Vol. 125, No. 3 Printed in U.SA.
JOURNAL OF BACTEROLOGY, Mar. 1976, p. 1207-1210 Copyright C 1976 American Society for Microbiology
NOTES Chromosomal Location of Antibiotic Resistance Genes in Neisseria gonorrhoeae G. BISWAS, S. COMER,
AND
P. F SPARLING*
Departments of Bacteriology and Immunology and Medicine, University ofNorth Carolina School of Medicine, Chapel Hill, North Carolina 27514
Received for publication 28 October 1975
Transformation with purified plasmid and chromosomal deoxyribonucleic acid from a clinical isolate of Neisseria gonorrhoeae showed that each of seven loci affecting drug resistance (penA, penB, ery, str, tet, chl, and env) was chromosomal.
Recently several groups have reported evidence of plasmids in Neisseria gonorrhoeae (3, 8, 9, 15). A small plasmid with a molecular weight of 2.37 x 106 to 2.9 x 106 has been found in 10 of the 11 tested strains (8, 9, 15), and an additional larger plasmid with a molecular weight of 24.5 x 106 was found in two of four strains studied by Stiffier et al. (15). No correlation has been noted between plasmid size or copy number and the antibiotic sensitivity of these strains (8, 9, 15). We have independently performed similar studies which confirm these results. In addition, we have used purified chromosomal and plasmid deoxyribonucleic acid (DNA) from a resistant clinical isolate in transformation experiments to decisively demonstrate the chromosomal location of genes for antibiotic resistance. Most bacterial strains, media, and methods of culture used in this study have been described previously (12, 14). Diphasic medium (13) consisted of a bottom phase of GC base agar (Difco) and an upper phase of GC base broth (11). For preparation of labeled DNA, mid-log phase cells from diphasic medium were inoculated into 25 to 50 ml of GC base broth containing either 2 ,uCi of [14C]adenine per ml or 10 ,uCi of [3H]adenine per ml and incubated with vigorous agitation for 6 to 8 h at 36 C in the presence of 5% CO2 to late-logarithmic phase. All media were supplemented with 1% (vol/vol) supplements 1 and 2 of Kellogg et al. (5). Cells were harvested in the cold by centrifugation, washed once, and stored at -70 C until use. Cultures of colony type 1 or type 2 (5) cells were always at least 80% pure for the particular colony type at the time of harvest. Defined
minimal medium (2) was used for transformation of nutritional requirements. Cell lysates were prepared essentially as described by Guerry et al. (4). Thawed cells suspended in 2 ml of 25% (wt/vol) sucrose in 0.05 M
tris(hydroxymethyl)aminomethane-hydrochloride, pH 8, were incubated with 100 gg of lysozyme (Sigma) per ml and 0.01 M ethylenediaminetetraacetate at 0 C for 10 min. Lysis was achieved by the addition of 1.2 ml of water and 0.4 to 1.2 ml of 1% (wt/vol) Sarkosyl solution, followed by incubation at 0 C for 20 min. NaCl (5 M) was then added to the crude lysate to a final concentration of 1 M. After storage at 4 C overnight, most chromosomal DNA was pelleted by centrifugation at 25,000 x g at 4 C for 30 min. The supernatant, which retained most of the plasmid DNA originally present in the cells, was referred to as salt-cleared lysate. Covalently closed circular plasmid DNA was separated from chromosomal DNA by dyebuoyant density equilibrium centrifugation by a modification of the method of Radloff et al. (10). Each centrifuge tube contained ethidium bromide (EB) (Calbiochem) in a final concentration of 330 ug/ml, 4.5 ml of lysate, and sufficient CsCl (Harshaw) in TE buffer [0.01 M tris(hydroxymethyl)aminomethane-hydrochloride, pH 8.0-0.001 M ethylenediaminetetraacetate, pH 8.0] to bring the final refractive index to 1.3888. After centrifugation in a Spinco Ti 65 fixed-angle rotor at 45,000 rpm for 60 h at 20 C, fractions of approximately 0.2 ml were collected by piercing the bottom of the tube. Selected fractions were extracted with isoamyl alcohol to remove EB and were dialyzed against TE buffer at 4 C.
