Genetic Diversity and Mosaicism at the por Locus of Neisseria gonorrhoeae

The por genes of the predominant serovars of Neisseria gonorrhoeae circulating in a high-frequency transmitter core group located in Nairobi, Kenya, were examined for nucleotide sequence polymorphism. The level of por gene diversity did not differ significantly between core group-derived gonococcal strains and gonococcal strains originating elsewhere. However, por mosaicism appeared to be more frequent among core group-derived strains, suggesting that recombination of different por sequences may be a important strategy by which N. gonorrhoeae generates por gene diversity within core group populations. Despite extensive sequence variability, por expressed by gonococcal isolates of different geographic origin exhibited conserved patterns of nucleotide change, suggesting that diversity among por alleles may also be finite.     

The lipopolysaccharide (R type) as a common antigen of Neisseria gonorrhoeae. II. Use of hen antiserum to gonococcal lipopolysaccharide in a rapid slide test for the identification of N. gonorrhoeae from primary isolates and secondary cultures.

An antiserum has been prepared in hens to R-type gonococcal lipopolysaccharide (LPS) and used in a simple slide-agglutination test for the identification of Neisseria gonorrhoeae. Anti-LPS serum agglutinated gonococcal cells representative of the four colony types of N. gonorrhoeae. Absorption of the antiserum with LPS removed the agglutinating activity. Secondary cultures (1120) were tested without observation of the colony type and all were agglutinated. No agglutination occurred with strains of Neisseria meningitidis, Neisseria lactamica, non-pathogenic Neisseria. Pseudomonas aeruginosa, Branhamella catarrhalis, or with species of lactobacilli and Acinetobacter. Cross-reactivity of the antiserum occurred with some streptococci. The anti-LPS serum was used to identify N. gonorrhoeae in primary isolates from the cervix, urethra, and pharynx. Of 251 gonococcal isolates tested, 249 were agglutinated by the antiserum, while all of the corresponding second cultures were agglutinated. The antiserum did not agglutinate N. meningitidis found in primary isolates from pharyngeal specimens. Anti-LPS hen serum should be useful for the rapid identification of N. gonorrhoeae in primary isolates or secondary cultures.     

Enzyme-linked immunosorbent assays for the detection of Neisseria gonorrhoeae specific antibodies.

An indirect enzyme-linked immunosorbent assay (ELISA) using rigid polystyrene microtiter plates was adapted to detect specific gonococcal antibodies against outer membrane-complex antigens extracted from Neisseria gonorrhoeae. The concentration of antigen to obtain maximum coating of the well was 10 micrograms protein per millilitre. The optimal binding of the primary antibody and enzyme-conjugated antimmunoglobulin was achieved after 1 h at 37 degrees C. Under these conditions using gonococcal antisera, no cross-reactivity was observed with outer membrane antigens extracted from Neisseria meningitidis serogroups B, C, X, Y, and W135. Neisseria meningitidis serogroup A demonstrated low levels of cross-reactivity. All the non-pathogenic Neisseria spp. tested were negative (absorbance value at 400 nm/30 min less than 0.15). The reaction of immune serum against outer membrane complex absorbed to the microwells was completely inhibited with soluble-specific antigen but not with purified N. gonorrhoeae lipopolysaccharide. Quantitative inhibition permitted the measurement of low levels of antigen (0.5 microgram/ml). The detection of N. gonorrhoeae antibody with ELISA is specific and highly sensitive.     

Penicillinase-producing Neisseria gonorrhoeae conjunctivitis on some Nigerian children.

During 21 month study of bacterial conjunctivitis among 121 children in two health care centres in Calabar, Nigeria, a total of 90 (74.4%) cases were culturally confirmed. Neonates had the highest age-specific attack rates with 48 (53.3%) cases. Neisseria gonorrhoeae, the predominant pathogen, was recovered from 32 (35.6%) infections; 21 (65.6%) of them from neonates. Cultures of genital swabs of consenting parents of infected neonates as well as those of three female children aged 2-12 years with concurrent vulvo-vaginitis yielded N. gonorrhoeae. Younger women, mostly primi-gravidae were more frequently found to have benefited from peri-natal health care services than older multi-gravidae. Nevertheless, such access to health care services did not appear to influence the frequency of gonococcal conjunctivitis in neonates from the two maternal groups (P < 0.01). Sexual abuse and contaminated fomites were the possible modes of gonococcal infection transmission to older children. Overall, 22 (68.8%) strains of gonococci were resistant to penicillin; 19 (59.4%) were penicillinase-producing Neisseria gonorrhoeae (PPNG), while 5 (15.6%) had chromosomally-mediated resistance. All isolates were sensitive to erythromycin. This study recommends a review of gonorrhoea surveillance in pregnancy to include routine examination of cervical swabs just before delivery.     

