rates observed in the presence and absence of pyocins. In some experiments, complex medium similar to that described previously (23) containing 5 mM glucose but lacking IsoVitaleX enrichment (BBL Microbiology Systems) was used as the diluent. Radioisotopes and cheiiicals. The following radioisotopes were obtained from the New England Nuclear Corp., Boston, Mass.: [8-'4C]hypoxanthine (specifilc activity, 42.4 mCi/mmol); [8-3H]adenine (specific activity, 18.9 Ci/mmol); [6-3H]uracil (specific activity, 30 Ci/mmol); and L-[ring-2,6-3H]tyrosine (specific activity, 42.9 Ci/mmol). [3H]acetic acid, sodium salt (specific activity, 1.5 Ci/mmol) was obtained from ICN, Irvine, Calif. All chemicals were of reagent grade and purchased from Sigma Chemical Co., St. Louis, Mo. Autolysis and leakage of labeled adenine. Autolysis was measured as the decrease in turbidity at 540 nm as previously described (35). To measure the leakage of [3H]adenine-labeled material, gonococci were grown for two generations in basal medium containing 2 ,uCi of [8-3H]adenine per ml of medium. Cells were harvested, resuspended in fresh medium lacking

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[8-3H]adenine, and incubated for 20 min at 37°C. Cultures were then centrifuged, and the cell pellet was resuspended in 50 mM HEPES (N-2-hydroxyethylpiperazine-N'-ethanesulfonic acid) buffer (pH 8.5). The cell suspensions were incubated at 37'C, and at intervals 5-ml aliquots were removed and centrifuged (4,100 x g, 5 min), and the supernatants were placed at 4°C. Total radioactivity was determined from 0.2-ml aliquots of the cell suspensions. To measure the activity associated with extracellular nucleic acid, triplicate 1ml aliquots of the supernatants were added to tubes containing 1 ml 20% trichloroacetic acid, incubated at 4°C for 30 to 60 min, and filtered onto Whatman GF/ C filter disks. Filters were processed and radioactivity was determined as previously described (35).

RESULTS Effect of pyocins on the growth of N. gonorrhoeae JW-31. Previous studies (27) showed that pyocin 611 131 inhibited the growth of N. gonorrhoeae in a complex medium. However, it was possible that unidentified components (e.g., long-chain fatty acids) in this medium acted synergistically with the pyocins. Therefore, various amounts of diethylaminoethyl-cellulose-purified pyocin 611 131 were added to mid-logarithmic-phase cultures of strain JW-31 growing in defined medium. Figure 1 shows that the addition of pyocins produced a rapid, concentration-dependent inhibition of growth. At lower concentrations of pyocins, turbidity increased after several hours, suggesting the growth of resistent cells in the population. These cells were inoculated into solid medium and subsequently were shown to be susceptible to pyocin 611 131. Thus, pyocins were the limiting factor at low concentrations. l

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FIG. 1. Effect of pyocin concentration on the growth of N. gonorrhoeae JW-31. Symbols: 0, none; A, 0.01 ml; 0a 0.05 ml; and K 2, 0.25 ml of a suspension of diethylaminoethyl-cellulose-purifiedpyocin 611 131 added to 50-ml cultures. Arrow indicates time of pyocin addition.

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MORSE, JONES, AND LYSKO

Quantitation of pyocin activity. Quantitation of pyocin activity by spotting various dilutions of pyocin 611 131 on a lawn of N. gonorrhoeae JW-31 yielded a titer of 1.2 x 105 U. This estimation was, at best, a relative measurement. Therefore, the procedure of Kageyama et al. (16) was used to determine the number of active pyocin particles. Log-phase cells of strain JW-31 (1.4 x 108 CFU/ml) were added to various dilutions of the pyocin preparation. After 10 min, the suspensions were diluted and plated to determine the number of viable organisms. Ideally, the logarithm of the survival fraction will yield a straight line when plotted against the amount of pyocin. However, the experimental results (Fig. 2) exhibit biphasic kinetics. By using the equation of Kageyama et al. (16), it was calculated that there were 0.92 pyocins per CFU at low pyocin concentrations (relative dose 1); at higher pyocin concentrations (relative dose 7), there were 2.8 pyocins per cell. Therefore, the total number of active pyocins in this preparation was between 1.5 x 1010 and 3.9 x 1010/ml. These values are ca. 2 x 105-fold greater than those determined by the spot-plate method. Time course of pyocin killing. Pyocins (MOI = 2/CFU) were added to a suspension of N. gonorrhoeae JW-31 in defined medium. At intervals aliquots were removed, diluted, and

