Growth responses of Neisseria gonorrhoeae auxotypes to required amino acids and bases in liquid medium

Neisseria gonorrhoeae strains can be grouped or differentiated (auxotyped) by their requirements for none, or any one or more of proline, uracil, hypoxanthine, and citrulline or ornithine. Most strains were readily assessed because they responded with growth or no growth on each defined auxanographic medium. Other strains gave indeterminate responses on agar and the reasons were not obvious. Liquid growth studies for quantifying the usual responses showed that yields of appropriate N. gonorrhoeae auxotrophs were proportional to replacement concentrations of any one of these amino acids or bases, of methionine, or of cysteine plus cystine. This type of response, where log growth rates and lag times were unaffected, is proposed as the basis for defining (simple) auxotrophy in gonococci. The formula of the defined medium was improved by increasing proline, uracil, and hypoxanthine beyond limiting concentrations, and decreasing citrulline or ornithine, and cysteine plus cystine. Fatty acid--free bovine albumin was used to ensure homogeneous growth in liquid media. In agar, it was superior to starch for the nonnutritive protective effect required by many strains.     

Nutritional requirements of anaerobic coryneforms.

The nutritional requirements of three species of anaerobic coryneforms and their serotypes (Propionibacterium acnes types I and II, P. avidum types I and II, and P. granulosum) were determined. Strains of P. avidum would consistently grow to a transmittance of 1 to 3% at 560 nm in a basal salts medium supplemented with glucose, pantothenate, biotin, thiamine, and 12 amino acids (alanine, arginine, cysteine, glutamine, glycine, histidine, isoleucine, methionine, phenylalanine, serine, tyrosine, and tryptophan). Strains of P. acnes and P. granulosum, however, failed to grow in this medium unless six additional amino acids were present (asparagine, leucine, lysine, proline, threonine, and valine). All three species grew equally well whether the 18 amino acids were supplied in the form of a casein hydrolysate supplemented with tryptophan or were added separately. Nicotinamide enhanced growth of P. acnes but had no effect on growth of P. avidum and P. granulosum. Other nutrients which were not absolute requirements, but which significantly improved growth of these species, included the purines guanine and/or adenine, Tween 80, which served as a source of oleic acid, sodium L-lactate, alpha-ketoglutarate, and pyruvate. Strains (86) comprising all five groups grew well in the defined medium, except four strains of P. acnes type II (29 tested), which failed to grow unless heme and vitamin K were added to the medium. One strain of P. granulosum (22 tested) failed to grow in any defined medium, suggesting an additional growth factor requirement.     

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.     

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.     

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.     

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.     

Ethyl carbamate precursor citrulline formation from arginine degradation by malolactic wine lactic acid bacteria

Major commercially available strains for induction of malolactic fermentation in wine were examined for arginine metabolism in a resting cell system at wine pH with the aim of evaluating their ability to excrete and utilize citrulline, a precursor of carcinogenic ethyl carbamate (urethane). All strains tested excreted citrulline from arginine degradation. Citrulline was stored intracellularly during growth in arginine rich medium and was released upon lysis of the cells. All strains were found to degrade citrulline as a sole amino acid and some of them were able to reutilize previously excreted citrulline.     

Studies on the Cecal Microflora of Commercial Broiler Chickens

A study was made of the cecal microflora isolated from broilers (5-week-old) reared under typical commercial husbandry conditions. Three hundred and twenty-five bacterial strains (randomly isolated from colonies representing 49 to 81% of the microscopic count) were isolated from cecal digesta of six animals on a rumen fluid roll tube medium (M98-5). Seventy-seven percent of these strains consisted of strict anaerobes: gram-negative, pleomorphic cocci (5.2%), Peptostreptococcus (1.5%), gram-positive rods (36.1% as Propionibacterium acnes and Eubacterium sp.), gram-negative rods (18.6% as Bacteroides clostridiiformis, B. hypermegas and B. fragilis) and sporeforming rods (15.7% as Clostridium sp.). Two types of facultatively anaerobic bacteria (gram-positive cocci and Escherichia coli) were also isolated and constituted 17.5% of the remaining flora. The distribution of the bacterial groups isolated from six cecal samples varied considerably. Data on the growth requirements of anaerobic strains indicated that many could be cultured in a simple medium consisting of an energy source, minerals, reducing agent, Trypticase, and yeast extract (or a vitamin mixture in place of yeast extract). The growth of some of these bacteria was also enhanced by CO(2) and rumen fluid. These preliminary data suggest that some of the more numerous anaerobes isolated from the chicken cecum may not require complex nutrients for growth and, in fact, may be nutritionally similar to rumen anaerobes.     

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.     