Influence of conserved and hypervariable genetic markers on genotyping circulating strains of Neisseria gonorrhoeae

Presently there is no vaccine against Neisseria gonorrhoeae and therefore accurate information on gonococcal transmission plays a crucial role for interventions designed to limit the spread of infections caused by this microorganism. We evaluated the impact of two different categories of genetic markers, (i) concatenated sequences of 10 housekeeping genes and (ii) hypervariable porB DNA sequences, on the genetic relatedness and subsequently on genotyping analysis of this human pathogen. Eighty gonococcal isolates from Canada, China, the US, Argentina, Venezuela and Chile, collected over different times, were analyzed. Our results show that the choice of genetic marker had a profound effect on the interpretation of genotyping results associated with N. gonorrhoeae. The concatenated sequences of the housekeeping genes preserved the genetic relatedness of closely related isolates, enabling detection of the predominant strains circulating within a community (Saskatchewan, Canada) over an extended period of time. In contrast, a genetic marker based on antigen gene, porB, may lead to a failure to detect these predominant circulating strains. Based on the analysis of the DNA sequences of the 10 housekeeping genes, we identified two major clonal complexes, CC33 and CC22, which comprised STs from China, and Argentina as well as two STs from Canada. Several minor clonal complexes were observed among isolates from Saskatchewan. eBURST analysis suggested that the majority of the tested gonococcal isolates from Saskatchewan, Canada were endemic, with only a couple of genotypes introduced.     

Properdin is critical for antibody-dependent bactericidal activity against Neisseria gonorrhoeae that recruit C4b-binding protein

Gonorrhea, a sexually transmitted disease caused by Neisseria gonorrhoeae, is an important cause of morbidity worldwide. A safe and effective vaccine against gonorrhea is needed because of emerging resistance of gonococci to almost every class of antibiotic. A gonococcal lipooligosaccharide epitope defined by the mAb 2C7 is being evaluated as a candidate for development of an Ab-based vaccine. Immune Abs against N. gonorrhoeae need to overcome several subversive mechanisms whereby gonococcus evades complement, including binding to C4b-binding protein (C4BP; classical pathway inhibitor) and factor H (alternative pathway [AP] inhibitor). The role of AP recruitment and, in particular, properdin in assisting killing of gonococci by specific Abs is the subject of this study. We show that only those gonococcal strains that bind C4BP require properdin for killing by 2C7, whereas strains that do not bind C4BP are efficiently killed by 2C7 even when AP function is blocked. C3 deposition on bacteria mirrored killing. Recruitment of the AP by mAb 2C7, as measured by factor B binding, occurred in a properdin-dependent manner. These findings were confirmed using isogenic mutant strains that differed in their ability to bind to C4BP. Immune human serum that contained bactericidal Abs directed against the 2C7 lipooligosaccharide epitope as well as murine antigonococcal antiserum required functional properdin to kill C4BP-binding strains, but not C4BP-nonbinding strains. Collectively, these data point to an important role for properdin in facilitating immune Ab-mediated complement-dependent killing of gonococcal strains that inhibit the classical pathway by recruiting C4BP.     

The lipopolysaccharide (R type) as a common antigen of Neisseria gonorrhoeae. I. Immunizing properties.

The ability of R-type lipopolysaccharide (LPS), isolated from Neisseria gonorrhoeae colony type 4, to protect against infection with N. gonorrhoea colony type 1 (T1 isolates) in the mouse and chicken embryo was investigated. C57 black mice were immunized intraperitoneally with 50 microgram of LPS, and challenged intracerebrally with 10-20 LD50's of N. gonorrhoeae colony type 1. Immunized mice were significantly protected (P less than 0.01 to less than 0.05) against challenge with different T1 isolates of N. gonorrhoeae when compared with non-immunized mice. Mice, injected with succinylated or alkali-treated LPS were not protected against gonococcal challenges. In a second animal model, leghorn hens were immunized intravenously with three injections of 500 microgram of LPS followed by a booster of 2.5 mg 2 weeks later. Embryonated eggs obtained from immunized hens were protected against challenge with 5 x 10(3) - 1 x 10(4) LD50's of three different T1 isolates. When hens were injected with the chemically modified LPS, the embryos were not resistant to gonococcal challenge. The results of this study demonstrate the ability of R-type gonococcal LPS to provide protection against different T1 isolates of N. gonorrhoeae.     