1207
1208
~.
J. BACTERIOL.
NOTES
microscopy (Fig. 1) of the satellite bands in four strains revealed a mean contour length of 1.20 to 1.30 ,um for >99% of the plasmid molecules.
Ribonucleic acid in adenine-labeled samples was hydrolyzed by the addition of 0.5 ml of 1 N NaOH to aliquots of each fraction, followed by overnight incubation at 37 C. A 0.5-ml amount of 1 N HCl was then added, followed by 50 ,ug of calf thymus DNA as carrier. DNA was precipitated with either 10% trichloroacetic acid or 95% ethanol, collected by filtration, dried, and counted in a toluene-based scintillant. Electron microscopy of plasmid DNA was done by the technique of Kleinschmidt et al. (7) using an AE1 electron microscope. Contour lengths were measured with a map-measuring device by projecting photographs of DNA molecules onto a screen. For control, Simian virus 40 DNA, kindly donated by J. Newbold, was similarly photographed and measured. Transformation was performed as previously described (11-14), using 0.3 ,ig of DNA per ml. Concentrations of DNA were determined by absorbance at 260 nm in an Acta CIII spectrophotometer, using calf thymus DNA as a stand-
Calculated plasmid molecular weights were 2.48 x 106 to 2.69 x 106. There were no differences in plasmid size in an antibiotic-resistant strain (FA5), an antibiotic-sensitive mutant of FA5 (FA52), or in two "wild-type" antibioticsensitive strains, FA19 and F62. Plasmid size was nearly identical in colony type 1 (piliated) and 4 (nonpiliated) cells of FA5, F62, and FA19. Approximately 4 to 6% of total DNA was isolated as covalently closed circular DNA by EBCsCl centrifugation of crude lysates (non-saltcleared) from strains FA5, FA52, F62, and FA19. Assuming a molecular weight of 109 for gonococcal chromosomal DNA (6), each strain therefore contained approximately 16 to 24 copies of plasmid per chromosome. These results are in agreement with those previously reported by others (8, 9, 15) and suggest that genes for drug resistance and piliation are not carried on plasmids. More conclusive evidence would be provided by transformation experiments with purified plasmid and chromosomal DNA, however. Accordingly, plasmid DNA was prepared from salt-cleared lysates of resistant clinical isolate FA5 by two
ard. Results of dye-buoyant density centrifugation showed that each of 11 strains examined contained a minor band of relatively dense satellite DNA in addition to the major band of chromosomal DNA. Examination by electron
40 Np .g J. .: ~~
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FIG. 1. Electron micrograph of2.57 x 10 'ldalton plasmid from N. gonorrhoeae strain FA5, colony type 2.
.
NOTES
VOL. 125, 1976
1209
cycles of CsCl-EB density gradient centrifugation. Chromosomal DNA was prepared from the same strain by redissolving the 1 M NaCl precipitate of the crude lysate in 50 mM tris(hydroxymethyl)aminomethane (pH 7.4) containing 5 mM ethylenediaminetetraacetate, followed by two cycles of CsCl-EB centrifugation. Plasmid DNA purified in this manner was free of linear molecules as observed by electron microscopy and sedimented as a single sharp peak in neutral 5 to 20% sucrose gradients. Chromosomal DNA was free of circular molecules as observed by electron microscopy and formed a single broad peak which sedimented more rapidly than plasmid DNA in neutral 5 to 20% sucrose gradients. Transformation of appropriate recipients with purified plasmid and chromosomal DNA revealed that each of seven drug resistance markers and a single studied nutritional marker was associated with chromosomal DNA (Table 1). The activity of chromosomal DNA
was essentially identical to that observed with bulk unfractionated DNA. The occasional activity seen with plasmid DNA in certain experiments was always less than 0.1% of that observed with chromosomal DNA. Our results do not exclude the possibility that drug resistance genes could be carried on plasmid DNA that is linearly integrated into chromosome. Moreover, we only studied one clinical isolate by these methods, and it did not contain the larger 24.5 x 106-molecular-weight plasmid observed in certain strains of N. gonorrhoeae by Stiffier et al. (15). With these limitations, our results provide the most direct evidence to date that drug resistance in clinical isolates of the gonococcus is not due to R factors or related plasmids but rather is due to chromosomal mutations. In this respect, gonococci are apparently an exception to the usual observation that antibiotic resistance of clinically isolated bacteria is due to drug resistance plasmids (1).