Accumulation of trehalose by Escherichia coli K-12 at high osmotic pressure depends on the presence of amber suppressors

When grown at high osmotic pressure, some strains of Escherichia coli K-12 synthesized substantial levels of free sugar and accumulated proline if it was present in the growth medium. The sugar was identified as trehalose by chemical reactivity, gas-liquid chromatography, and nuclear magnetic resonance spectroscopy. Strains of E. coli K-12 could be divided into two major classes with respect to osmoregulation. Those of class A showed a large increase in trehalose levels with increasing medium osmolarity and also accumulated proline from the medium, whereas those in class B showed no accumulation of trehalose or proline. Most class A strains carried suppressor mutations which arose during their derivation from the wild type, whereas the osmodefective strains of class B were suppressor free. When amber suppressor mutations at the supD, supE, or supF loci were introduced into such sup0 osmodefective strains, they became osmotolerant and gained the ability to accumulate trehalose in response to elevated medium osmolarity. It appears that the original K-12 strain of E. coli carries an amber mutation in a gene affecting osmoregulation. Mutants lacking ADP-glucose synthetase (glgC) accumulated trehalose normally, whereas mutants lacking UDP-glucose synthetase (galU) did not make trehalose and grew poorly in medium of high osmolarity. Trehalose synthesis was repressed by exogenous glycine betaine but not by proline.     

Antiviral Effect of Diammonium Glycyrrhizinate on Cell Infection by Porcine Parvovirus

The goal of this study was to evaluate how two new hydrolysates from poultry by-products act on ten lactobacilli growth kinetics when supplemented to the growth medium. These effects were compared with ones induced by two most common commercial hydrolysates, i.e., tryptone and peptone. Growth medium, supplemented with one of new hydrolysates, 78T, as only nitrogen source, can sustain the maximum growth rate and the biomass yield in the same way of MRS, reach of different nitrogen sources. Moreover aminopeptidase activities (AA) of each strain were determined to investigate the effect of the growth condition on the modulation of aminopeptidase pattern. Five cell extracts of each ten strains, obtained from their cultivation in MRS and in the presence of the two common hydrolysates and the two new ones, were considered. AA was investigated against five different chromogenic substrates: β-naphthyl amide derivatives of l-anomers of leucine, lysine, proline, glycine–proline, and phenilalanine–proline. A great variability of AA was observed among the strains: also strains belonging to the same species showed peculiar AA profile.     

Growth, D-glucose utilization and malolactic fermentation by Leuconostoc œnos strains in 18 media deficient in one amino acid

Six Leuconostoc œnos strains were used to study the effect of the deficiency of one amino acid on growth, heterofermentative pathway and malolactic fermentation. All strains had an absolute requirement for four amino acids (isoleucine, glutamic acid, tryptophan and arginine) and needed six other amino acids (valine, methionine, cysteine, leucine, aspartic acid and histidine) for optimum growth. Each deficiency in one amino acid had a particular effect on D-glucose utilization. Overproduction and underproduction of D-lactic acid were observed. The rate of L-malic acid consumption in media deficient in one amino acid was lower than in the complete medium with all amino acids.
Although some deficiencies (glycine, phenylalanine, proline or tyrosine) had no influence on the growth, they noticeably limited the malolactic fermentation.     

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.     

Comparison of hup trait and intrinsic antibiotic resistance for assessing rhizobial competitiveness axenically and in soil

A defined medium for growth of 24 strains of Moraxella (Branhamella) catarrhalis was devised. This medium (medium B4) contains sodium lactate as a partial carbon source, proline as both a partial carbon source and a partial nitrogen source, aspartate as a partial nitrogen source, and the growth factors arginine, glycine, and methionine. Either aspartate, glutamate, or proline could serve as sole nitrogen source, but growth occurred at a significantly better rate if proline was present together with either aspartate or glutamate, or with both aspartate and glutamate. With the exception of strain ATCC 23246, all the strains had an absolute requirement for arginine and either a partial or absolute requirement for glycine. The concentration of glycine required for optimal growth was found to be relatively high for an amino acid growth factor. Heart infusion broth was found to be growth inhibitory for spontaneous mutants of one strain able to grow in the absence of arginine, and such mutants reverted readily to arginine dependence accompanied by the ability to grow faster on the complex medium. Growth rates in the defined medium B4 were enhanced by the simultaneous addition of asparagine, glutamate, glutamine, leucine, lysine, histidine, and phenylalanine.     

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.     