Immune-enhanced phagocytosis of Neisseria gonorrhoeae by macrophages: characterization of the major antigens to which opsonins are directed

antisera were prepared in rabbits against whole organisms of colony type 1 Neisseria gonorrhoeae strains F62 and B (fron gonococcal urethritis) and 7122 (a strain typical of those associated with disseminated gonococcal infection), and against purified outer membrane components from the same strains including pili and principal outer membrane protein. Antibody levels to pili, principal outer membrane protein and lipopolysaccharide were determined using a quantitative enzyme-linked immunosorbent assay. Each antiserum was heat-inactivated and tested for opsonic for its homologous strain, and this immune-enhanced phagocytosis was decreased by adsorption with homologous purified outer membrane components: pili greater than lipopolysaccharide greater than principal outer membrane protein. Opsonic activity was approximately equal for antiserum to purified pili and antiserum to the whole organisms for each of the three strains, and purified antibody to pili was highly opsonic. The F(ab')2 fragments of antibody to pili were not opsonic, indicating a role for the Fc receptor on the phagocyte membrane in immune-enhanced phagocytosis of gonococci.     

Characterization of Neisseria gonorrhoeae strains isolated from patients with conjunctivitis

The conjunctivitis produced by Neisseria gonorrhoeae is the less frequently reported clinical form of gonococcal infection. We aim to phenotypically characterize N. gonorrhoeae isolated from conjunctivae sites. A total of six cases of this disease were notified in the Camagüey province, Cuba. All the strains isolated were penicillin-producing, showed the serogroup WI and exhibited the same antimicrobial susceptibility pattern and plasmid profile (2.6-3. 2-24.5). The results contribute to the characterization of N. gonorrhoeae strains circulating in our environment.     

Immunological heterogeneity of pili of Neisseria gonorrhoeae.

Sera of rabbits immunized with pili or formalized cultures of Neisseria gonorrhoeae contained pili antibodies. The reaction between pili and specific gamma globulin was followed by direct visual observation with an electron microscope. Using pili from 31 strains and antisera against three strains, only a few crossreactions between pili were observed. From this it is concluded that gonococcal pili are antigenically heterogeneous. However, serum raised with a T3 culture (with no detectable pili) contained antibodies against pili of the homologous T2 strain. It is proposed that the pilus antigen may exist in a form different from the typical pilus in gonococci.     

Auxotypes of Neisseria gonorrhoeae isolated from localized and disseminated infections in Montreal.

A survey recently made in the United States on the regional distribution of auxotypes of Neisseria gonorrhoeae suggested that isolates from different geographic areas often differ in auxotype. A subsequent auxotyping study in Montreal of 901 isolates of N. gonorrhoeae, 15 from patients with disseminated gonococcal infection, proved interesting in many regards. Gonococcal genetic medium, modified by the addition of other amino acids, was used. Most (93%) of the strains isolated from patients with localized infection belonged to one of the following three phenotypes: arginine-, hypoxanthine- and uracil-dependent (44%); prototrophic (33%); and proline-dependent (16%). Of the 15 strains responsible for disseminated infection 14 required arginine, hypoxanthine and uracil for growth.     

Identification of epitopes recognized by monoclonal antibodies SM1 and SM2 which react with all pili of Neisseria gonorrhoeae but which differentiate between two structural classes of pili expressed by Neisseria meningitidis and the distribution of their encoding sequences in the genomes of Neisseria spp

