Purine metabolism in Leishmania donovani and Leishmania braziliensis

We have studied purine metabolism in the culture forms of Leishmania donovani and Leishmania braziliensis. These organisms are incapable of synthesizing purines de novo from glycine, serine, or formate and require an exogenous purine for growth. This requirement is better satisfied by adenosine or hypoxanthine than by guanosine. Bothe adenine and inosine are converted to a common intermediate, hypoxanthine, before transformation to nucleotides. This is due to the activity of an adenine aminohydrolase (EC 3.5.4.2), a rather unusual finding in a eukaryotic cell. There is a preferential synthesis of adenine nucleotides, even when guanine or xanthine are used as precursors.The pathways of purine nucleotide interconversions in these Leishmania resemble those found in mammalian cells except for the absence of de novo purine biosynthesis and the presence of an adenine-deaminating activity.     

Guanosine metabolism in Neurospora crassa

Two aspects of guanosine metabolism in Neurospora have been investigated. (a) The inability of adenine mutants (blocked prior to IMP synthesis) to use guanosine as a nutritional supplement; and (b) the inhibitory effect of guanosine on the utilization of hypoxanthine as a purine source for growth by these mutants. Studies on the utilization of guanosine indicated that the proportion of adenine derived from guanosine may be limiting for the growth of adenine mutants. In wild type, adenine is produced through the biosynthetic pathway when grown in the presence of guanosine. The amount of adenine produced through the de novo biosynthesis in wild type increases with increasing concentrations of guanosine in the medium. However, the total purine synthesis does not increase. Guanosine inhibits the uptake of hypoxanthine severely. In addition, guanosine and its nucleotide derivatives also inhibit the hypoxanthine phosphoribosyltransferase activity, at the same time stimulating the adenine phosphoribosyltransferase activity. Guanosine's effects on the uptake of hypoxanthine and its conversion to the nucleotide form may be the reasons why guanosine inhibits the utilization of hypoxanthine but not adenine by these mutants.     

Purine salvage to adenine nucleotides in different skeletal muscle fiber types.

Rates of purine salvage of adenine and hypoxanthine into the adenine nucleotide (AdN) pool of the different skeletal muscle phenotype sections of the rat were measured using an isolated perfused hindlimb preparation. Tissue adenine and hypoxanthine concentrations and specific activities were controlled over a broad range of purine concentrations, ranging from 3 to 100 times normal, by employing an isolated rat hindlimb preparation perfused at a high flow rate. Incorporation of [(3)H]adenine or [(3)H]hypoxanthine into the AdN pool was not meaningfully influenced by tissue purine concentration over the range evaluated (approximately 0.10-1.6 micromol/g). Purine salvage rates were greater (P < 0.05) for adenine than for hypoxanthine (35-55 and 20-30 nmol x h(-1) x g(-1), respectively) and moderately different (P < 0.05) among fiber types. The low-oxidative fast-twitch white muscle section exhibited relatively low rates of purine salvage that were approximately 65% of rates in the high-oxidative fast-twitch red section of the gastrocnemius. The soleus muscle, characterized by slow-twitch red fibers, exhibited a high rate of adenine salvage but a low rate of hypoxanthine salvage. Addition of ribose to the perfusion medium increased salvage of adenine (up to 3- to 6-fold, P < 0.001) and hypoxanthine (up to 6- to 8-fold, P < 0.001), depending on fiber type, over a range of concentrations up to 10 mM. This is consistent with tissue 5-phosphoribosyl-1-pyrophosphate being rate limiting for purine salvage. Purine salvage is favored over de novo synthesis, inasmuch as delivery of adenine to the muscle decreased (P < 0.005) de novo synthesis of AdN. Providing ribose did not alter this preference of purine salvage pathway over de novo synthesis of AdN. In the absence of ribose supplementation, purine salvage rates are relatively low, especially compared with the AdN pool size in skeletal muscle.     

Nature of 6-methylpurine inhibition and characterization of two 6-methylpurine-resistant mutants of Neurospora crassa.

