Physiology and metabolism of pathogenic Neisseria: partial characterization of the respiratory chain of Neisseria gonorrhoeae.

The cell membrane-associated respiratory electron transport chain of Neisseria gonorrhoeae was examined using electron paramagnetic spectroscopy (EPR) at liquid helium temperatures and optical spectroscopy at liquid nitrogen and room temperatures. EPR spectra of dithionite-reduced particles indicated the presence of centers N-1 and N-3 in the site I region of the respiratory chain, whereas reduction with succinate revealed the existence of center S-1 from the succinate cytochrome c reductase segment. Free radical(s) resembling that due to falvin semiquinone were observed with both reductants. Low temperature (77 K) optical difference spectra indicated the presence of cytochromes with alpha band maxima at 549, 557, and 562. Bands at 567, 535, and 417 nm, characteristic of the CO compound of cytochrome o, were also identified. Cytochromes a1 and a3 were not detected; however, a broad but weak absorbance with an alpha band maximun at 600 nm and a Soret shoulder at 440 nm was observed. Hence the respiratory chain of N. gonorrhoeae appears to contain several nonheme iron centers, cytochrome c, two b cytochromes, with cytochrome o which probably serves as the terminal oxidase.     

Purine metabolism in high- and low-uric acid lines of chickens: hypoxanthine/guanine phosphoribosyltransferase activities

The activity of hypoxanthine/guanine phosphoribosyltransferase (HGPRT) was examined in the livers and kidneys of two genetic lines of chickens selected for different plasma uric acid levels. Previous work demonstrated that the high-uric acid line (HUA) had significantly greater de novo uric acid synthesis rates in kidney tissue compared to the low-uric acid line (LUA). In addition, phosphoribosylpyrophosphate (PRPP) synthetase and xanthine dehydrogenase activities in livers and kidneys were significantly higher in the HUA compared to the LUA line. PRPP pool sizes were also significantly higher in both livers and kidneys of HUA birds. HGPRT activities in livers of HUA birds were significantly (P less than 0.05) greater than in LUA birds. The mean value of liver HGPRT was 7.36 +/- 0.25 pmole inosine-5'-monophosphate (IMP) and 6.05 +/- 0.27 pmole IMP produced/micrograms protein/hr, respectively, for the HUA and LUA lines. There were no significant differences (P greater than 0.05) in kidney HGPRT activities between the two groups. The mean value of kidney HGPRT was 52.87 +/- 1.62 pmole IMP and 50.72 +/- 1.62 pmole IMP produced/micrograms protein/hr, respectively, for the HUA and LUA line. Elevated liver HGPRT may serve to enhance the regeneration of PRPP in the HUA liver. Elevated liver PRPP synthetase and PRPP pool size suggest an increased flux through the de novo purine biosynthetic pathway in HUA birds. The resulting additional pyrophosphate from the glutamine PRPP amidotransferase reaction would stimulate recovery of PRPP and spare the system from a substantial loss of energy.     

The effect of iron on the metabolism and pathogenicity of Neisseria gonorrhoeae

Abstract Neisseria gonorrhoeae can undergo phenotypic variation in cell surface structure and virulence, depending on the level of available iron in the environment. Studies performed in vitro show that under conditions of iron limitation, gonococci express new outer membrane proteins, and possibly produce siderophores, while an increase in available iron may enhance the lethality of gonococci for chicken embryos and induce resistance to inactivation by serum. These findings suggest that iron metabolism may be important in the pathogenesis of gonococcal infection.     

Genome plasticity in Neisseria gonorrhoeae

Abstract The pathogenic Neisseria have exploited the processes of horizontal DNA transfer and genetic recombination as mechanisms for the generation of extensive protein variation and modulation of gene expression. Localized recombinations have been well documented in members of multigene families as have alterations in short repetitive sequences. Here we report an analysis of the chromosomal structure of a defined lineage of Neisseria gonorrhoeae strain MS 11 pilin variants. This study reveals the occurrence of large rearrangements, including the amplification of a 26 kb region and an inversion involving more than a third of the chromosome. Additionally, a restriction site polymorphism that correlates with pilin expression has been observed. These findings highlight the flexibility of the gonococcal genome.     

Glucose Metabolism in Neisseria gonorrhoeae

The metabolism of glucose was examined in several clinical isolates of Neisseria gonorrhoeae. Radiorespirometric studies revealed that growing cells metabolized glucose by a combination on the Entner-Doudoroff and pentose phosphate pathways. A portion of the glyceraldehyde-3-phosphate formed via the Entner-Doudoroff pathway was recycled by conversion to glucose-6-phosphate. Subsequent catabolism of this glucose-6-phosphate by either the Entner-Doudoroff or pentose phosphate pathways yielded CO(2) from the original C6 of glucose. Enzyme analyses confirmed the presence of all enzymes of the Entner-Doudoroff, pentose phosphate, and Embden-Meyerhof-Parnas pathways. There was always a high specific activity of glucose-6-phosphate dehydrogenase (EC 1.1.1.49) relative to that of 6-phosphogluconate dehydrogenase (EC 1.1.1.44). The glucose-6-phosphate dehydrogenase utilized either nicotinamide adenine dinucleotide phosphate or nicotinamide adenine dinucleotide as electron acceptor. Acetate was the only detectable nongaseous end product of glucose metabolism. Following the disappearance of glucose, acetate was metabolized by the tricarboxylic acid cycle as evidenced by the preferential oxidation of [1-(14)C]acetate over that of [2-(14)C]acetate. When an aerobically grown log-phase culture was subjected to anaerobic conditions, lactate and acetate were formed from glucose. Radiorespirometric studies showed that under these conditions, glucose was dissimilated entirely by the Entner-Doudoroff pathway. Further studies determined that this anaerobic dissimilation of glucose was not growth dependent.     

