Auxanographic grouping and typing of Neisseria gonorrhoeae.

Nearly 96% of 1297 Neisseria gonorrhoeae isolates in Hamilton were assigned to six major auxanographic groups (non-requiring or NR, Pro-, Orn-, Pro-Cit-Ura-, Orn-Ura-Hyx-, Cit-Ura-Hyx-) as established by requirements for none, or any one or more of proline, uracil, hypoxanthine, citrulline (Cit-), or citrulline replaceable by ornithine (Orn-) on chemically defined medium modified from Catlin (1973). Seven other groups, and strains not growing, accounted for 4.2%. The most common groups were Orn-Ura-Hyx- (25.6%) and Pro-Cit-Ura (31.8%). This latter group has not been previously described. These "Pro Cit/Orn Ura Hyx" criteria were among the most unequivocal to interpret; with rare exceptions for proline, a requirement was shown by absence of growth at any time in the zones of inoculation from a replicator. For some strains, some of the possible additional requirements (leucine, valine, isoleucine, glutamine, threonine, serine, histidine, etc.) could be less readily ascertained, because an occasional manifestation was to reduce the amount of growth, or to slow down the rate of growth, compared to complete medium. The first four of the six groups were the least fastidious, in that few strains had any additional requirements. About 2% of strains were inhibited by 0.25 mM phenylalanine.     

Biochemical and genetic studies with arginine and proline auxotrophs of Neisseria gonorrhoeae

The prevalence of specific arginine biosynthesis gene defects was studied for 319 arginine-requiring clinical isolates of Neisseria gonorrhoeae by using the ability of the strains to utilize intermediates of arginine biosynthesis. Only 11% of the uracil-requiring strains defective in the carbamylation of ornithine to yield citrulline had a defective carbamoylphosphate synthetase gene (carAB). Strains defective in carAB were of auxotype CUH. The other strains (89%) having a dual requirement for citrulline and uracil, which were mostly of auxotype PCU, were defective in the ornithine transcarbamoylase gene (argF). Over 90% of the strains were defective either in argJ (174 strains) or in argF (126 strains). Three argininosuccinate-requiring strains (i.e., defective in argG) of auxotype PAU were identified. Some of the arginine auxotrophs of N. gonorrhoeae defective in carAB, argJ, argF, or argG were complemented by genetic transformation with DNA from recombinant bacteriophages carrying characterized gonococcal arginine biosynthesis genes. Gene defects in proA (five strains) and in proB (six strains) were identified by gonococcal transformation assays with recombinant bacteriophages or plasmids carrying proline biosynthesis genes from N. gonorrhoeae. None of the 11 proline-requiring strains tested was defective in proC.     

Arginine and pyrimidine biosynthetic defects in Neisseria gonorrhoeae strains isolated from patients

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- Ura- strains. Activities of glutamate acetyltransferase, aspartate transcarbamylase, and orotate phosphoribosyltransferase, encoded respectively by argE, pyrB, and pyrE, were absent in some Arg- Ura- isolates. Gonococci that were unable to utilize ornithine for growth in place of citrulline lacked activity of carbamyl phosphate synthetase (encoded by car). Defects of car imposed requirements for both citrulline (or arginine) and a pyrimidine because of the dual role of carbamyl phosphate in the two pathways. Defects of argE, car, pyrB, and pyrE were separately introduced by genetic transformation into representatives of a gonococcal strain which initially was prototrophic. Results of enzyme assays of these isogenic auxotrophic transformants confirmed the gene-enzyme relationships.     

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.     

