Many of the Neisseria gonorrhoeae strains isolated from patients require arginine for growth in a defined medium. As a basis for genetic studies of these Arg-strains, we examined two biosynthetic enzymes of Arg+ (nonrequiring) gonococci. Cell-free extracts contained (i) glutamate acetyltransferase, which catalyzes the formation of L-ornithine from a-N-acetyl-L-ornithine, and (ii) ornithine transcarbamylase, which catalyzes the reaction between L-ormithine and carbamyl phosphate, yielding L-citrulhine. Arg-strains which were unable to utilize a-N-acetyl-L-ornithine for growth lacked significant activity of glutamate acetyltransferase, and activity was gained by Arg+ clones derived by DNAmediated transformation. Some of the Arg-patient isolates were unable to use either a-N-acetyl-L-ornithine or L-ornithine in place of arginine, and two separate steps of genetic transformation were required to yield Arg+ cells. Extracts of these doubly auxotrophic cells lacked glutamate acetyltransferase activity, but, unexpectedly, they displayed normal ornithine transcarbamylase activity. This finding illustrates the importance of identifying the products specified by arg loci during genetic studies of arginine auxotrophy.on August 10, 2020 by guest
Neisseria gonorrhoeae strains with nutritional requirements that include arginine (Arg-), uracil (Ura-), and hypoxanthine have attracted attention because of their tendency to cause disseminated infections. As a basis for genetic studies of arginine and pyrimidine biosynthesis, we examined the activities of four enzymes of these pathways in cell-free extracts of both prototrophic and Arg-Urastrains. Activities of glutamate acetyltransferase, aspartate transcarbamylase, and orotate phosphoribosyltransferase, encoded respectively by argE, pyrB, and
The inheritance of epitopes of protein I, the principal protein of the outer membrane of Neisseria gonorrhoeae, was investigated by DNA-mediated transformation. Protein I transformants were isolated by selection for the linked spectinomycin-resistance determinant. Twelve monoclonal antibodies used in coagglutination tests identified epitopes of the two forms of protein I (P.IA and P.IB). A given gonococcal culture from patients expresses epitopes of either P.IA or P.IB and rarely, if ever, exhibits hybrid P.IA/P.IB reactivities. Nevertheless, we found 35 P.IA/P.IB recombinants among 1506 transformants. Transmission by DNA of the hybrid reactivities and the apparent molecular mass characteristic of a given P.IA/P.IB species verified the genetic basis of the phenotypic changes. A recombinant that expressed six P.IA and two P.IB epitopes is of interest as a possible component of a gonococcal vaccine, because one or more of these epitopes are shared with 99.8% of a worldwide collection of 1858 clinical strains.
SUMMARY The responses to vancomycin and 11 other antibacterial drugs and the nutritional requirements of gonococci recovered from two selective media were determined. Single urogenital specimens from 508 patients attending a social hygiene clinic in 1975 yielded 97 strains of Neisseria gonorrhoeae; 95 were recovered on VCNT (a modification of Thayer-Martin medium), always inoculated first, and 69 on LC medium containing lincomycin (4 l.g/ml) and colistin (5 Mg/ml).The two drugs at these concentrations in LC medium were not inhibitory for isolates from either medium. Unexpectedly, three isolates on VCNT were susceptible to vancomycin at the concentration (3 ,ug/ml) in VCNT medium; these three were typically sensitive to penicillins but were hypersusceptible to erythromycin (inhibited by (0 05 ,Ag/ml) and rifampin ((0-02 yg/ml). Resistance to streptomycin (>500 jig/ml) (22% of the strains) was correlated with increased resistance to penicillins, erythromycin, and rifampin in most instances. All streptomycin-resistant gonococci required proline, or arginine, or none of the test compounds. Strains requiring arginine, hypoxanthine, and uracil were uniformly sensitive to antibiotics but not hypersusceptible. In contrast, six strains of N gonorrhoeae isolated in Denmark required arginine (not satisfied by ornithine), hypoxanthine, and uracil and were hypersusceptible to vancomycin (inhibited by 0S5 M4g/ml), erythromycin, and rifampin. DNA-mediated transformation showed that all three hypersusceptibilities of one Danish strain were introduced together into a wild-type gonococcus, suggesting that a mutation of an env (envelope) locus might be responsible for the atypical permeability.
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