6-Methylpurine, an analog of adenine, inhibits the growth of Neurospora crassa. From kinetic studies it was found that 6-methylpurine is converted to its nucleotide form by adenine phosphoribosyltransferase (EC 2.4.2.7), and inhibits the de novo purine biosynthesis. Adenine relieves the growth inhibition caused by 6-methylpurine, whereas hypoxanthine is not very effective. Studies dealing with hypoxanthine utilization in the presence of 6-methylpurine indicated a severely reduced uptake of hypoxanthine and a general slowdown in its further metabolism. Two mutants (Mepr-3 and Mepr-10) which are resistant to 6-methylpurine were characterized. Studies of purine base uptake and the in vivo and in vitro conversion to nucleotides indicated that Mepr-10 may be an adenine phosphoribosyltransferase-defective mutant, whereas Mepr-3 may be a mutant with altered feedback response to 6-methylpurine. Both mutants showed a severely lowered hypoxanthine phosphoribosyltransferase activity, but because 6-methylpurine did not have any effect on the conversion of hypoxanthine to IMP in the wild type, it was concluded that 6-methylpurine resistance in these mutants cannot be due to lowered hypoxanthine phosphoribosyltransferase activity, but rather that the lowering of enzyme activity may be a secondary effect.     

The potentiation of adenine toxicity to Chinese hamster cells by coformycin: Suppression in mutants with altered regulation of purine biosynthesis or increased adenylate-deaminase activity

When added to medium containing coformycin (2 μM or above), adenine is lethal to Chinese hamster fibroblasts at the concentration inhibiting de novo purine biosynthesis (Debatisse and Buttin, '77b). Rescue by hypoxanthine suggested that cells die of IMP starvation when the analog can turn off deamination of both adenosine and adenylate. As predicted from this hypothesis, two classes of variants resistant to the mixture of coformycin + adenine have been isolated: Class 1 variants have altered control of de novo IMP biosynthesis; they fall into two subclasses on the basis of their resistance to adenosine. Class 2 variants have a 6–10-fold increased level of AMP-deaminase (E.C.: 3.5.4.6); their growth in the selective medium is temperature-dependent, a property accounted for by the observation that cell growth in the presence of coformycin imposes a gradual thermodependent decay of specific AMP-deaminase activity in both wild-type and variant lines. This control by coformycin of AMP-deaminase activity isunaltered in mutants deficient in the four activities of adenosine-kinase, APRT, HGPRT and deoxycytidine-kinase. Most of the resistant variants are unstable and exhibit either increased or reduced resistance, depending on prolonged growth in selective or normal medium.     

Transport of adenine, hypoxanthine and uracil into Escherichia coli.

Uptake of adenine, hypoxanthine and uracil by an uncA strain of Escherichia coli is inhibited by uncouplers or when phosphate in the medium is replaced by less than 1 mM-arsenate, indicating a need for both a protonmotive force and phosphorylated metabolites. The rate of uptake of adenine or hypoxanthine was not markedly affected by a genetic deficiency of purine nucleoside phosphorylase. In two mutants with undetected adenine phosphoribosyltransferase, the rate of adenine uptake was about 30% of that in their parent strain, and evidence was obtained to confirm that adenine had then been utilized via purine nucleoside phosphorylase. In a strain deficient in both enzymes adenine uptake was about 1% of that shown by wild-type strains. Uptake of hypoxanthine was similarly limited in a strain lacking purine nucleoside phosphorylase, hypoxanthine phosphoribosyltransferase and guanine phosphoribosyltransferase. Deficiency of uracil phosphoribosyltransferase severely limits uracil uptake, but the defect can be circumvented by addition of inosine, which presumably provides ribose 1-phosphate for reversal of uridine phosphorylase. The results indicate that there are porter systems for adenine, hypoxanthine and uracil dependent on a protonmotive force and facilitated by intracellular metabolism of the free bases.     

Lack of enhanced purine biosynthesis in HGPRT- and Lesch-Nyhan cells.

The rate of de novo purine biosynthesis was measured in a series of hypoxanthine guanine phosphoribosyl transferase deficient (HGPRT-) cells from a variety of sources, including human Lesch-Nyhan cells. Under optimum growth conditions, no enhanced purine biosynthesis was detected (in contrast to previous reports). An 'elevated' level of de novo purine biosynthesis could be detected in mutants following starvation for glutamine. However, this was the result of depression of purine biosynthesis in normal cells, with a resulting artifactual overproduction in mutants.     