Nutritional induction and suppression of fruiting in Myxococcus xanthus FBa

A defined agar medium (A agar) containing 15 amino acids in concentrations between 0.5 and 2 mm was developed for studying the fruiting cycle of Myxococcus xanthus FBa. Cells grew only vegetatively in this medium unless the initial concentration of one of nine required or stimulatory amino acids was lowered about 50-fold. In the latter circumstance, fruiting bodies developed after several days of vegetative growth. The conclusion was that fruiting occurred when any amino acid required for normal growth became limiting in the environment. High concentrations (10 mm) of phenylalanine, tryptophan, or methionine prevented fruiting without affecting growth. Mutants requiring arginine, thymidine, or adenine could not be induced to fruit by limiting their unique requirement although they responded to the same deprivations which brought about fruiting of the wild type. A histidine auxotroph formed fruiting bodies when histidine was lowered to growth-limiting concentrations, provided that the medium was supplemented with purines. A uracil auxotroph was isolated that, perhaps secondarily, had lost some of the mechanisms which control the formation of fruiting bodies; if uracil was present, it formed fruits even when no amino acid was limiting. No concentration of uracil was sufficient to prevent fruiting. Fruiting bodies were formed when mixtures of the uracil auxotroph and wild-type cells were inoculated on A agar plus uracil, even when 75% of the cells were wild type. Microcysts of both strains were present in the fruiting bodies.     

Development of a defined minimal medium for the growth of Neisseria gonorrhoeae

This investigation describes the development of a solid and a liquid medium (Gonococcal Genetic Medium; GGM) which support the rapid growth of 41 gonococcal clinical isolates and laboratory strains with a minimum number of nutritional components. The complete medium contains minimal salts, eight amino acids, two nitrogen bases, vitamins, coenzymes, key metabolic intermediates, and some miscellaneous components. Results indicate that GGM can be modified and simplified even further than we described. In liquid GGM, several gonococcal strains grew logarithmically after a 2- to 3-h lag period with generation times ranging from 72 to 115 min, reaching optical densities of 175 to 320 Klett units in the presence of seven amino acids and in the absence of a CO(2) atmosphere. The development of a solid and a liquid defined minimal medium such as GGM should greatly broaden the avenues of experimentation for biochemical genetic studies with N. gonorrhoeae, especially gonococcal genetic transformation. N. gonorrhoeae can be classified into eight major and minor phenotypic groups, depending on its growth responses on GGM to just five amino acids: cysteine and cystine, arginine, proline, isoleucine, and serine. Such results demonstrate the feasibility of using GGM as a simple, sensitive, rapid probe for investigating the epidemiological patterns of gonorrhea.     

Polyamines and regulation of ornithine biosynthesis in Escherichia coli.

The growth rate of several polyamine-deficient mutants of Escherichia coli was very low in minimal medium and increased markedly upon the addition of putrescine, spermidine, arginine, citrulline, or argininosuccinic acid. The endogenous content of polyamines was not significantly altered by the supplementation of polyamine-starved cultures with arginine or its precursors. In contrast, these compounds as well as putrescine or spermidine caused a 40-fold reduction in intracellular ornithine levels when added to polyamine-depleted bacteria. In vivo experiments with radioactive glutamic acid as a precursor and in vitro assays of the related enzymes showed that the decrease in ornithine levels was due to the inhibition of its biosynthesis rather than to an increase in its conversion to citrulline or delta 1-pyrroline-5-carboxylic acid and proline. High endogenous concentrations of ornithine were toxic for the E. coli strains tested. The described results indicate that the stimulatory effect of putrescine and spermidine on the growth of certain polyamine-starved bacteria may be partially due to the control of ornithine biosynthesis by polyamines.     

General approach to bacterial nutrition: growth factor requirements of Moraxella nonliquefaciens.

A general procedure was devised for the determination of growth factor requirements of heterotrophic bacteria based upon identification of individual nutrients as they are successively depleted from a limited quantity of complex medium. By using this approach, it was possible to develop a defined medium for growth of Moraxella nonliquefaciens that contained nine amino acids and three vitamins. Three of the amino acids, proline, serine, and cysteine, were required in unusually high concentrations to obtain optimal growth. Methionine had a sparing action on the requirements for serine and cysteine. Glycine could substitute for serine. Although a required nutrient, cysteine was inhibitory for growth, but this inhibitory action was antagonized by valine or leucine. The requirement for cysteine was satisfied by cystine, glutathione, or sodium sulfide. M. nonliquefaciens could not use ammonia as a nitrogen source but could use glutamate or aspartate for this purpose. With the exception of 1 auxotrophic strain, the growth factor requirements of 23 independently isolated strains of M. nonliquefaciens were essentially the same.     