The pili expressed by all isolates of Neisseria gonorrhoeae react with two monoclonal antibodies, SM1 and SM2. In contrast, although many isolates of Neisseria meningitidis also express pili (class I) which react with antibodies SM1 and SM2, a proportion express pili (class II) which fail to react. In order to define the epitopes recognized by these antibodies, a series of overlapping peptides corresponding to the amino acid sequence of conserved regions of gonococcal pili have been synthesized. The minimum epitope recognized by antibody SM1 was found to comprise a linear peptide EYYLN, corresponding to residues 49-53 of mature pilin. In contrast, antibody SM2 reacted with a number of peptides from around the cysteine residue (Cys 1) at position 120, suggesting that an extended region may contribute to a conformational epitope recognized by this antibody in the native protein. The identification of the two epitopes defines structural differences between the classes of pili expressed by meningococci. In order to determine the distribution of pilin gene sequences in Neisseria we used as hybridization probes an oligonucleotide (PS1) with the sequence 5'-GAGTATTACCTGAATCA-3' which spans the coding region for the SM1 epitope, and a fragment of the 3' end of the gonococcal pilE gene which contains conserved sequences flanking the two Cys codons and encodes the SM2 epitope. All strains of N. gonorrhoeae and N. meningitidis tested, regardless of piliation phenotype, harboured DNA sequences homologous to those encoding the carboxy-terminus of meningococcal class I pilin. Furthermore, all gonococci and all meningococci producing class I pili hybridized with oligonucleotide probe PS1. Non-reverting non-piliated derivatives of previously class I pilus-producing strains showed reduced hybridization signals with this probe, but nevertheless retained sequences homologous to the coding sequence for the SM1 epitope. However, meningococci producing class II pili could be divided into two groups on the basis of their reaction with the PS1 probe: half the strains tested failed to react, which is consistent with our previous analysis of silent class I pilin sequences; the remainder reacted (relatively weakly) with the probe, suggesting that the silent pil sequences in these strains extend further towards the 5' end of the pilin gene than in strains studied previously. Some strains of Neisseria lactamica reacted weakly with both types of probe but failed to produce SM1-reactive pili. In contrast, isolates of Neisseria flava, Neisseria pharyngis, Neisseria sicca and a series of unrelated bacteria failed to react with both SM1 antibody and the DNA probes. This confirms that possession of 'gonococcal' pilin sequences is limited to the pathogenic neisseriae.     

临床分离耐甲氧西林溶血性葡萄球菌的SCCmec分型及同源性分析

目的了解临床分离耐甲氧西林溶血性葡萄球菌（MRSH）的SCCmec基因型别及相同SCCmec型别菌株的同源性。方法多重PCR进行SCCmec分型,ERIC-PCR法对相同SCCmec型别菌株进行同源性分析。结果83株临床分离MRSH菌株中,SCCmecI型有23株（27．7％）,SCCmecⅡ型有10株（12．1％）,SCCmecm型有24株（28．9％）,SCCmecIV型有1株（1．2％）,I、Ⅱ混合型有8株（9．6％）,I、Ⅲ混合型有6株（7．2％）,Ⅱ、11混合型有5株（6．0％）,I、Ⅱ、Ⅲ混合型有3株（3．6％）,未分型3株（3．6％）。ERIC—PCR结果显示,23株SCCmecI型分为11型,其中A型5株,B型5株,C型3株,其余8株各为1型,2株未分型;10株SCCmecⅡ型分为6型,其中D型4株,E型2株,3株各为1型,1株未分型;24株SCCmecm型分为9型,其中F型11株,G型2株,H型2株,I型2株,5株各为1型,2株未分型。结论临床分离MRSH中,SCCmecI、Ⅲ型为多,部分菌株呈混合型别;相同SCCmec型别的部分菌株之间可能存在克隆传播。     

Transformation of leucine and rifampin traits in Neisseria gonorrhoeae with deoxyribonucleic acid from homologous and heterologous origins.

A leucine-requiring, rifampin-sensitive strain of Neisseria gonorrhoeae was transformed to a leucine-nonrequiring, rifampin-resistant phenotype with deoxyribonucleic acid (DNA) obtained from both N. meningitidis and N. gonorrhoeae. The transforming efficiency of the meningococcal DNA was about 10- to 100-fold less than that of the homologous gonococcal DNA. A chemically defined medium that would support growth of most gonococcal isolates was used as a complete medium. A minimal medium was used for selection of Leu+ transformants. N-methyl-N'-nitro-N-nitrosoguanidine was used as a mutagen for isolating leucine prototrophs from leucine-requiring isolates of N. gonorrohoeae.     