plated to determine the surviving fraction. The results (Fig. 3) indicate that 80% of the cells absorbed pyocin within 5 min. With an MOI of 200/CFU, 99% of the cells absorbed pyocins by 5 min (data not shown). Attempts to reverse pyocin inhibition by washing the cell pellets were unsuccessful. No significant decrease in viability was observed in the absence of pyocins during the same incubation period. Effect of pyocins on macromolecular synthesis. The incorporation of either [3H]acetate, ['4C]hypoxanthine, [3H]uracil, or [3H]tyrosine was used to measure macromolecular synthesis. Approximately 92% of the acetate associated with the trichloroacetic acid-insoluble material was found in the lipid-containing cell fractions (ethanol-soluble and ethanol-ether soluble fractions). Previous studies (3, 4) determined that gonococci incorporated acetate primarily into the fatty acid acyl groups of phospholipids. Hypoxanthine is converted to adenine and guanine by N. gonorrhoeae (24) and was incorporated primarily into the nucleic acid-containing cell fraction (94% of the trichloroacetic acid-insoluble material). Uracil was incorporated almost exclusively into the nucleic acid-containing cell fraction (98% of the trichloroacetic acid-insoluble material). Tyrosine was incorporated primarily into the protein-containing cell fraction (77% of the trichloroacetic acid-insoluble material). However, a significant percentage was

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FIG. 2. Relationship of survival to pyocin dose. Amounts (0.01 ml) of pyocin dilutions were added to 1.4 x 108 CFU of N. gonorrhoeae JW-31. After incubation for 20 min at 37°C, organisms were diluted and plated for the determination of viable counts. Values represent average of four experiments.

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FIG. 3. Time course of pyocin killing of N. gonorrhoeae JW-31. Pyocin 611 131 (MOI 2/CFU) was added to a suspension of cells in defined medium. At intervals samples were removed, diluted, and plated to determine the surviving fraction. =

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found in the lipid-containing cell fraction. This may represent alcohol-soluble proteins (30) or the catabolism and subsequent incorporation of the tyrosine ring into lipid. The effect of pyocin 611 131 on protein, nucleic acid, and lipid synthesis by N. gonorrhoeae JW-31 was examined. The results are shown in Fig. 4. Pyocin 611 131 completely inhibited the incorporation of [3H]tyrosine (Fig. 4B), [3H]acetate (Fig. 4C), and ['4C]hypoxanthine (Fig. 4D) within 2 min after the addition of pyocins (zero time). No significant decrease in the counts was observed, suggesting that there was no degradation of these macromolecules. The incorporation of [3H]uracil (Fig. 4A) was inhibited between 2 and 5 min after pyocin addition. However, the time difference and the decrease in counts associated with the trichloroacetic acidinsoluble material may not be significant. The previous experiments did not establish the point at which cell lysis began. To answer this question, the effect of pyocins on the release of nucleic acid was determined. The results (Fig. 5) show that nucleic acid was released between 5 and 10 min after pyocin addition. This release corresponds with a decrease in the turbidity of the culture. No appreciable release of nucleic

acid or decrease in turbidity was observed in the absence of pyocins. Effect of pyocins on oxygen uptake. Pyocin 611 131 markedly inhibited oxygen uptake by cells of strain JW-31 suspended in defined 12

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FIG. 5. Release of nucleic acid after the addition of pyocin 611 131 to N. gonorrhoeae JW-31: pyocin added at zero time (arrow). Symbols: release of 14Clabeled nucleic acid by (0) control and (0) pyocintreated culture; A, decrease in turbidity after pyocin addition.

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20 15 10 5 0 20 15 10 5 OM Minutes Milutes FIG. 4. Effect of pyocin 611 131 on macromolecular synthesis in N. gonorrhoeae JW-31. After addition of the labeled precursors (0.4 t,Ci/ml) to mid-log phase cultures (Klett 50 to 60) of strain JW-31, 25-ml volumes were pipetted into two flasks. Pyocins (MOI = 10/CFU) were added to one flask; an equal volume of pyocin buffer (3) was added to the control flask. Symbols: 0, control; 0, pyocin-treated.