Neisseria gonorrhoeae Auxotyping: Differentiation of Clinical Isolates Based on Growth Responses on Chemically Defined Media

A system is described for differentiating clin?cal isolates of Neisseria gonorrhoeae based on their growth or absence of growth on a set of 11 chemically defined agar media. The complete medium, NEDA, contains all of the compounds required for gonococcal growth; but isolates differ in their ability to grow on NEDA from which selected compounds are individually omitted. The differential compounds include L-proline, L-arginine, L-ornithine, L-methionine, hypoxanthine, uracil, thiamine, and thiamine pyrophosphate. A distinctive pattern of growth responses on the standard media defines an auxotype. Twenty auxotypes were found among a group of 251 gonococci which were isolated from patients examined in the clinics of one city during a 3-month span of time. Another collection of 74 strains from several different countries yielded two additional auxotypes. The stability of the nutritional requirements on which the auxotyping depends was verified in two ways. Cultures isolated from different anatomic sites of a patient or from sexual partners represented the same auxotype, as did cultures which were repeatedly isolated from cases of presumptive treatment failures. Also, the auxotypes of gonococci remained the same after numerous subcultures. The reproducibility of results and the variety and number of auxotypes indicate the potential value of the auxotyping system as an epidemiological tool.     

Nutritional studies of histoplasma capsulatum

Summary A chemically defined medium, composed of inorganic salts, glucose, asparagine, cystine, and a vitamin supplement, has been devised for growth of the yeast phase ofHistoplasma capsulatum. Growth in this medium was abundant and compared favorably with that in media containing complex natural material. Conversion of each of the 20 strains examined was accomplished by one or more passages on agar slants of the medium and incubation of the cultures at 37° C. Yeast phase cultures on this medium have been stored for 6 months or more at approximately 4° C without conversion or loss of viability. Of the 20 strains examined for vitamin requirements of the yeast phase, all were partially deficient for thiamine; nine for inositol; five, either partially or completely deficient for niacin; and one, completely deficient for biotin.No specific amino acid was required for growth of the yeast phase, but an organic source of sulfur and one of nitrogen were essential. Cystine and cysteine were equally effective for growth of the yeast phase when supplied on an equivalent sulfur basis and very little difference in growth occurred in media which contained equal amounts of nitrogen in any one of the following compounds: asparagine, glutamic acid, aspartic acid, and proline,Of the 20 strains, all but one, which requires biotin, were capable of continued growth in the mycelial phase when subcultured on an agar medium containing only inorganic salts and dextrose, but growth was improved significantly by asparagine or casein hydrolysate.This investigation was supported in part by a PHS research grant (AI-03524) from the National Institute of Allergy and Infectious Diseases, Public Health Service.     

Induced changes in the surface of Neisseria gonorrhoeae

Growth of Neisseria gonorrhoeae strain F62 on medium containing pyruvate and a high ratio of cysteine to cystine resulted in functional and structural changes that are consistent with phenotypic changes in lipopolysaccharide. Both transparent (O-) and moderately opaque (O+) variants became more sensitive to killing by normal human serum and resistant to killing by pyocin G, a bacteriocin from Pseudomonas aeruginosa. Electrophoresis of outer membranes in the presence of sodium dodecyl sulfate demonstrated differences also dependent upon the growth medium. When gels were treated with periodic acid and stained with silver, lanes containing outer membranes obtained after growth in the modified medium demonstrated two bands in addition to those independent of the growth medium. The enhancement of these additional bands by periodate treatment indicated that they represent material containing carbohydrate. The mechanism by which the changes in the growth medium affected the surface of N. gonorrhoeae is not known; however, the changes demonstrated by electrophoresis were dependent upon either the high concentration of cysteine or the high ratio of cysteine to cystine.     

Isolation and characterization of Pseudomonas putida mutants affected in arginine, ornithine and citrulline catabolism: function of the arginine oxidase and arginine succinyltransferase pathways.