TABLE 1. Transforming ability ofplasmid and chromosomal DNA prepared from FA5, colony type
M. Maness and G. C. Foster performed several preliminary experiments. This work was supported by Public Health Service grant AI10646 and by Public Health Service Research Career Development Award A133032 to P. F. S., both from the National Institute of Allergy and Infectious Diseases.
3a
Recipient strain
Donor marker e-
FA19
str-1c ery-1 tet-I chl-1 penAl pro+
Transformants (per mi)b Pmid Chromosomal
4 x 101 4.13 x 104 2 x 101 1.33 x 105 0 2.40 x 104 0 3.0 x 104 8 x 101 1.16 x 105 Z-1 0 9.57 x 103 ery-l 0 2.53 x 104 FA36 penBI 0 6.20 x 103 FA52 env+ 0 5.22 x 104 a Plasmid DNA was prepared from the supernatant fluid remaining after precipitation of high-molecular-weight DNA with 1 M NaCl and purified by two cycles of dye-buoyant density centrifugation. Chromosomal DNA was extracted from the precipitate formed after addition of 1 M NaCl to a crude lysate and purified by two cycles of dye-buoyant density centrifugation. bTransformants standardized to number per 108 colony-forming units exposed to DNA. DNA predigested with deoxyribonuclease was used as control; net transformants were obtained by subtracting the background frequency of spontaneous mutants. c Concentrations of drugs used in selecting transformants were 0.03 or 0.37 ,ug of penicillin G per ml for penA or penB (respectively); 0.37 to 0.50 jig of erythromycin per ml for ery-1; 0.37 ,ug of tetracycline per ml for tet-l; 0.75 Ag of chloramphenicol per ml for chl-l; and 200 ug of streptomycin per ml for str-l. The env+ locus was selected with 0.5 ug of erythromycin per ml and then scored for resistance to erythromycin, tetracycline, and chloramphenicol (each 1.0 ,ug/ml) and penicillin (0.5 Ag/ml) (12).
LITERATURE CITED 1. Benveniste, R., and J. Davies. 1973. Mechanisms of antibiotic resistance in bacteria. Annu. Rev. Biochem. 42:471-506. 2. Catlin, B. W. 1973. Nutritional profiles of Neisseria
gonorrhoeae, Neisseria meningitidis, and Neisseria lactamica in chemically defined media, and the use of growth requirements in gonococcal typing. J. Infect. Dis. 128:178-194. 3. Engelkirk, P. G., and D. E. Schoenhard. 1973. Physical evidence of plasmids in Neisseria gonorrhoeae. J. Infect. Dis. 127:197-200. 4. Guerry, P., D. J. LeBlanc, and S. Falkow. 1973. General method for isolation of plasmid deoxyribonucleic acid. J. Bacteriol. 116:1064-1066. 5. Kellogg, D. S., Jr., W. L. Peacock, Jr., W. E. Deacon, L. Brown, and C. I. Pirkle. 1963. Neisseria gonorrhoeae. I. Virulence genetically linked to clonal variation. J. Bacteriol. 85:1274-1279. 6. Kingsbury, D. T. 1969. Estimate of genome size in various microorganisms. J. Bacteriol. 98:1400-1404. 7. Kleinschmidt, A. K., D. Lang, D. Jacherts, and R. K. Zahn. 1962. Darstellung und L&ngenmessungen des gesamten desoxyribonuclein-Saure-Inhaltes von T2Bakteriophagen. Biochim. Biophys. Acta 61:857-864. 8. Mayer, L. W., K. K. Holmes, and S. Falkow. 1974. Characterization of plasmid deoxyribonucleic acid from Neisseria gonorrhoeae. Infect. Immun. 10:712717. 9. Paichaudhuri, S., E. Bell, and M. R. J. Salton. 1975. Electron microscopy of plasmid deoxyribonucleic acid from Neisseria gonorrhoeae. Infect. Immun. 11:11411146. 10. Radloff, R., W. Bauer, and J. Vinograd. 1967. A dyebuoyant density method for the detection and isolation of closed circular duplex DNA: the closed circular DNA in HeLa cells. Proc. Natl. Acad. Sci. U.S.A. 57:1514-1521.