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ANTIMICROB. AGENTS CHEMOTHER.

medium (Fig. 6). Inhibition began within 2 min after the addition of pyocins and was complete by 5 min. Heat-inactivated (100°C for 10 min) pyocins had no effect upon oxygen uptake by strain JW-31. Also, active pyocins had no effect upon oxygen uptake by the pyocin-resistant strain JW-31R. Pyocins were not as effective in inhibiting oxygen uptake by gonococci suspended in complex medium (Table 1). This difference may be caused by binding of pyocins to unidentified medium components or a reduced binding efficiency. Table 1 shows that L-lactate did not appreciably stimulate the uptake of 02 by intact cells. After pyocin treatment, L-lactate stimulated 02 uptake by strain JW-31 but not by strain CS-7. These data suggested that the cell lysis observed with strain JW-31 enabled L-lactate to interact with components of the electron transport chain. The nonautolytic strain CS-7 did not become permeable to L-lactate. Previous studies have shown that the cytochromes of strain CS-7 are reduced by L-lactate (37). Therefore, this strain would have responded had the membrane been permeable. Role of pyocin 611 131 in cell lysis. The cell lysis observed after pyocin addition may

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JW-31

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result directly from interaction with the pyocin or through the activity of endogenous gonococcal autolysins. To differentiate between the possibilities, autolytic (JW-31) and non-autolytic (CS-7) strains were compared (Fig. 7). When suspended in HEPES buffer at pH 8.5, strain JW-31 underwent autolysis, resulting in the release of nucleic acid (Fig. 7A). In contrast, strain CS-7 did not exhibit marked autolysis; most of the labeled material released was of low molecular weight and not precipitable by trichloroacetic acid (Fig. 7C). Addition of pyocin to a log-phase culture of strain JW-31 resulted in cell lysis (Fig. 7B; see also Fig. 1). However, the addition of pyocin to a log-phase culture of strain CS-7 resulted in growth inhibition but not cell lysis (Fig. 7D).

DISCUSSION Kageyama et al. (16) showed that killing of P. aeruginosa by the R-type pyocin R-1 was a single-hit process. In contrast, log-phase gonococci exhibit differential susceptibility to killing by the R-type pyocin 611 131. Approximately 85% of the CFU are killed after interaction with 1 pyocin, whereas the remainder are killed after interaction with 2 to 3 pyocins. This observation is not surprising, since gonococci exist as diplococci which would require interaction with 2 pyocins to prevent formation of a colony. Furthermore, Westling-Haggstrom et al. (36) observed that the majority of actively dividing gonococci exhibit only partially developed septa and, thus, would be killed by a single pyocin. R-type pyocins interact with receptors on the lipopolysaccharide of P. aeruginosa (14) and recognize similar receptors on the gonococcal lipopolysaccharide (31). The binding of pyocin 611 131 to N. gonorrhoeae JW-31 was rapid and essentially complete by 5 min. Pyocins contracted after binding to the cell surface (27, 32); the resultant inhibitory effects could not be reversed by extensive washing of the cells. Kaziro and Tanaka (17) and Iijima (13) reported that R-type pyocins caused a rapid and complete cessation of ribonucleic acid, deoxyribonucleic acid, and protein synthesis after their adsorption to the surface of susceptible cells of P. aeruginosa. The degradation of ribosomes but not deoxyribonucleic acid was observed, and the activities of phenylalanyl-transfer ribonucleic acid synthetase and ribonucleic acid nucleotidyltransferase were not affected (18). Additionally, active transport of amino acids was markedly inhibited (13). However, in the presence of high concentrations of Mg2+, the degradation of ribosomes (17) and leakage of amino acid pools (13) did not occur. In the present

VOL. 18, 1980


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FIG. 7. Relationship between autolysis and pyocin-induced cell lysis. (A and C) Percentage of initial turbidity and percent of initial cell-associated [3H]adenine released into the supernatant when gonococci were suspended in 50 mM HEPES buffer. Symbols; 0, turbidity; 0, [3H]adenine in supernatant: A, trichloroacetic acid-insoluble [3H]adenine in supernatant. (B and D) Effect ofpyocin 611 131 on the growth of N. gonorrhoeae strains JW-31 and CS-7. Symbols: 0, control; 0, pyocin-treated. Arrows indicate time of pyocin addition.