Biochemical and genetic studies with arginine and proline auxotrophs of Neisseria gonorrhoeae

The prevalence of specific arginine biosynthesis gene defects was studied for 319 arginine-requiring clinical isolates of Neisseria gonorrhoeae by using the ability of the strains to utilize intermediates of arginine biosynthesis. Only 11% of the uracil-requiring strains defective in the carbamylation of ornithine to yield citrulline had a defective carbamoylphosphate synthetase gene (carAB). Strains defective in carAB were of auxotype CUH. The other strains (89%) having a dual requirement for citrulline and uracil, which were mostly of auxotype PCU, were defective in the ornithine transcarbamoylase gene (argF). Over 90% of the strains were defective either in argJ (174 strains) or in argF (126 strains). Three argininosuccinate-requiring strains (i.e., defective in argG) of auxotype PAU were identified. Some of the arginine auxotrophs of N. gonorrhoeae defective in carAB, argJ, argF, or argG were complemented by genetic transformation with DNA from recombinant bacteriophages carrying characterized gonococcal arginine biosynthesis genes. Gene defects in proA (five strains) and in proB (six strains) were identified by gonococcal transformation assays with recombinant bacteriophages or plasmids carrying proline biosynthesis genes from N. gonorrhoeae. None of the 11 proline-requiring strains tested was defective in proC.     

Immunoreactivity of intact virions of human immunodeficiency virus type 1 (HIV-1) reveals the existence of fewer HIV-1 immunotypes than genotypes

In order to protect against organisms that exhibit significant genetic variation, polyvalent vaccines are needed. Given the extreme variability of human immunodeficiency virus type 1 (HIV-1), it is probable that a polyvalent vaccine will also be needed for protection from this virus. However, to understand how to construct a polyvalent vaccine, serotypes or immunotypes of HIV must be identified. In the present study, we have examined the immunologic relatedness of intact, native HIV-1 primary isolates of group M, clades A to H, with human monoclonal antibodies (MAbs) directed at epitopes in the V3, C5, and gp41 cluster I regions of the envelope glycoproteins, since these regions are well exposed on the virion surface. Multivariate analysis of the binding data revealed three immunotypes of HIV-1 and five MAb groups useful for immunotyping of the viruses. The analysis revealed that there are fewer immunotypes than genotypes of HIV and that clustering of the isolates did not correlate with either genotypes, coreceptor usage (CCR5 and CXCR4), or geographic origin of the isolates. Further analysis revealed distinct MAb groups that bound preferentially to HIV-1 isolates belonging to particular immunotypes or that bound to all three immunotypes; this demonstrates that viral immunotypes identified by mathematical analysis are indeed defined by their immunologic characteristics. In summary, these results indicate (i) that HIV-1 immunotypes can be defined, (ii) that constellations of epitopes that are conserved among isolates belonging to each individual HIV-1 immunotype exist and that these distinguish each of the immunotypes, and (iii) that there are also epitopes that are routinely shared by all immunotypes.     

Vigorous response of human innate functioning IgM memory B cells upon infection by Neisseria gonorrhoeae

Neisseria gonorrhoeae, the cause of the sexually transmitted infection gonorrhea, elicits low levels of specific Ig that decline rapidly after the bacteria are cleared. Reinfection with the same serovar can occur, and prior gonococcal infection does not alter the Ig response upon subsequent exposure, suggesting that protective immunity is not induced. The mucosal Ig response apparent during gonorrhea does not correlate with that observed systemically, leading to a suggestion that it is locally generated. In considering whether N. gonorrhoeae directly influences B cells, we observed that gonococcal infection prolonged viability of primary human B cells in vitro and elicited robust activation and vigorous proliferative responses in the absence of T cells. Furthermore, we observed the specific expansion of IgD(+)CD27(+) B cells in response to gonococcal infection. These cells are innate in function, conferring protection against diverse microbes by producing low-affinity, broadly reactive IgM without inducing classical immunologic memory. Although gonococcal infection of B cells produced small amounts of gonococcal-specific IgM, IgM specific for irrelevant Ags were also produced, suggesting a broad, polyspecific Ig response. The gonococci were effectively bound and engulfed by B cells. TLR9-inhibitory CpGs blocked B cell responses, indicating that intracellular bacterial degradation allows for innate immune detection within the phagolysosome. To our knowledge, this is the first report of a bacterial pathogen having specific affinity for the human IgM memory B cells, driving their potent activation and polyclonal Ig response. This unfocused T-independent response explains the localized Ig response that occurs, despite an absence of immunologic memory elicited during gonorrhea.     