1210
NOTES
11. Sarubbi, F. A., Jr., E. Blackman, and P. F. Sparling. 1974. Genetic mapping of linked antibiotic resistance loci in Neisseria gonorrhoeae. J. Bacteriol. 120:12841292. 12. Sarubbi, F. A., Jr., P. F. Sparling, E. Blackman, and E. Lewis. 1975. Loss of low-level antibiotic resistance in Neisseria gonorrhoeae due to env mutations. J. Bacteriol. 124:750-756. 13. Sparling, P. F. 1966. Genetic transformation of Neisseria gonorrhoeae to streptomycin resistance. J. Bac-
J. BACTERIOL. teriol. 92:1364-1371. 14. Sparling, P. F., Sarubbi, F. A., Jr., and E. Blackman. 1975. The inheritance of low-level resistance to penicillin, tetracycline, and chloramphenicol in Neisseria gonorrhoeae. J. Bacteriol. 124:740-749. 15. Stiffler, P. W., S. A. Lerner, M. Bohnhoff, and J. A. Morello. 1975. Plasmid deoxyribonucleic acid in clinical isolates of Neisseria gonorrhoeae. J. Bacteriol. 122:1293-1300.
JOURNAL OF BACTEROLOGY, Mar. 1976, p. 1207-1210 Copyright C 1976 American Society for Microbiology
NOTES Chromosomal Location of Antibiotic Resistance Genes in Neisseria gonorrhoeae G. BISWAS, S. COMER,
AND
P. F SPARLING*
Departments of Bacteriology and Immunology and Medicine, University ofNorth Carolina School of Medicine, Chapel Hill, North Carolina 27514
Received for publication 28 October 1975
Transformation with purified plasmid and chromosomal deoxyribonucleic acid from a clinical isolate of Neisseria gonorrhoeae showed that each of seven loci affecting drug resistance (penA, penB, ery, str, tet, chl, and env) was chromosomal.
Recently several groups have reported evidence of plasmids in Neisseria gonorrhoeae (3, 8, 9, 15). A small plasmid with a molecular weight of 2.37 x 106 to 2.9 x 106 has been found in 10 of the 11 tested strains (8, 9, 15), and an additional larger plasmid with a molecular weight of 24.5 x 106 was found in two of four strains studied by Stiffier et al. (15). No correlation has been noted between plasmid size or copy number and the antibiotic sensitivity of these strains (8, 9, 15). We have independently performed similar studies which confirm these results. In addition, we have used purified chromosomal and plasmid deoxyribonucleic acid (DNA) from a resistant clinical isolate in transformation experiments to decisively demonstrate the chromosomal location of genes for antibiotic resistance. Most bacterial strains, media, and methods of culture used in this study have been described previously (12, 14). Diphasic medium (13) consisted of a bottom phase of GC base agar (Difco) and an upper phase of GC base broth (11). For preparation of labeled DNA, mid-log phase cells from diphasic medium were inoculated into 25 to 50 ml of GC base broth containing either 2 ,uCi of [14C]adenine per ml or 10 ,uCi of [3H]adenine per ml and incubated with vigorous agitation for 6 to 8 h at 36 C in the presence of 5% CO2 to late-logarithmic phase. All media were supplemented with 1% (vol/vol) supplements 1 and 2 of Kellogg et al. (5). Cells were harvested in the cold by centrifugation, washed once, and stored at -70 C until use. Cultures of colony type 1 or type 2 (5) cells were always at least 80% pure for the particular colony type at the time of harvest. Defined
minimal medium (2) was used for transformation of nutritional requirements. Cell lysates were prepared essentially as described by Guerry et al. (4). Thawed cells suspended in 2 ml of 25% (wt/vol) sucrose in 0.05 M
