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.     

Molecular characterization of two beta-lactamase-specifying plasmids isolated from Neisseria gonorrhoeae.

The molecular nature of two distinct gonococcal R plasmids, 4.4 X 10(6) and 3.2 X 10(6) daltons, encoding beta-lactamase activity were examined. Both plasmids contained about 40% of the transposable ampicillin resistance sequence Tn2. Deoxyribonucleic acid-deoxyribonucleic acid polynucleotide sequence studies have shown that the two gonococcal plasmids share about 70% of their sequences and are closely related to RSF0885, a 4.1 X 10(6)-dalton plasmid found in a beta-lactamase-producing strain of Haemophilus influenzae. All three of these R plasmids possess a guanine-plus-cytosine content of 0.40 to 0.41 mol fraction and are present as multicopy gene pools in their bacterial hosts.     

Transformation-derived Neisseria gonorrhoeae plasmids with altered structure and function.

Plasmid deoxyribonucleic acid from Neisseria gonorrhoeae containing a 7.1-kilobase (kb) (4.7-megadalton) penicillinase (Pcr) plasmid transformed homogenic gonococci to penicillinase production at a low frequency. About 25% of the penicillinase-producing gonococcal transformants contained Pcr plasmids which were either larger or smaller than the 7.1 kb donor plasmid; these Pcr plasmids varied in size from 3.45 to 42 kb. Some of these altered plasmids differed from the donor plasmid in stability or in frequency of mobilization by a 36-kb (24-megadalton) conjugative plasmid. A restriction endonuclease cleavage map of the 7.1-kilobase Pcr plasmid and several of the smaller deleted plasmids was constructed. The most common size of altered Pcr plasmid was 5.1 kb (3.4 megadaltons). A Pcr plasmid isolated from a gonococcus in London, England, was identical with these 5.1-kb transformant plasmids in both size and restriction endonuclease cleavage profiles, suggesting that the 5.1-kb Pcr plasmid could have arisen from a 7.1-kb Pcr plasmid by a transformation-associated deletion in nature.     

Conjugal transfer of beta-lactamase-producing plasmids of Neisseria gonorrhoeae to Neisseria meningitidis

Twenty clinical isolates of beta-lactamase-producing Neisseria gonorrhoeae from Japanese sources were studied to define their ability to serve as donors for their plasmids in conjugation with Neisseria meningitidis. These twenty strains of N. gonorrhoeae harbored the 4.5-megadalton (Mdal) beta-lactamase-producing plasmids and the 24.5-Mdal conjugative plasmids. We found that only three of twenty N. gonorrhoeae strains showed a detectable conjugation frequency (greater than 10(-5)) with N. meningitidis as the recipient although all strains were capable of mobilizing beta-lactamase-producing plasmids to N. gonorrhoeae and to Escherichia coli. The 4.5-Mdal beta-lactamase-producing plasmid was maintained in N. meningitidis, but the large 24.5-Mdal conjugative plasmid has not been found in N. meningitidis transconjugants.     

Penicillinase-producing Neisseria gonorrhoeae: a molecular comparison of 5.3-kb and 7.4-kb beta-lactamase plasmids.

Restriction endonuclease analysis and heteroduplex studies indicate that the only difference between the 5.3-kilobase (kb) and 7.4-kb plasmids from beta-lactamase-producing Neisseria gonorrhoeae is that the latter is the 5.3-kb plasmid with a 2.1-kb insertion. The insertion is bounded by inverted repeats of approximately 300 base pairs. Several plasmids from beta-lactamase-producing N. gonorrhoeae isolated at different times and in different countries were compared. Nine 5.3-kb plasmids were examined and found to be indistinguishable, as were sixteen 7.4-kb plasmids.     

Origin of small beta-lactamase-specifying plasmids in Haemophilus species and Neisseria gonorrhoeae.

Fifty-nine percent of unselected strains of Haemophilus parainfluenzae were found to carry small, phenotypically cryptic plasmid DNA species. Using filter blot hybridization, we found several plasmids which were homologous to the small beta-lactamase-specifying plasmids pJB1 and pFA7, which were originally isolated from Haemophilus ducreyi and Neisseria gonorrhoeae, respectively. Detailed filter hybridization studies combined with electron microscope heteroduplex analysis suggested that three cryptic plasmids are completely homologous to the non-TnA sequences of pJB1. One cryptic plasmid was found to be highly homologous to pJB603, a small beta-lactamase plasmid previously found in two isolates of H. influenzae. A second group of plasmids were found to carry sequences homologous to pJB1 and other sequences homologous to pJB603. These results strongly suggest that small beta-lactamase plasmids found in Haemophilus species and N. gonorrhoeae may have arisen by insertion of the transposable beta-lactamase-specifying element TnA into small, phenotypically cryptic replicons resident in H. parainfluenzae. Attempts to reproduce such a recombination event in the laboratory were not successful.     