PilC of Neisseria meningitidis is involved in class II pilus formation and restores pilus assembly, natural transformation competence and adherence to epithelial cells in PilC-deficient gonococci

Type 4 pili produced by the pathogenic Neisseria species constitute primary determinants for the adherence to host tissues. In addition to the major pilin subunit (PilE), neisserial pili contain the variable PilC proteins represented by two variant gene copies in most pathogenic Neisseria isolates. Based upon structural differences in the conserved regions of PilE, two pilus classes can be distinguished in Neisseria meningitidis . For class I pili found in both Neisseria gonorrhoeae and N. meningitidis , PilC proteins have been implicated in pilus assembly, natural transformation competence and adherence to epithelial cells. In this study, we used primers specific for the pilC2 gene of N. gonorrhoeae strain MS11 to amplify, by the polymerase chain reaction, and clone a homologous pilC gene from N. meningitidis strain A1493 which produces class II pili. This gene was sequenced and the deduced amino acid sequence showed 75.4% and 73.8% identity with the gonococcal PilC1 and PilC2, respectively. These values match the identity value of 74.1% calculated for the two N. gonorrhoeae MS11 PilC proteins, indicating a horizontal relationship between the N. gonorrhoeae and N. meningitidis pilC genes. We provide evidence that PilC functions in meningococcal class II pilus assembly and adherence. Furthermore, expression of the cloned N. meningitidis pilC gene in a gonococcal pilC1,2 mutant restores pilus assembly, adherence to ME-180 epithelial cells, and transformation competence to the wild-type level. Thus, PilC proteins exhibit indistinguishable functions in the context of class I and class II pili.     

Molecular characterization of the interaction between porins of Neisseria gonorrhoeae and C4b-binding protein

Neisseria gonorrhoeae, the causative agent of gonorrhea, is a natural infection only in humans. The resistance of N. gonorrhoeae to normal human serum killing correlates with porin (Por)-mediated binding to the complement inhibitor, C4b-binding protein (C4BP). The entire binding site for both porin molecules resides within complement control protein domain 1 (CCP1) of C4BP. Only human and chimpanzee C4BPs bind to Por1B-bearing gonococci, whereas only human C4BP binds to Por1A strains. We have now used these species-specific differences in C4BP binding to gonococci to map the porin binding sites on CCP1 of C4BP. A comparison between human and chimpanzee or rhesus C4BP CCP1 revealed differences at 4 and 12 amino acid positions, respectively. These amino acids were targeted in the construction of 13 recombinant human mutant C4BPs. Overall, amino acids T43, T45, and K24 individually and A12, M14, R22, and L34 together were important for binding to Por1A strains. Altering D15 (found in man) to N15 (found in rhesus) introduced a glycosylation site that blocked binding to Por1A gonococci. C4BP binding to Por1B strains required K24 and was partially shielded by additional glycosylation in the D15N mutant. Only those recombinant mutant C4BPs that bound to bacteria rescued them from 100% killing by rhesus serum, thereby providing a functional correlate for the binding studies and highlighting C4BP function in gonococcal serum resistance.     

Deoxyribonucleic acid modifications and restriction endonuclease production in Neisseria gonorrhoeae.

Modification of gonococcal deoxyribonucleic acid (DNA) was investigated, and the relationship with endonuclease production was explored. Both chromosomal and plasmid DNA from different gonococcal strains, irrespective of their plasmid content, was poorly cleaved by the restriction endonucleases HaeII, HaeIII, SacII, and BamHI. The fragment pattern of the Tn3 segment present on the 7.2-kilobase gonococcal resistance plasmid, when compared to its known DNA sequence, allowed us to conclude that the HaeIII and BamHI resistance was due to modification of these sites. A comparison of the fragment pattern of the resistance plasmid, when isolated from Escherichia coli or Neisseria gonorrhoeae, revealed that the resistance of HaeII must also be due to modification of its recognition sequence. Isoschizomers of HaeII and HaeIII can be found in isolates of N. gonorrhoeae (NgoI and NgoII, respectively). A new restriction endonuclease in gonococci, NgoIII, with a specificity similar to SacII, is reported here. High-pressure liquid chromatography of gonococcal DNA showed the presence of 5-methylcytosine. It is suggested that the methylation of cytosine residues in the HaeII (NgoI), HaeIII (NgoII), and SacII (NgoIII) recognition sites is the basis for the resistance of gonococcal DNA to cleavage by these enzymes. This methylation may be part of a host restriction modification system. In two out of five gonococcal strains the sequence -GATC- was modified. One strain unable to modify this sequence was a spontaneous mutant of a strain carrying such a modifying function.