tris(hydroxymethyl)aminomethane-hydrochloride, pH 8, were incubated with 100 gg of lysozyme (Sigma) per ml and 0.01 M ethylenediaminetetraacetate at 0 C for 10 min. Lysis was achieved by the addition of 1.2 ml of water and 0.4 to 1.2 ml of 1% (wt/vol) Sarkosyl solution, followed by incubation at 0 C for 20 min. NaCl (5 M) was then added to the crude lysate to a final concentration of 1 M. After storage at 4 C overnight, most chromosomal DNA was pelleted by centrifugation at 25,000 x g at 4 C for 30 min. The supernatant, which retained most of the plasmid DNA originally present in the cells, was referred to as salt-cleared lysate. Covalently closed circular plasmid DNA was separated from chromosomal DNA by dyebuoyant density equilibrium centrifugation by a modification of the method of Radloff et al. (10). Each centrifuge tube contained ethidium bromide (EB) (Calbiochem) in a final concentration of 330 ug/ml, 4.5 ml of lysate, and sufficient CsCl (Harshaw) in TE buffer [0.01 M tris(hydroxymethyl)aminomethane-hydrochloride, pH 8.0-0.001 M ethylenediaminetetraacetate, pH 8.0] to bring the final refractive index to 1.3888. After centrifugation in a Spinco Ti 65 fixed-angle rotor at 45,000 rpm for 60 h at 20 C, fractions of approximately 0.2 ml were collected by piercing the bottom of the tube. Selected fractions were extracted with isoamyl alcohol to remove EB and were dialyzed against TE buffer at 4 C.
1207
1208
~.
J. BACTERIOL.
NOTES
microscopy (Fig. 1) of the satellite bands in four strains revealed a mean contour length of 1.20 to 1.30 ,um for >99% of the plasmid molecules.
Ribonucleic acid in adenine-labeled samples was hydrolyzed by the addition of 0.5 ml of 1 N NaOH to aliquots of each fraction, followed by overnight incubation at 37 C. A 0.5-ml amount of 1 N HCl was then added, followed by 50 ,ug of calf thymus DNA as carrier. DNA was precipitated with either 10% trichloroacetic acid or 95% ethanol, collected by filtration, dried, and counted in a toluene-based scintillant. Electron microscopy of plasmid DNA was done by the technique of Kleinschmidt et al. (7) using an AE1 electron microscope. Contour lengths were measured with a map-measuring device by projecting photographs of DNA molecules onto a screen. For control, Simian virus 40 DNA, kindly donated by J. Newbold, was similarly photographed and measured. Transformation was performed as previously described (11-14), using 0.3 ,ig of DNA per ml. Concentrations of DNA were determined by absorbance at 260 nm in an Acta CIII spectrophotometer, using calf thymus DNA as a stand-
Calculated plasmid molecular weights were 2.48 x 106 to 2.69 x 106. There were no differences in plasmid size in an antibiotic-resistant strain (FA5), an antibiotic-sensitive mutant of FA5 (FA52), or in two "wild-type" antibioticsensitive strains, FA19 and F62. Plasmid size was nearly identical in colony type 1 (piliated) and 4 (nonpiliated) cells of FA5, F62, and FA19. Approximately 4 to 6% of total DNA was isolated as covalently closed circular DNA by EBCsCl centrifugation of crude lysates (non-saltcleared) from strains FA5, FA52, F62, and FA19. Assuming a molecular weight of 109 for gonococcal chromosomal DNA (6), each strain therefore contained approximately 16 to 24 copies of plasmid per chromosome. These results are in agreement with those previously reported by others (8, 9, 15) and suggest that genes for drug resistance and piliation are not carried on plasmids. More conclusive evidence would be provided by transformation experiments with purified plasmid and chromosomal DNA, however. Accordingly, plasmid DNA was prepared from salt-cleared lysates of resistant clinical isolate FA5 by two
ard. Results of dye-buoyant density centrifugation showed that each of 11 strains examined contained a minor band of relatively dense satellite DNA in addition to the major band of chromosomal DNA. Examination by electron
40 Np .g J. .: ~~
~
~
.~
.-
.'