A comprehensive model of purine uptake by the malaria parasite Plasmodium falciparum: identification of four purine transport activities in intraerythrocytic parasites

Plasmodium falciparum is incapable of de novo purine biosynthesis, and is absolutely dependent on transporters to salvage purines from the environment. Only one low-affinity adenosine transporter has been characterized to date. In the present study we report a comprehensive study of purine nucleobase and nucleoside transport by intraerythrocytic P. falciparum parasites. Isolated trophozoites expressed (i) a high-affinity hypoxanthine transporter with a secondary capacity for purine nucleosides, (ii) a separate high-affinity transporter for adenine, (iii) a low-affinity adenosine transporter, and (iv) a low-affinity/high-capacity adenine carrier. Hypoxanthine was taken up with 12-fold higher efficiency than adenosine. Using a parasite clone with a disrupted PfNT1 (P. falciparum nucleoside transporter 1) gene we found that the high-affinity hypoxanthine/nucleoside transport activity was completely abolished, whereas the low-affinity adenosine transport activity was unchanged. Adenine transport was increased, presumably to partly compensate for the loss of the high-affinity hypoxanthine transporter. We thus propose a model for purine salvage in P. falciparum, based on the highly efficient uptake of hypoxanthine by PfNT1 and a high capacity for purine nucleoside uptake by a lower affinity carrier.     

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.     

Helicobacter pylori relies primarily on the purine salvage pathway for purine nucleotide biosynthesis

Helicobacter pylori is a chronic colonizer of the gastric epithelium and plays a major role in the development of gastritis, peptic ulcer disease, and gastric cancer. In its coevolution with humans, the streamlining of the H. pylori genome has resulted in a significant reduction in metabolic pathways, one being purine nucleotide biosynthesis. Bioinformatic analysis has revealed that H. pylori lacks the enzymatic machinery for de novo production of IMP, the first purine nucleotide formed during GTP and ATP biosynthesis. This suggests that H. pylori must rely heavily on salvage of purines from the environment. In this study, we deleted several genes putatively involved in purine salvage and processing. The growth and survival of these mutants were analyzed in both nutrient-rich and minimal media, and the results confirmed the presence of a robust purine salvage pathway in H. pylori. Of the two phosphoribosyltransferase genes found in the H. pylori genome, only gpt appears to be essential, and an Δapt mutant strain was still capable of growth on adenine, suggesting that adenine processing via Apt is not essential. Deletion of the putative nucleoside phosphorylase gene deoD resulted in an inability of H. pylori to grow on purine nucleosides or the purine base adenine. Our results suggest a purine requirement for growth of H. pylori in standard media, indicating that H. pylori possesses the ability to utilize purines and nucleosides from the environment in the absence of a de novo purine nucleotide biosynthesis pathway.     

Effects of methotrexate on nucleotide pools in normal human T cells and the CEM T cell line

It has been proposed that the clinical utility of methotrexate (MTX) in the treatment of rheumatoid arthritis may be due, in part, to inhibition of 5-amino imidazole-4-carboxamide ribonucleotide formyltransferase (AICARFT) by polyglutamated forms of MTX. AICARFT is the second folate dependent enzyme in de novo purine biosynthesis. In this study, the effects of MTX on de novo purine biosynthesis as well as total nucleotide pools were evaluated in both the human T cell line, CEM, and phytohemagglutinin-activated normal human T lymphocytes. De novo synthesized purines were metabolically labeled with 14C-glycine after MTX treatment and analyzed by HPLC. In normal T cells, MTX produced a dose-dependent reduction in de novo adenosine and guanosine pools with maximal effects (>50%) at 1 microM MTX. In CEM cells, de novo purine synthesis was almost completely blocked by 1 microM MTX. Total purine pools were also reduced in both cell types after MTX treatment. Since 1 microM MTX caused almost complete growth inhibition in CEM cells, we evaluated whether growth could be reconstituted with exogenous purine bases and pyrimidine nucleosides which can be utilized via salvage pathways. The combination of hypoxanthine and thymidine substantially reversed growth inhibition with 1 microM MTX in CEM cells. Taken together, these results demonstrate that MTX inhibits de novo nucleotide synthesis in T cells and suggest that AICARFT inhibition may be one aspect of the multi-site mechanism of MTX action in the treatment of rheumatoid arthritis.     