Pseudomonas putida mutants impaired in the utilization of arginine are affected in either the arginine succinyltransferase pathway, the arginine oxidase route, or both. However, mutants affected in one of the pathways still grow on arginine as sole carbon source. Analysis of the products excreted by both wild-type and mutant strains suggests that arginine is mainly channelled by the oxidase route. Proline non-utilizing mutants are also affected in ornithine utilization, confirming the role of proline as an intermediate in ornithine catabolism. Mutants affected in ornithine cyclodeaminase activity still grow on proline and become unable to use ornithine. Both proline non-utilizing mutants and ornithine-cyclodeaminase-minus mutants are unable to use citrulline. These results, together with induction of ornithine cyclodeaminase when wild-type P. putida is grown on citrulline, indicate that utilization of citrulline as a carbon source proceeds via proline with ornithine as an intermediate. Thus in P. putida, the aerobic catabolism of arginine on the one hand and citrulline and ornithine on the other proceed by quite different metabolic segments.     

Amino acid requirements of Eikenella corrodens

The amino acid requirements of asaccharolytic Eikenella corrodens strains were investigated and a minimal amino acid medium was developed. Single amino acid deletions performed in a chemically defined medium indicated that these strains required arginine, cysteine, histidine, lysine, and proline, and partially required tyrosine. These six amino acids plus aspartic acid, glutamic acid, and glycine supported growth of E. corrodens in a medium containing only inorganic salts and vitamins.     

Tritrichomonas foetus: Partly defined cultivation medium for study of the purine and pyrimidine metabolism

A partly defined medium was successfully designed for the cultivation of Tritrichomonas foetus, an anaerobic protozoan parasite of cattle. The medium consists of hypoxanthine, uracil, and thymidine as the sole precursors of nucleotides in T. foetus. Elimination of any one of the three precursors from the medium led to cessation of T. foetus growth. The information provided by this medium verifies our previous observations that T. foetus is incapable of de novo purine and pyrimidine synthesis, that hypoxanthine can be converted to AMP and GMP, that uracil is incorporated into all pyrimidine ribonucleotides including UDP-glucose—the precursor of glycogen synthesis, and that thymidine is the only precursor of TMP. The omission of folate from the medium, without affecting growth of T. foetus, also supports our previous finding that the parasite does not have functioning dihydrofolate reductase or thymidylate synthetase. The successful plating of T. foetus on agar plates incorporating the partly defined medium with near 100% plating efficiency makes it possible to isolate T. foetus mutants for further studies of purine and pyrimidine metabolism in this parasite.     

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.     

Biphasic resin system for growing concentrated cultures of microorganisms and its application to the cultivation of Neisseria gonorrhoeae.

A technique is described in which a polyester casting resin is used instead of agar for the growth of Neisseria gonorrhoeae in a biphasic system. In complex medium, higher concentrations and stability of T1 colony type were obtained with resin than with either agar or broth alone after 24 h of incubation. In defined medium, the growth of gonococci and colony type stability were similar in single-phase and resin systems and superior to the growth and stability occurring in the biphasic agar system.     

Effect of radioprotective agents in sporulation medium on Bacillus subtilis spore resistance to hydrogen peroxide, wet heat and germicidal and environmentally relevant UV radiation

Aims: To determine the effects of cysteine, cystine, proline and thioproline as sporulation medium supplements on Bacillus subtilis spore resistance to hydrogen peroxide (H₂O₂), wet heat, and germicidal 254 nm and simulated environmental UV radiation. Methods and Results: Bacillus subtilis spores were prepared in a chemically defined liquid medium, with and without supplementation of cysteine, cystine, proline or thioproline. Spores produced with thioproline, cysteine or cystine were more resistant to environmentally relevant UV radiation at 280–400 and 320–400 nm, while proline supplementation had no effect. Spores prepared with cysteine, cystine or thioproline were also more resistant to H₂O₂ but not to wet heat or 254‐nm UV radiation. The increases in spore resistance attributed to the sporulation supplements were eliminated if spores were chemically decoated. Conclusions: Supplementation of sporulation medium with cysteine, cystine or thioproline increases spore resistance to solar UV radiation reaching the Earth’s surface and to H₂O₂. These effects were eliminated if the spores were decoated, indicating that alterations in coat proteins by different sporulation conditions can affect spore resistance to some agents. Significance and Impact of the Study: This study provides further evidence that the composition of the sporulation medium can have significant effects on B. subtilis spore resistance to UV radiation and H₂O₂. This knowledge provides further insight into factors influencing spore resistance and inactivation.     