Structural comparison of Neisseria gonorrhoeae outer membrane proteins.

Outer membranes from opaque colonia variants of Neisseria gonorrhoeae P9 contain a major outer membrane protein (protein I) together with one or more of a series of heat-modifiable proteins (proteins II). Proteins I. II, and IIa have been isolated by detergent extraction of outer membranes. Amino acid analysis showed proteins II and IIa to have a very similar composition. Cyanogen bromide cleavage of proteins II and IIa produced a pair of fragments with identical molecular weight and a pari which differed by an amount (0.5K) equivalent to the difference between the intact proteins. Tryptic peptide maps of 125I-labeled proteins II, IIa, and IIb showed many similarities, with only a few peptides unique to any one protein. Peptide maps of protein IIa from cells which had been surface labeled showed that the unique peptides were exposed on the surface. The heat-modifiable proteins thus appear to form a family of proteins with closely related structure probably differing in that part which is exposed on the bacterial surface.     

淋球菌耐药性与耐药质粒的研究

为了解湛江地区淋球菌耐药现状及耐药质粒的分布,并初步探讨其相互关系,对１９９８￣１９９９年广东省湛江地区健康鉴定出的９８株淋球菌流行株,应用纸片扩散法测定细胞对１０种抗生素的敏感性,并采用碱裂解法进行质粒抽提,分析耐药质粒的分布情况,选取ＮＧ４、ＮＧ３１、ＮＧ４３、ＮＧ７０进行细菌的接合试验,以观察耐药质粒能否通过接合进行传递以及传递后受体菌耐药性的变化。结果显示６．１２％菌株对全部抗生素敏感,４８．９６％的菌株对３种及３种以上的抗生素耐药;共检出四种不同分子量的质闰,７．４ｋｂ和４．２ｋｂ质粒检出率较高,分别为５９．１６％和６７．３２％,质粒谱型１２种,以７．４ｋｇ＋４．２ｋｇ和３９．５ｋｇ＋７．４ｋｇ＋４．２ｋｇ为主,占３８．７６％。在细菌的接合传递试验中,ＮＧ４３可通过接合传递将其耐药性传递给淋球菌及大肠     

Sequence analysis of the family of penicillinase-producing plasmids of Neisseria gonorrhoeae

The exact nature of the sequence differences between the medically important family of gonococcal penicillinase-producing plasmids has been ascertained. The entire DNA sequence of the Asia-type plasmid, pJD4, demonstrated that it is 7426 bp and contains two direct repeats (DR30) that are implicated in the formation of deletion variant plasmids, such as the Africa-type plasmid. We have identified putative DnaA and IHF binding sites, various open reading frames that are thought to specify functional proteins, and some important DNA sequences involved with conjugative transfer of gonococcal beta-lactamase plasmids. The deletion in the Africa-type plasmid is 1827 bp and one of the DR30 repeats is also missing. The deletion in the Rio-type plasmid and several Toronto-type plasmids was determined to be 2273 bp and the sequence spanning the deletion was identical irrespective of geographic or temporal origin. The &Ncirc;imes-type plasmid is an Africa-type plasmid and also contains an IS5 insertion sequence. Since IS5 has not been identified in gonococcal isolates, we suggest that this sequence may have been inserted after the original gonococcal plasmid was transformed into Escherichia coli. The New Zealand plasmid is an Asia-type plasmid that contains an endogenous tandem duplication of 1883 bp and the direct DR2 is implicated in this duplication. The nature of the defined truncation of Tn2 present in the various plasmids is also discussed.     

Biochemical comparison of pili from variants of Neisseria gonorrhoeae P9

Four different types of pili produced by variants of Neisseria gonorrhoeae P9 were isolated and characterized. The pili differed in subunit molecular weight with SDS-PAGE and in subunit isoelectric point on agarose gels. Isoelectric points of the major molecular species in Triton-agarose gels of octylglucoside solubilized pili were: delta, pI 6.5; alpha, pI 6.0; beta, pI 5.3 and gamma, pI 5.5. Amino acid analyses of pili showed close homology between different types but a reduction in the content of aspartate and serine was notable in the low molecular weight delta pili; also beta and gamma maps of tryptic/chymotryptic digests of pili with several major peptides apparently common to all four pilus types.     

High-frequency mobilization of broad-host-range plasmids into Neisseria gonorrhoeae requires methylation in the donor.