-C6 0
~
.
.
:
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-~ ~~ ~ ~
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FIG. 1. Electron micrograph of2.57 x 10 'ldalton plasmid from N. gonorrhoeae strain FA5, colony type 2.
.
NOTES
VOL. 125, 1976
1209
cycles of CsCl-EB density gradient centrifugation. Chromosomal DNA was prepared from the same strain by redissolving the 1 M NaCl precipitate of the crude lysate in 50 mM tris(hydroxymethyl)aminomethane (pH 7.4) containing 5 mM ethylenediaminetetraacetate, followed by two cycles of CsCl-EB centrifugation. Plasmid DNA purified in this manner was free of linear molecules as observed by electron microscopy and sedimented as a single sharp peak in neutral 5 to 20% sucrose gradients. Chromosomal DNA was free of circular molecules as observed by electron microscopy and formed a single broad peak which sedimented more rapidly than plasmid DNA in neutral 5 to 20% sucrose gradients. Transformation of appropriate recipients with purified plasmid and chromosomal DNA revealed that each of seven drug resistance markers and a single studied nutritional marker was associated with chromosomal DNA (Table 1). The activity of chromosomal DNA
was essentially identical to that observed with bulk unfractionated DNA. The occasional activity seen with plasmid DNA in certain experiments was always less than 0.1% of that observed with chromosomal DNA. Our results do not exclude the possibility that drug resistance genes could be carried on plasmid DNA that is linearly integrated into chromosome. Moreover, we only studied one clinical isolate by these methods, and it did not contain the larger 24.5 x 106-molecular-weight plasmid observed in certain strains of N. gonorrhoeae by Stiffier et al. (15). With these limitations, our results provide the most direct evidence to date that drug resistance in clinical isolates of the gonococcus is not due to R factors or related plasmids but rather is due to chromosomal mutations. In this respect, gonococci are apparently an exception to the usual observation that antibiotic resistance of clinically isolated bacteria is due to drug resistance plasmids (1).
TABLE 1. Transforming ability ofplasmid and chromosomal DNA prepared from FA5, colony type
M. Maness and G. C. Foster performed several preliminary experiments. This work was supported by Public Health Service grant AI10646 and by Public Health Service Research Career Development Award A133032 to P. F. S., both from the National Institute of Allergy and Infectious Diseases.
3a
Recipient strain
Donor marker e-
FA19
str-1c ery-1 tet-I chl-1 penAl pro+
Transformants (per mi)b Pmid Chromosomal
4 x 101 4.13 x 104 2 x 101 1.33 x 105 0 2.40 x 104 0 3.0 x 104 8 x 101 1.16 x 105 Z-1 0 9.57 x 103 ery-l 0 2.53 x 104 FA36 penBI 0 6.20 x 103 FA52 env+ 0 5.22 x 104 a Plasmid DNA was prepared from the supernatant fluid remaining after precipitation of high-molecular-weight DNA with 1 M NaCl and purified by two cycles of dye-buoyant density centrifugation. Chromosomal DNA was extracted from the precipitate formed after addition of 1 M NaCl to a crude lysate and purified by two cycles of dye-buoyant density centrifugation. bTransformants standardized to number per 108 colony-forming units exposed to DNA. DNA predigested with deoxyribonuclease was used as control; net transformants were obtained by subtracting the background frequency of spontaneous mutants. c Concentrations of drugs used in selecting transformants were 0.03 or 0.37 ,ug of penicillin G per ml for penA or penB (respectively); 0.37 to 0.50 jig of erythromycin per ml for ery-1; 0.37 ,ug of tetracycline per ml for tet-l; 0.75 Ag of chloramphenicol per ml for chl-l; and 200 ug of streptomycin per ml for str-l. The env+ locus was selected with 0.5 ug of erythromycin per ml and then scored for resistance to erythromycin, tetracycline, and chloramphenicol (each 1.0 ,ug/ml) and penicillin (0.5 Ag/ml) (12).