study, pyocin 611 131 inhibited the ribonucleic acid, deoxyribonucleic acid and protein synthesis of N. gonorrhoeae JW-31 as measured by the incorporation of labeled precursors. The addition of 20 mM Mg2" reduced the decrease in turbidity but did not prevent cell death (data not shown). Little is known about transport systems in N. gonorrhoeae (21). However, studies on the effect of pyocins on the uptake and leakage of various molecules are in progress. Lipid synthesis by N. gonorrhoeae was also inhibited immediately after the addition of pyocin 611 131. S-type pyocins have been shown to inhibit lipid synthesis of susceptible P. aeruginosa 10 to 20 min before protein and nucleic acid synthesis are inhibited (28). However, no information is available as to whether R-type pyocins such as 611 131 inhibit lipid synthesis of P. aeruginosa. Kageyama (15) showed that the electron transport system of P. aeruginosa was not directly affected by pyocin RI, but as a result of inhibition of active transport, oxygen uptake was inhibited when the substrate was glucose. Maximum inhibition of oxygen uptake occurred after

a lag of 30 to 60 min. In contrast, gonococcal respiration was immediately affected. Inhibition began ca. 1 min after the addition of pyocins; oxygen uptake was completely inhibited by 5 min. This is about the time at which maximum pyocin binding occurred. The inhibition of macromolecular synthesis occurred before the complete inhibition of oxygen uptake. Inhibition of gonococcal oxygen uptake by pyocins is probably independent of the inhibition of active transport. Since the oxygen uptake of pyocin-treated N. gonorrhoeae strain JW-31 was stimulated by L-lactate, pyocins do not appear to directly inhibit electron transport components. However, further studies are required. The addition of pyocin 611 131 to a culture of a susceptible strain of P. aeruginosa resulted in inhibition of growth. However, lysis was not evident for several hours (data not shown). Similar results were obtained by Kaziro and Tanaka (12) with another R-type pyocin. In marked contrast, growth inhibition of gonococcal strain JW-31 by pyocin 611 131 was followed shortly by cell lysis. Lysis, as measured by release of ['4C]hypoxanthine-labeled material or by a de-

422


crease in turbidity, was evident 10 to 15 min after pyocin addition. This lysis may be mediated by a pyocin-associated lytic enzyme (16) or due to endogenous gonococcal autolysins (21, 35). To differentiate between these possibilities, a nonautolytic strain and an autolytic strain were compared. N. gonorrhoeae strain CS-7 did not undergo extensive autolysis when suspended in buffer. Furthermore, this strain probably possesses a suppressed autolytic system (6, 33) since the addition of penicillin to exponential phase cultures inhibited growth but did not promote lysis (data not shown). Similarly, the addition of pyocin 611 131 inhibited the growth of strain CS-7, but did not produce cell lysis. Cultures of the autolytic strain JW-31 underwent lysis after the addition of penicillin (data not shown), and exhibited growth inhibition and lysis following the addition of pyocins. Thus, it is likely that lysis is mediated by endogenous gonococcal autolysins and is not a direct consequence of pyocin inhibition. However, the cell envelopes of the two strains could be different enough to prevent access of autolytic pyocin activity to an active site in CS-7. The mechanism by which pyocin 611 131 inhibits gonococci is unknown at present. It is likely that the inhibition of deoxyribonucleic acid, ribonucleic acid, protein, and lipid synthesis are secondary events that occur after a. primary event at the cytoplasmic membrane (34). R-type pyocins are composed of a contractile sheath surrounding a hollow core (10) and structurally resemble bacteriophage tails. Moreover, they are serologically related to temperate bacteriophage found in P. aeruginosa (12). Contraction of the pyocin is required for inhibition (9). Thus, it is likely that the mechanism of inhibition is similar to that of bacteriophage ghosts (5). Bacteriophage specific for N. gonorrhoeae have not been isolated (8). The availability of transducing phage(s) for N. gonorrhoeae would be a useful tool in the study of the genetic basis of virulence. It is tempting to speculate that the bacteriophage of P. aeruginosa which are serologically related to the R-type pyocins, may propagate on N. gonorrhoeae and serve as a transducing vehicle. ACKNOWLEDGMENTS This research was supported by a grant from Miles Laboratories, Inc. S.A.M. is the recipient of Public Health Service Research Career Development Award AI-00140 from the National Institute of Allergy and Infectious Diseases. P.G.L. is a recipient of an N.L. Tartar research fellowship and a Venereal Diseases Research Fund fellowship from the American Social Health Association.