Neisseria gonorrhoeae: Acquisition of auxotrophy in the mouse

During passage in the normal mouse, a strain ofNeisseria gonorrhoeae (F62) has acquired genetic defects phenotypically expressed in requirements for arginine and uracil and a partial requirement for hypoxanthine. In addition, the mouse-passaged strain exhibits slower growth and colonial morphology that differs from any of the four parental colony types. Multiple changes in the mouse-passaged strain suggest several mutations. The new auxotrophic requirements and some of the colony characteristics of the mouse-derived strain resemble gonococci isolated from disseminated human infections. A normal mouse model for gonorrhea may contribute greatly to future understanding of the adaptive events occurring in the gonococcus as a result of selective host pressures.     

Physiology and metabolism of pathogenic neisseria: tricarboxylic acid cycle activity in Neisseria gonorrhoeae.

Tricarboyxlic acid cycle activity was examined in Neisseria gonorrhoeae CS-7. The catabolism of glucose in N. gonorrheae by a combination of the Entner-Doudoroff and pentose phosphate pathways resulted in the accumulation of acetate, which was not further catabolized until the glucose was depleted or growth became limiting. Radiorespirometric studies revealed that the label in the 1 position of acetate was converted to CO2 at twice the rate of the label in the 2 position, indicating the presence of a tricarboxylic acid cycle. Growth on glucose markedly reduced the levels of all tricarboxylic acid cycle enzymes except citrate synthase (EC 4.1.3.7). Extracts of glucose-grown cells contained detectable levels of all tricarboxylic acid cycle enzymes except aconitase (EC 4.2.1.3), isocitrate dehydrogenase (EC 1.1.1.42), and a pyridine nucleotide-dependent malate dehydrogenase (EC 1.1.1.37). Extracts of cells capable of oxidizing acetate lacked only the pyridine nucleotide-dependent malate dehydrogenase. In lieu of this enzyem, a particulate pyridine nucleotide-independent malate oxidase (EC 1.1.3.3) was present. This enzyme required flavin adenine dinucleotide for activity and appeared to be associated with the electron transport chain. Radiorespirometric studies utilizing labeled glutamate demonstrated that a portion of the tricarboxylic acid cycle functioned during glucose catabolism. In spite of the presence of all tricarboxylic acid cycle enzymes, N. gonorrhoeae CS-7 was unable to grow in medium supplemented with cycle intermediates.     

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.     

Survival of Neisseria gonorrhoeae in the Mail

Cultures of Neisseria gonorrhoeae, which used Thayer-Martin slants as transport medium, survived at least 1 day in the mail.     

Coagglutination as a test for Neisseria gonorrhoeae

After growth on Thayer-Martin medium, 196 strains of freshly isolated Neisseria gonorrhoeae were subjected to a coagglutination reaction. The sensitivity of the test was 94% and did not vary much in the hands of four consecutive technicians. In a group of 99 strains tested by one of the technicians non-interpretable results were obtained with 17% of the strains when the test was performed with cells taken from the first or primary plate, against 9% when cells from the secondary (subcultured) plate were used. The lowest number of non-interpretable results was found with a modified Thayer-Martin medium, which also showed the lowest number of false negatives (2%).No non-interpretable results were obtained when the bacterial suspension was first heated to 100°C for 3 min. In a group of 14 recently isolated strains of non-gonococcal species there was only one, preventable, false-positive strain and there were none in a group of 12 meningococci (all of them laboratory strains).In comparison with the fermentation test with Lingelsheim's sugars, the coagglutination test with cells taken from the primary plate with Thayer-Martin medium yielded a conclusive result more often. The test is simple and rapid and does not require special technical equipment. It seems to deserve a place as a confirmative test in the search for gonococci in samples from the urogenital-anal area.     

Modification of the Rapid Fermentation Test for Neisseria gonorrhoeae

A modification of the rapid sugar fermentation procedure has been developed because of difficulties encountered in reproducing the reported results. Changes in the method have made the test more sensitive and reproducible.     

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.     

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.     

Purine synthesis de novo and salvage in hypoxanthine phosphoribosyltransferase-deficient mice

Extreme degrees of hypoxanthine phosphoribosyltransferase (HPRT) deficiency in man are associated with gross sex-linked neurological dysfunction, gout and urinary stones (the Lesch-Nyhan or 'complete HPRT-deficiency' syndrome). The less severe degrees of enzyme deficiency (sex-linked recessive gout and/or urolithiasis or the 'partial HPRT-deficiency' syndrome) may be associated with minor neurological manifestations. Whole body purine synthesis de novo is accelerated in both these groups of patients. A strain of mice with an experimentally produced mutation at the HPRT locus showed some residual 'apparent HPRT activity' in brain, liver, testicular, splenic, kidney and ovarian tissues but not in erythrocyte haemolysates. The mutation removes exons 1 and 2 of the coding region of the gene together with the promotor and about 10 kb of upstream sequence from the gene. It is therefore possible that the observed 'apparent HPRT activity' in these mice is due to the operation of an alternative metabolic pathway. Purine synthesis de novo was markedly accelerated in their brain, testicular, splenic and kidney tissues. It was not accelerated in the liver tissue of male mice hemizygous for the mutation and the degree of acceleration in the female homozygotes only just reached statistical significance at the p = 0.02 level. This observation casts doubt on the importance of modulations in the rate of hepatic purine synthesis de novo as a mechanism for maintaining a steady supply of purines for translocation to other organs.