Purine metabolism in cultured aortic and coronary endothelial cells

Purine salvage pathways in cultured endothelial cells of macrovascular (pig aorta) and microvascular (guinea pig coronary system) origin were investigated by measuring the incorporation of radioactive purine bases (adenine or hypoxanthine) or nucleosides (adenosine or inosine) into purine nucleotides. These precursors were used at initial extracellular concentrations of 0.1, 5, and 500 microM. In both types of endothelial cells, purine nucleotide synthesis occurred with all four substrates. Aortic endothelial cells salvaged adenine best among purines and nucleosides when applied at 0.1 microM. At 5 and 500 microM, adenosine was the best precursor. In contrast, microvascular endothelial cells from the coronary system used adenosine most efficiently at all concentrations studied. The synthetic capacity of salvage pathways was greater than that of the de novo pathway. As measured using radioactive formate or glycine, de novo synthesis of purine nucleotides was barely detectable in aortic endothelial cells, whereas it readily occurred in coronary endothelial cells. Purine de novo synthesis in coronary endothelial cells was inhibited by physiological concentrations of purine bases and nucleosides, and by ribose or isoproterenol. The isoproterenol-induced inhibition was prevented by the beta-adrenergic receptor antagonist propranolol. The end product of purine catabolism in aortic endothelial cells was found to be hypoxanthine, whereas coronary endothelial cells degraded hypoxanthine further to xanthine and uric acid, a reaction catalyzed by the enzyme xanthine dehydrogenase.     

Regulation of purine de novo synthesis in cultured human fibroblasts: the role of P-ribose-PP.

Procedures for assaying the rate of purine de novo synthesis in cultured fibroblast cells have been compared. These were (i) the incorporation of [(14)C]-glycine or [(14)C]formate in alpha-N-formylglycinamide ribonucleotide (an intermediate in the purine synthetic pathway) and (ii) the incorporation of [(14)C]-formate into newly synthesised cellular purines and purines excreted by the cell into the medium. Fibroblast cells, derived from patients with a deficiency of hypoxanthine phosphoribosyltransferase (HPRT-) (EC 2.4.2.8) and increased rates of purine de novo synthesis, were compared with fibroblasts from healthy subjects (HPRT+). Fetal calf serum, which was used to supplement the assay and cell growth medium, was found to contain sufficient quantities of the purine base hypoxanthine to inhibit purine de novo synthesis in HPRT+ cells. This inhibition was the basis of differentiation between HPRT- and HPRT+ cells. In the absence of added purine base, both cell types had similar capacities for purine de novo synthesis. This result contrasts with the increased rates of purine de novo synthesis reported for a number of human HPRT- cells in culture but conforms recent studies made on human HPRT- lymphoblast cells. The intracellular concentration and utilisation of 5-phosphoribosyl-1-pyrophosphate (P-Rib-PP), a substrate and potential controlling factor for purine de novo synthesis, were determined in HPRT- and HPRT+ cells. The rate of utilisation of P-Rib-PP in the salvage of free purine bases was far greater than that in purine de novo synthesis. Although HPRT- cells had a 3-fold increase in P-Rib-PP content, the rate of P-Rib-PP generation was similar to HPRT+ cells. Thus, in fibroblasts, the concentration of P-Rib-PP appears to be critical in the control of de novo purine synthesis and its preferential utilisation in the HPRT reaction limits its availability for purine de novo synthesis. In vivo, HPRT+ cells, in contrast to HPRT- cells, may be operating purine de novo synthesis at a reduced rate because of their ability to reutilise hypoxanthine.     

Regulation of de novo purine biosynthesis in Chinese hamster cells

Regulation of de novo purine biosynthesis was examined in two Chinese hamster cell lines, CHO and V79. De novo purine biosynthesis is inhibited at low concentrations of adenine. The mechanism of inhibition was studied using the RNA and protein synthesis inhibitors actinomycin D, cycloheximide, and azacytidine. Although all three inhibitors rapidly inhibited de novo purine biosynthesis in vivo, neither adenine nor the RNA and protein synthesis inhibitors could be found to have an effect in vitro on either phosphoribosylpyrophosphate (PRPP) synthetase or amido phosphoribosyltransferase, the first enzymes of the de novo pathway. However, in the presence of actinomycin D, cycloheximide, and azacytidine, there was a 50% or greater reduction in PRPP concentrations. This reduction in PRPP levels is correlated with a 2-fold increase in purine nucleotides in the acid-soluble pool. It is proposed that in the presence of the metabolic inhibitors there is an increase in nucleotide pools due to degradation of RNA, with a resulting feedback inhibition on de novo purine biosynthesis. In contrast to a previous report (Martin, D. W., Jr., and Owen, N. T. (1972) J. Biol. Chem. 247, 5477-5485), we could find no evidence for a repressor type mechanism in these cells.     