LITERATURE CITED 1. Benveniste, R., and J. Davies. 1973. Mechanisms of antibiotic resistance in bacteria. Annu. Rev. Biochem. 42:471-506. 2. Catlin, B. W. 1973. Nutritional profiles of Neisseria
gonorrhoeae, Neisseria meningitidis, and Neisseria lactamica in chemically defined media, and the use of growth requirements in gonococcal typing. J. Infect. Dis. 128:178-194. 3. Engelkirk, P. G., and D. E. Schoenhard. 1973. Physical evidence of plasmids in Neisseria gonorrhoeae. J. Infect. Dis. 127:197-200. 4. Guerry, P., D. J. LeBlanc, and S. Falkow. 1973. General method for isolation of plasmid deoxyribonucleic acid. J. Bacteriol. 116:1064-1066. 5. Kellogg, D. S., Jr., W. L. Peacock, Jr., W. E. Deacon, L. Brown, and C. I. Pirkle. 1963. Neisseria gonorrhoeae. I. Virulence genetically linked to clonal variation. J. Bacteriol. 85:1274-1279. 6. Kingsbury, D. T. 1969. Estimate of genome size in various microorganisms. J. Bacteriol. 98:1400-1404. 7. Kleinschmidt, A. K., D. Lang, D. Jacherts, and R. K. Zahn. 1962. Darstellung und L&ngenmessungen des gesamten desoxyribonuclein-Saure-Inhaltes von T2Bakteriophagen. Biochim. Biophys. Acta 61:857-864. 8. Mayer, L. W., K. K. Holmes, and S. Falkow. 1974. Characterization of plasmid deoxyribonucleic acid from Neisseria gonorrhoeae. Infect. Immun. 10:712717. 9. Paichaudhuri, S., E. Bell, and M. R. J. Salton. 1975. Electron microscopy of plasmid deoxyribonucleic acid from Neisseria gonorrhoeae. Infect. Immun. 11:11411146. 10. Radloff, R., W. Bauer, and J. Vinograd. 1967. A dyebuoyant density method for the detection and isolation of closed circular duplex DNA: the closed circular DNA in HeLa cells. Proc. Natl. Acad. Sci. U.S.A. 57:1514-1521.
1210
NOTES
11. Sarubbi, F. A., Jr., E. Blackman, and P. F. Sparling. 1974. Genetic mapping of linked antibiotic resistance loci in Neisseria gonorrhoeae. J. Bacteriol. 120:12841292. 12. Sarubbi, F. A., Jr., P. F. Sparling, E. Blackman, and E. Lewis. 1975. Loss of low-level antibiotic resistance in Neisseria gonorrhoeae due to env mutations. J. Bacteriol. 124:750-756. 13. Sparling, P. F. 1966. Genetic transformation of Neisseria gonorrhoeae to streptomycin resistance. J. Bac-
J. BACTERIOL. teriol. 92:1364-1371. 14. Sparling, P. F., Sarubbi, F. A., Jr., and E. Blackman. 1975. The inheritance of low-level resistance to penicillin, tetracycline, and chloramphenicol in Neisseria gonorrhoeae. J. Bacteriol. 124:740-749. 15. Stiffler, P. W., S. A. Lerner, M. Bohnhoff, and J. A. Morello. 1975. Plasmid deoxyribonucleic acid in clinical isolates of Neisseria gonorrhoeae. J. Bacteriol. 122:1293-1300.
