NgoII, a restriction endonuclease from Neisseria gonorrhoeae.

EndoR . NgoII, a class II restriction endonuclease isolated from Neisseria gonorrhoeae, was purified to electrophoretic homogeneity. We were able to separate it from another restriction endonuclease of N. gonorrhoeae, NgoI, by phosphocellulose chromatography. NgoII is an isoschizomer of HaeIII, a restriction endonuclease of Haemophilus aegyptius, and was found to recognize the deoxyribonucleic acid nucleotide base sequence GGCC. NgoII was able to digest phage lambda deoxyribonucleic acid over a wide pH range, with optimal activity at pH 8.5. The enzyme has an absolute requirement for Mg2+; maximal enzyme activity was observed at 1 mM Mg2+. The active enzyme has a molecular weight of 65,000 and appears to be composed of six subunits of identical molecular weight (11,000). No methylase activity could be detected in the purified enzyme preparation.     

Deoxyribonucleic acid repair capacities of Neisseria gonorrhoeae: absence of photoreactivation.

No difference in survival was observed when ultraviolet-irradiated gonococcal cells were subsequently incubated in the dark or exposed to photoreactivating light. This observation indicates that photoreactivation is absent in Neisseria gonorrhoeae.     

Nmel, a restriction endonuclease from Neisseria meningitidis

A restriction endonuclease, Nmel, present in Neisseria meningitidis was partially purified by passing through a blue 2-cross linked agarose column; no contaminating nucleases remained detectable. This enzyme cleaved phage lambda, adenovirus type 2 and phi x 174 DNA but did not cleave SV40 DNA. It had an absolute requirement for Mg2+ for its activity and was inhibited by high concentrations of NaCl or MgCl2. Nmel activity was completely abolished after 1 h of incubation at 65 degrees C. S-adenosyl-L-methionine and ATP had no effect on its activity suggesting that Nmel is a type II restriction endonuclease enzyme. It is the first report of a restriction enzyme present in N. meningitidis.     

Multivariate analysis of Neisseria DNA restriction endonuclease patterns

Chromosomal DNA was extracted from eleven Neisseria meningitidis and seven Neisseria gonorrhoeae isolates and cleaved with the restriction enzyme HindIII. The DNA fragments were separated according to their size, using a 4% polyacrylamide gel. The band patterns obtained were digitized and statistically analysed by the SIMCA method. To develop the models for N. meningitidis (class 1) and N. gonorrhoeae (class 2), all eleven meningococci and seven gonococci, were used. All strains were classified correctly and showed an extremely good class separation.     

Penicillin resistance in Neisseria gonorrhoeae due to beta-lactamase production.

Two isolates of Neisseria gonorrhoeae from two patients who did not respond to repeated treatment with penicillin were found to contain an active beta-lactamase. This enzyme has not been previously found in other gonococci isolated in the United States. Compared to other gonococci, these isolates had higher penicillin minimal inhibitory concentrations, and gave very small or no zones of inhibition in the disc agar diffusion test. The enzyme was demonstrated with three different rapid tests for beta-lactamase, by disc diffusion assay methods, and by gas-liquid chromatography-mass spectrometry determination of penicilloic acid--the enzymatic end product from penicillin.     

Identification of a new sequence-specific endonuclease, NgoII, from Neisseria gonorrhoeae.

A class II restriction endonuclease which recognizes the same nucleotide sequence as EndoR.HaeIII has been found in four of seven isolates of Neisseria gonorrhoeae tested.     

Plasmid deoxyribonucleic acid in clinical isolates of Neisseria gonorrhoeae.

Two different sizes of circular covalently closed deoxyribonucleic acid plasmids have been identified in four independent clinical isolates of eisseria gonorrhoeae. All four strains contained a small plasmid with a molecular weight of 2.8 X 10-6 and two of the four stains also contained a large plasmid with a molecular weight of 24.5 X 10-6. The avirulent derivative of each of these four strains had the same plasmid complement as its virulent parent. There was no correlation between the presence of these plasmids and antibiotic resistance, piliation, and colony type associated with virulence, or ability to grow without seven specific amino acid supplements.     

Autoplaquing in Neisseria gonorrhoeae.

Irregularly shaped autoplaques were observed on a lawn of two different strains of Neisseria gonorrhoeae. Autoplaquing occurred on gonococcal genetic medium lacking arginine and was noninducible on complete gonococcal genetic medium. The cell density, incubation temperature, and agar base influenced autoplaquing. Single-colony suspensions varied in plaque morphology. We were unable to isolate a stable nonplaquing variant but separated strain RUN5287 into two plaquing phenotypes.     

Construction of a Neisseria gonorrhoeae MS11 derivative deficient in NgoMI restriction and modification.

We have cloned from Neisseria gonorrhoeae MS11 the gene encoding a methylase that modifies the sequence GCCGGC. The corresponding restriction enzyme was also encoded by this clone. Sequence analysis demonstrated that the methylase shares sequence similarities with other cytosine methylases, but the sequence organization of M.NgoMI is different from that seen for other cytosine methylases. A deletion was introduced into the chromosome of N. gonorrhoeae MS11 to produce strain MUG701, a strain that is inactivated in both the methylase and the restriction genes. Although this strain no longer methylated its DNA at the NgoMI recognition sequence, cells were viable and had no other significant phenotypic changes. Transformation data indicated that MS11 does not produce enough restriction activity to block plasmid transformation in the gonococcus, even though restriction activity could be demonstrated in E. coli containing the cloned gene.     