Studies on purine transport and on purine content in vacuoles isolated from Saccharomyces cerevisiae.

The transport of purine derivatives into vacuoles isolated from Saccharomyces cerevisiae was studied. Vacuoles which conserved their ability to take up purine compounds were prepared by a modification of the method of polybase-induced lysis of spheroplasts. Guanosine greater than inosine = hypoxanthine greater than adenosine were taken up with decreasing initial velocities, respectively; adenine was not transported. Guanosine and adenosine transporting systems were saturable, with apparent Km values 0.63 mM and 0.15 mM respectively, while uptake rates of inosine and of hypoxanthine were linear functions of their concentrations. Adenosine transport in vacuoles appeared strongly dependent on the growth phase of the cell culture. The system transporting adenosine was further characterized by its pH dependency optimum of 7.1 and its sensitivity to inhibition by S-adenosyl-L-methionine. In the absence of adenosine in the external medium, [14C]adenosine did not flow out from preloaded vacuoles. However, in the presence of external adenosine, a very rapid efflux of radioactivity was observed, indicating an exchange mechanism for the observed adenosine transport in the vacuoles. In isolated vacuoles the only purine derivative accumulated was found to be S-adenosyl-L-homocysteine.     

Alteration of purine metabolism by AICA-riboside in human B lymphoblasts.

The effect of 5-amino-4-imidazole-carboximide (AI-CA)-riboside on different pathways of purine metabolism (biosynthesis de novo, salvage pathways, adenosine metabolism, ATP catabolism) was studied in human B lymphoblasts (WI-L2). AICA-Riboside markedly decreased intracellular levels of 5-phosphoribosyl-1-pyrophosphate and in consequence affected purine biosynthesis de novo and purine salvage pathways. AICA-riboside inhibited incorporation of glycine into purine nucleotides, but when formate was used as the precursor of purine biosynthesis de novo, a biphasic effect was observed. The incorporation of formate into purine nucleotides was increased by AICA-riboside at concentrations up to 2 mM but decreased at higher concentrations. Salvage of the purine bases adenine, hypoxanthine, and guanine was markedly inhibited and utilization of extracellular adenosine in B lymphoblasts was reduced by AICA-riboside. AICA-riboside increased ribose 1-phosphate concentrations and increased degradation of prelabeled ATP. No effect on the intracellular levels of orthophosphate was found. Proliferation of WI-L2 lymphoblasts was only slightly affected at concentrations of AICA-riboside below 500 microM but markedly inhibited by higher concentrations.     

Regulation of Purine Metabolism in Intact Leaves of Coffea arabica

The capacity of Coffea arabica leaves (5- x 5-mm pieces) to synthesize de novo and catabolize purine nucleotides to provide precursors for caffeine (1,3,7-trimethylxanthine) was investigated. Consistent with de novo synthesis, glycine, bicarbonate, and formate were incorporated into the purine ring of inosine 5[prime]-monophosphate (IMP) and adenine nucleotides ([sigma]Ade); azaserine, a known inhibitor of purine de novo synthesis, inhibited incorporation. Activity of the de novo pathway in C. arabica per g fresh weight of leaf tissue during a 3-h incubation period was 8 [plus or minus] 4 nmol of formate incorporated into IMP, 61 [plus or minus] 7 nmol into [sigma]Ade, and 150 nmol into caffeine (the latter during a 7-h incubation). Coffee leaves exhibited classical purine catabolism. Radiolabeled formate, inosine, adenosine, and adenine were incorporated into hypoxanthine and xanthine, which were catabolized to allantoin and urea. Urease activity was demonstrated. Per g fresh weight, coffee leaf squares incorporated 90 [plus or minus] 22 nmol of xanthine into caffeine in 7 h but degraded 102 [plus or minus] 1 nmol of xanthine to allantoin in 3 h. Feedback control of de novo purine biosynthesis was contrasted in C. arabica and Cucurbita pepo, a species that does not synthesize purine alkaloids. End-product inhibition was demonstrated to occur in both species but at different enzyme reactions.     