LITERATURE CITED 1. Blackwell, C., H. Young, and I. Anderson. 1979. Sensitivity of Neisseria gonorrhoeae to partially purified

ANTIMICROB. AGENTS CHEMOTHER. R-type pyocins and a possible approach to epidemiological typing. J. Med. Microbiol. 12:321-335. 2. Bradley, D. E. 1967. Ultrastructure of bacteriophages and bacteriocins. Bacteriol. Rev. 31:230-314. 3. Cacciapuoti, A. F., W. S. Wegener, and S. A. Morse. 1978. Cell envelope of Neisseria gonorrhoeae: phospholipase activity and its relationship to autolysis. Infect. Immun. 20:418-420. 4. Cacciapuoti, A. F., W. S. Wegener, and S. A. Morse. 1979. Phospholipid metabolism in Neisseria gonorrhoeae: phospholipid hydrolysis in nongrowing cells. Lipids 14:718-726. 5. Duckworth, D. H. 1970. Biological activity of bacteriophage ghosts and "take-over" of host cell functions by bacteriophage. Bacteriol. Rev. 34:344-363. 6. Forsberg, C., and H. J. Rogers. 1971 Autolytic enzymes in growth of bacteria. Nature (London) 229:272-273. 7. Geizer, E. 1968. Antibacterial substances produced by different bacteria inhibiting the growth of Neisseria gonorrhoeae. J. Hyg. Epidemiol. Microbiol. Immunol. 12:241-243. 8. Goldberg, I. D., V. I. Steinberg, A. Siddiqui, E. J. Hart, and D. Schaper. 1978. Attempts to isolate a bacteriophage specific for Neisseria gonorrhoeae, p. 55-59. In G. F. Brooks, E. C. Gotschlich, K. K. Holmes, W. D. Sawyer, and F. E. Young (ed.), Immunobiology of Neisseria gonorrhoeae. American Society for Microbiology, Washington, D.C. 9. Govan, J. R. W. 1974. Studies on the pyocins of Pseudomonas aeruginosa: morphology and mode of action of contractile pyocins. J. Gen. Microbiology 80:1-15. 10. Higerd, T. B., C. A. Baechler, and R. S. Berk. 1969. Morphological studies on relaxed and contracted forms of purified pyocin particles. J. Bacteriol. 98:1378-1389. 11. Holloway, B. W., and V. Krishnapillai. 1975. Bacteriophages and bacteriocins, p. 99-132. In P. H. Clarke and M. H. Richmond (ed.), Genetics and biochemistry of Pseudomonas. John Wiley and Sons, New York. 12. Homma, J. Y., and H. Shionoya. 1967. Relationship between pyocine and temperate phage of Pseudomonas aeruginosa. III. Serological relationship between pyocins and temperate phages. Jpn. J. Exp. Med. 37:395421. 13. lijima, M. 1978. Mode of action of pyocin RI. J. Biochem. 83:395-402. 14. Ikeda, K., and F. Egami. 1969. Receptor substance for pyocin R. I. Partial purification and chemical properties. J. Biochem. 65:603-609. 15. Kageyama, M. 1978. Effect of pyocin R on the glucose metabolism of sensitive cells of Pseudomonas aeruginosa. J. Biochem. 84:1373-1379. 16. Kageyama, M., K. Ikeda, and F. Egami. 1964. Studies of a pyocin. III. Biological properties of the pyocin. J. Biochem. 55:59-64. 17. Kaziro, Y., and M. Tanaka. 1965. Studies on the mode of action of pyocin. I. Inhibition of macromolecular synthesis in sensitive cells. J. Biochem. 57:689-694. 18. Kaziro, Y., and M. Tanaka. 1965. Studies on the mode of action of pyocin. II. Inactivation of ribosomes. J. Biochem. 58:357-363. 19. Konisky, J. 1978. The bacteriocins, p. 71-136. In L. N. Ornston and J. R. Sokatch (ed.), The bacteria, vol. VI. Academic Press, Inc., New York. 20. Kuroda, K., and M. Kageyama. 1979. Biochemical properties of a new flexuous bacteriocin, pyocin Fl, produced by Pseudomonas aeruginosa. J. Biochem. 85:7-19. 21. Morse, S. A. 1979. The biology of the gonococcus. CRC Crit. Rev. Microbiol. 7:92-189. 22. Morse, S. A., and L. Bartenstein. 1974. Factors affecting autolysis of Neisseria gonorrhoeae. Proc. Soc. Exp. Biol. Med. 145:1418-1421. 23. Morse, S. A., and L. Bartenstein. 1976. Adaptation of the Minitek system for the rapid identification of Neisseria gonorrhoeae. J. Clin. Microbiol. 3:8-13.

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