Conjugative plasmids in Neisseria gonorrhoeae.

A conjugation system initially discovered in beta-lactamase-producing gonococci mobilized small non-selftransmissible R plasmids encoding beta-lactamase (penicillinase) production into other gonococci, Neisseria, and Escherichia coli. This conjugation system was mediated by a separate selftransmissible plasmid of 23.9 X 10(6) daltons, pFA2. Conjugative plasmids capable of mobilizing R plasmids were also found in nearly 8% of the non-penicillinase-producing gonococci. These were similar to pFA2 in size, buoyant density, and restriction endonuclease digest patterns but were less efficient than pFA2 in mobilization of the penicillinase plasmid pFA3. The presence of conjugative plasmids in gonococci isolated before the appearance of penicillinase-producing strains indicates that a conjugation system for plasmid transfer predated the appearance of R plasmids in gonococci.     

Penicillinase-producing Neisseria gonorrhoeae in Canada.

The incidence of infection with penicillinase-producing Neisseria gonorrhoeae (PPNG) reported to the Laboratory Centre for Disease Control in Ottawa has steadily increased since the first Canadian isolation of such a strain in 1976. As of September 1980 a total of 66 PPNG isolates had been referred for biological and genetic characterization as well as for central documentation of the epidemiologic aspects of each case. Over 90% of the infections were firmly traced to patients or contacts who had acquired the infection abroad; this indicates that Canada does not, as yet, have an epidemic focus of PPNG infection. This report includes a synopsis of the biological characteristics of these isolates and an analysis of the results of primary antibiotic treatment that illustrates the importance of considering spectinomycin as the antibiotic of choice for PPNG infections.     

Autolysis of Neisseria gonorrhoeae.

Autolysis of Neisseria gonorrhoeae was studied under different conditions. It was found that low pH and temperature, as well as the presence of divalent cations, spermine, sucrose, and polyvinylpyrrolidone, stabilized nongrowing gonococci. Ethylenediaminetetraacetic acid alone promoted lysis, whereas lysozyme had only a limited additive effect. The autolytic behavior of gonococci appears to be connected with their prolonged cell division process. The relative dependence on the outer membrane and the peptidoglycan layer for the mechanical stability of gonococci is discussed.     

Autolysis of Neisseria gonorrhoeae.

Physiological conditions that would provide maximal rates of autolysis of Neisseria gonorrhoeae were examined. Autolysis was found to occur over a broad pH range with the optimum at pH 9.0 IN 0.05 M tris(hydroxymethyl)amino-methane-maleate buffer. The temperature optimum was found to be 40 C. Potassium ions greatly stimulated autolysis at a concentration of 0.01 M. Exposure of growing N. gonorrhoeae cells to penicillin, vancomycin, or D-cycloserine influenced the susceptibility to the autolysis, whereas chloramphenicol afforded some protection against autolysis. The primary structure of the peptidoglycan is composed of muramic acid/glutamic acid/alanine/diaminopimelic acid/glucosamine in approximate molar ratios of 1:1:2:1:1, respectively. Exogenous radioactive diaminopimelic acid, D-glucosamine, and D-alanine were incorporated into peptidoglycan. During autolysis these radioactive fragments were released from cells.     

Entry of double-stranded deoxyribonucleic acid during transformation of Neisseria gonorrhoeae.

The state of donor deoxyribonucleic acid after entry into competent cells was examined by assaying the transformed cell lysates for donor-marker transforming activity and density of donor deoxyribonucleic acid in CsCl gradients. The experiments showed that deoxyribonucleic acid entered in native, double-stranded form.     

Genetic exchange mechanisms in Neisseria gonorrhoeae.

There are two mechanisms for genetic exchange in Neisseria gonorrhoeae. Plasmid deoxyribonucleic acid can be transferred by conjugation, which is dependent on the presence of a 24.5-megadalton plasmid in the donor cell. We have shown that chromosomal deoxyribonucleic acid can be exchanged between all colonial variants by transformation, but not by conjugation. In the nonpiliated variants, however, this exchange was dependent on the presence of the 24.5-megadalton plasmid in the recipient cell.     

Evolutionary analysis of the 7.1-kb beta-lactamase-specifying R-plasmid of Neisseria gonorrhoeae by restriction endonucleases.

A physical map of restriction enzyme sites was made for the large beta-lactamase-specifying gonococcal R-plasmid (pMR0360) isolated in 1976. Single sites in the plasmid were mapped for 11 endonucleases, and multiple sites of cleavage were mapped for 17 other enzymes. Conclusions about the structure and origin of this plasmid are discussed.     

Temperature-sensitive mutants of Neisseria gonorrhoeae.

Nine temperature-sensitive (Ts) mutants of Neisseria gonorrhoeae strain 49191 were isolated. They were proven to be different from each other by results of transformation experiments. None of the Ts mutations appeared to be linked to antibiotic resistance genes from strain 24392. However, Ts-9 demonstrated 8% linkage with a nalidixic acid resistance marker from strain RW-2.