Catabolism of arginine, citrulline and ornithine by Pseudomonas and related bacteria

The distribution of the arginine succinyltransferase pathway was examined in representative strains of Pseudomonas and related bacteria able to use arginine as the sole carbon and nitrogen source for growth. The arginine succinyltransferase pathway was induced in arginine-grown cells. The accumulation of succinylornithine following in vivo inhibition of succinylornithine transaminase activity by aminooxyacetic acid showed that this pathway is responsible for the dissimilation of the carbon skeleton of arginine. Catabolism of citrulline as a carbon source was restricted to relatively few of the organisms tested. In P. putida, P. cepacia and P. indigofera, ornithine was the main product of citrulline degradation. In most strains which possessed the arginine succinyltransferase pathway, the first step of ornithine utilization as a carbon source was the conversion of ornithine into succinylornithine through an ornithine succinyltransferase. However P. cepacia and P. putida used ornithine by a pathway which proceeded via proline as an intermediate and involved an ornithine cyclase activity.     

Roles of arginine in growth of Clostridium botulinum Okra B.

Group I strains of Clostridium botulinum are known to degrade arginine by the arginine deiminase pathway. We have found that C. botulinum Okra B consumed a level of arginine (20 g/liter) higher than the basal requirement for growth (3 g/liter). Arginine was probably the preferred source of nitrogen for biosynthesis but did not serve as a major source of energy. Citrulline and proline were produced as major fermentation products in media containing high levels of arginine, but in media with basal amounts of arginine these products were produced in lower quantities during growth and were later reassimilated. The results indicate that C. botulinum Okra B changes its metabolism during consumption of surplus arginine, and this change is associated with toxin repression, formation of citrulline and proline as end products, and possibly resistance to environmental stresses such as increased acidity and osmolarity.     

Growth and spore germination factors of various strains of Bacillus sphaericus

The work was aimed at studying the requirements of sixteen Bacillus sphaericus strains with a different larvicidal activity in amino acids and some other compounds necessary for their growth and spore germination. Most of the strains were found to require arginine, glutamate, methionine, threonine, serine, glycine, alanine and lysine, but they did not assimilate phenylalanine and proline. Arginine, methionine and glutamate were shown to be the most effective inductors of spore germination. Specific differences were detected in the requirements of virulent and avirulent strains. Glucose repressed both spore germination and spore formation.     

Improvement of a Chemically Defined Medium for the Sustained Growth of <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis>: Nutritional Requirements

The aims of this work were to improve a basal synthetic medium (BM) for the growth of Lactobacillus plantarum strains and to establish their amino-acid requirements. Amino-acid use was analyzed in the most nutritionally demanding bacterium. First, the improved BM (L. plantarum synthetic medium [LPSM]) was created by increasing some vitamins in the BM, especially p-aminobenzoic acid, vitamin B₁₂, and biotin; 5-fold phenylalanine, histidine, isoleucine, leucine, lysine, methionine, proline, serine, threonine, and tryptophan; and 10-, 60-, and 75-fold valine, arginine, and tyrosine, respectively. With these additions, the N8 and N4 strains of L. plantarum grew rapidly to reach final cell densities similar to those obtained in Mann–Rogosa–Sharpe medium. When cysteine, leucine, valine, isoleucine, threonine, and glutamic acid were individually removed from this medium, bacterial growth significantly decreased or ceased, indicating that these amino acids are essential for growth. The N4 strain also required lysine and tryptophan in addition to the six amino acids necessary for growth. L. plantarum N4 mainly consumed essential amino acids, such as valine, lysine, cysteine, and threonine as well as the stimulatory amino acid, arginine. Thus, the BM was improved mainly on the basis of annulling limitations with respect to amino acids. With this, improved medium cell densities in the order of 10⁹ colony-forming units/mL have been achieved, indicating that LPSM medium could be used for conducting metabolic and genetic studies on L. plantarum. Their low levels in orange juice suggest that these amino acids may not satisfy the total nitrogen requirement for the development of L. plantarum in the natural environment.     

Differential characteristics of catalase-positive campylobacters correlated with DNA homology groups

Eighty-four strains of catalase-positive campylobacters could be placed into seven distinct DNA homology groups (species), corresponding to Campylobacter fetus, "C. hyointestinalis," C. jejuni, C. coli, "C. laridis," "C. fecalis," and aerotolerant campylobacters. The biochemical and physiological characteristics of the strains were examined for their correlation with the homology groups. The characterization tests that provided the most reliable differentiation at the species and subspecies level were growth at 25 and 42 degrees C, sensitivity to cephalothin and nalidixic acid, growth in semisolid media containing 1% glycine and 3.5% NaCl, growth on plates containing 1.5% NaCl, growth in a semisolid minimal medium, anaerobic growth in the presence of 0.1% trimethylamine-N-oxide, hydrogen sulfide production in SIM medium and triple-sugar iron agar, hippurate hydrolysis, nitrite reduction, and growth on plates under an air atmosphere.