Antibiotic resistance in Neisseria gonorrhoeae has been associated with the acquisition of R plasmids from heterologous organisms. The broad-host-range plasmids of incompatibility groups P (IncP) and Q (IncQ) have played a role in this genetic exchange in nature. We have utilized derivatives of RSF1010 (IncQ) and RP1 (IncP) to demonstrate that the plethora of restriction barriers associated with the gonococci markedly reduces mobilization of plasmids from Escherichia coli into strains F62 and PGH 3-2. Partially purified restriction endonucleases from these gonococcal strains can digest RSF1010 in vitro. Protection of RSF1010-km from digestion by gonococcal enzymes purified from strain F62 is observed when the plasmid is isolated from E. coli containing a coresident plasmid, pCAL7. Plasmid pCAL7 produces a 5'-MECG-3' cytosine methylase (M.SssI). The M.SssI methylase only partially protects RSF1010-km from digestion by restriction enzymes from strain PGH 3-2. Total protection of RSF1010-km from PGH 3-2 restriction requires both pCAL7 and a second coresident plasmid, pFnuDI, which produces a 5'-GGMECC-3' cytosine methylase. When both F62 and PGH 3-2 are utilized as recipients in heterospecific matings with E. coli, mobilization of RSF1010 from strains containing the appropriate methylases into the gonococci occurs at frequencies 4 orders of magnitude higher than from strains without the methylases. Thus, protection of RSF1010 from gonococcal restriction enzymes in vitro correlates with an increase in the conjugal frequency. These data indicate that restriction is a major barrier against efficient conjugal transfer between N. gonorrhoeae and heterologous hosts.     

Characterization of carbonic anhydrase from Neisseria gonorrhoeae.

We have investigated the steady state and equilibrium kinetic properties of carbonic anhydrase from Neisseria gonorrhoeae (NGCA). Qualitatively, the enzyme shows the same kinetic behaviour as the well studied human carbonic anhydrase II (HCA II). This is reflected in the similar pH dependencies of the kinetic parameters for CO(2) hydration and the similar behaviour of the kinetics of (18)O exchange between CO(2) and water at chemical equilibrium. The pH profile of the turnover number, k(cat), can be described as a titration curve with an exceptionally high maximal value of 1.7 x 10(6) s(-1) at alkaline pH and a pK(a) of 7.2. At pH 9, k(cat) is buffer dependent in a saturable manner, suggesting a ping-pong mechanism with buffer as the second substrate. The ratio k(cat)/K(m) is dependent on two ionizations with pK(a) values of 6.4 and 8.2. However, an (18)O-exchange assay identified only one ionizable group in the pH profile of k(cat)/K(m) with an apparent pK(a) of 6.5. The results of a kinetic analysis of a His66-->Ala variant of the bacterial enzyme suggest that His66 in NGCA has the same function as a proton shuttle as His64 in HCA II. The kinetic defect in the mutant can partially be overcome by certain buffers, such as imidazole and 1,2-dimethylimidazole. The bacterial enzyme shows similar K(i) values for the inhibitors NCO(-), SCN(-) and N(3)(-) as HCA II, while CN(-) and the sulfonamide ethoxzolamide are considerably weaker inhibitors of the bacterial enzyme than of HCA II. The absorption spectra of the adducts of Co(II)-substituted NGCA with acetazolamide, NCO(-), SCN(-), CN(-) and N(3)(-) resemble the corresponding spectra obtained with human Co(II)-isozymes I and II. Measurements of guanidine hydrochloride (GdnHCl)-induced denaturation reveal a sensitivity of the CO(2) hydration activity to the reducing agent tris(2-carboxyethyl)phosphine (TCEP). However, the A(292)/A(260) ratio was not affected by the presence of TCEP, and a structural transition at 2.8--2.9 M GdnHCl was observed.     

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.     

Beta-lactamase-specifying plasmids isolated from Neisseria gonorrhoeae have retained an intact right part of a Tn3-like transposon.

In three beta-lactamase-producing strains of Neisseria gonorrhoeae, ampicillin resistance is due to the presence of a 7.4-kilobase plasmid. Heteroduplex analysis has shown that the R plasmid contains a 1.6-kilobase segment homologous to the right part (the region coding for the beta-lactamase) of the Tn3-like transposon Tn2301 the 1.6-kilobase DNA segment is not transposable, but it can give rise to a functional transposon, when linked to the left part of TN2301. This provides strong evidence that the R plasmids of N. gonorrhoeae are deletion derivatives of a plasmid that contained an entire TN3-like transposon.     

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.     

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.     

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.     

Origin and direction of in vitro replication of Haemophilus ducreyi and Neisseria gonorrhoeae ampicillin resistance plasmids.

The origin of replication of Haemophilus ducreyi and Neisseria gonorrhoeae ampicillin resistance plasmids was located by cloning BamHI restriction fragments into vector plasmid pAT153 and a derivative plasmid, pAT2. Selection was made for plasmid maintenance in a polA mutant. Direction of replication was determined by in vitro replication of plasmid DNA in the presence of radiolabeled deoxynucleotide.