Defined Physiological Conditions for the Induction of the L-Form of Neisseria gonorrhoeae

Defined conditions are described which allowed luxuriant growth over continuous subculture of strains of Neisseria gonorrhoeae in broth and on agar. Growth was equal to or surpassed that observed in Mueller-Hinton broth or on Mueller-Hinton blood agar. The final medium adopted consisted of medium 199 and a supplemental mixture of cysteine, glucose, and various salts. Addition of sodium bicarbonate or CO(2) enrichment was not required. For solidification, only agarose allowed growth of all strains; glutamic acid stimulated growth of two strains but was inhibitory for a third. The addition of 8% polyvinylpyrrolidone (PVP), 2% purified albumin, and penicillin resulted in induction of all three strains to the L-form with frequencies up to 0.3%. At present no induction to the L-form has been achieved in the absence of albumin. Various lots of PVP proved toxic in the defined medium, and extensive dialysis was required for good growth and L-form induction. Substitution of PVP with sucrose indicated a sucrose toxicity for the parental gonococcus even on the addition of albumin. L-form induction did occur on sucrose L-medium but at significantly lower frequencies. The colonies appeared 1 week later than those on PVP L-medium but at significantly lower frequencies. The colonies appeared 1 week later than those on PVP L-medium and remained very small and poorly developed.     

Arginine metabolism in Halobacterium salinarium, an obligately halophilic bacterium

Dundas, Ian E. D. (University of Illinois, Urbana), and H. Orin Halvorson. Arginine metabolism in Halobacterium salinarium, an obligately halophilic bacterium. J. Bacteriol. 91:113-119. 1966.-Arginine was shown to be essential for growth of Halobacterium salinarium strain 1 in a chemically defined medium. Citrulline was the only compound which could substitute for arginine without affecting growth. Resting cells of H. salinarium converted arginine to citrulline and citrulline to ornithine. Cells grown in an arginine-free medium with C(14)-ureido-labeled citrulline incorporated the isotope mainly into the arginine of their proteins. The enzymes arginine desimidase and ornithine transcarbamylase were found and studied in cell-free extracts of H. salinarium. Experiments indicated that arginine was degraded in H. salinarium by arginine desimidase to citrulline, and that citrulline was further degraded by ornithine transcarbamylase to carbamyl phosphate and ornithine. Synthesis of arginine from citrulline seems to occur via the formation of argininosuccinic acid.     

Nutritional requirements of Plasmodium falciparum in culture. I. Exogenously supplied dialyzable components necessary for continuous growth

Continuous cultivation of Plasmodium falciparum presently requires the nutritionally complex medium, RPMI 1640. A basal medium of KCl, NaCl, Na2HPO4, Ca(NO3)2, MgSO4, glucose, reduced glutathione, HEPES buffer, hypoxanthine, phenol red (in RPMI 1640 concentrations), and 10% (v/v) exhaustively dialyzed pooled human serum was used to determine which vitamins and amino acids had to be exogenously supplied for continuous cultivation. Supplementation of basal medium with calcium pantothenate, cystine, glutamate, glutamine, isoleucine, methionine, proline, and tyrosine was necessary for continuous growth. This semi-defined minimal medium supported continuous growth of four isolates of P. falciparum at rates slightly less than those obtained with RPMI 1640. Adding any other vitamin or amino acid did not improve growth. Incorporation of several non-essential amino acids, particularly phenylalanine and leucine, into proteins was markedly enhanced in the minimal medium compared to RPMI 1640.