Effect of L-methionine and S-adenosylmethionine on growth of an adenine mutant of Saccharomyces cerevisiae

A pink, adenine-requiring yeast utilized adenine, hypoxanthine, or S-adenosylmethionine (SAM), in quantities up to 3 mumoles per 100 ml of medium, as equivalent sources of purine for cell growth, but not methylthioadenosine or S-adenosylhomocysteine. Utilization of SAM for growth was inhibited by the presence of l-methionine in quantities greater than 0.6 mumole per 100 ml of medium. However, 6 mumoles of l-methionine had no effect on growth when adenine or hypoxanthine was the source of purine. These sources also reversed the inhibitory effects of 6 mumoles of the amino acid on the utilization of SAM. The presence of 400 mumoles of the amino acid resulted in some inhibition of growth when the organisms were grown with adenine, hypoxanthine, or adenine plus SAM but had no effect on the total uptake of adenine-8-(14)C. Studies on the uptake of radioactivity from a mixture of SAM-adenine-8-(14)C and (3)H-labeled SAM-methyl indicated that these components were taken into the cells at different rates which were altered by the presence of l-methionine. The fixation of (35)S from (35)S-labeled adenosylmethionine into the cells was inhibited by the presence of the amino acid. The cells synthesized and accumulated SAM in the presence of 400 mumoles of l-methionine plus adenine even when exogenous SAM was supplied. Approximately 47% of radioactivity fixed from exogenous SAM-adenine-8-(14)C and 12% from (3)H-labeled SAM-methyl were found in reisolated SAM.     

Purine metabolism in Saccharomyces cerevisiae.

The synthesis, interconversion, and catabolism of purine bases, ribonucleosides, and ribonucleotides in wild-type Saccharomyces cerevisiae were studied by measuring the conversion of radioactive adenine, hypoxanthine, guanine, and glycine into acid-soluble purine bases, ribonucleosides, and ribonucleotides, and into nucleic acid adenine and guanine. The pathway(s) by which adenine is converted to inosinate is (are) uncertain. Guanine is extensively deaminated to xanthine. In addition, some guanine is converted to inosinate and adenine nucleotides. Inosinate formed either from hypoxanthine or de novo is readily converted to adenine and guanine nucleotides.     

Genetic Transformation of Biosynthetically Defective Neisseria gonorrhoeae Clinical Isolates

Deoxyribonucleic acid (DNA) and chemically defined media were used in transformation tests of 51 strains of Neisseria gonorrhoeae which exhibited various biosynthetic defects when isolated from patients. These auxotrophic gonococci had one or more nutritional requirements involving proline, methionine, arginine, hypoxanthine, uracil, and thiamine pyrophosphate (THPP). DNA from a clinical isolate which did not require these compounds for growth on defined medium transformed each of the auxotrophic markers of all 51 recipient populations. Ten isolates had defects involving the synthesis of THPP; four strains (designated Thp(-)) had a growth requirement that was satisfied only by THPP, whereas the requirement of six strains (designated Thi(-)) was satisfied by either thiamine or THPP. DNA from Thp(-) donors elicited transformation of Thp(-) as well as Thi(-) recipients. Reciprocally, DNA from a Thi(-) donor transformed both Thi(-) and Thp(-) recipients. Furthermore, DNA from other auxotrophic gonococci had transforming activity for some phenotypically similar auxotrophic recipients. The findings indicate the existence of various nonidentical genetic defects which block reactions in the biosynthesis of proline, methionine, arginine, hypoxanthine, and THPP. Routine cultures from patients with gonorrhea were the source of these auxotrophic strains of N. gonorrhoeae; the various nutritional requirements were identified by a recently described system of gonococcal auxotyping. The transformation test results verify the hereditary basis of the auxotypes, establish that many different mutations exist in potentially virulent gonococci, and illustrate the value of these auxotrophic mutants for studies of the genetic structure and evolution of natural populations of gonococci.