Synthesis of a compound with folic acid activity by Lactobacillus arabinosus 17-5

Summary A compound with folic acid activity is synthesized by growing as well as respiring cells of Lactobacillus arabinosus in the presence of p-aminobenzoic acid. The essentiality of glutamic acid is seen in studies with respiring cells.The free folic acid activity elaborated by Lactobacillus arabinosus reaches its maximum in about 48 hrs. and is present mainly in the culture filtrate.Additions of Tween 80, or biotin and of xanthine show marked stimulation of the synthesis of folic acid activity.With the organisms Streptococcus faecalis R and Lactobacillus casei, requiring exogenous folic acid for growth, it is seen that the entire folic acid activity resides in the cells and as citrovorum factor.Sulphanilamide inhibits the synthesis of folic acid activity by Lactobacillus arabinosus.     

Synthesis of a substance exhibiting citrovorum factor activity by Lactobacillus arabinosus 17-5

Summary Growing and respiring cells of Lactobacillus arabinosus elaborate citrovorum factor activity from p-aminobenzoic acid. Pteroylglutamic acid is a step in this conversion.That the activity synthesized is apparently different from the citrovorum factor is inferable from observations on inhibition analysis, differential distribution between immiscible solvents at various p_H and bioautographic studies.Ascorbic acid aids in the synthesis of citrovorum factor active substance. Formate and formyl precursors have stimulatory effect on this synthesis but only in presence of ascorbic acid.     

Nutritional Requirements of Methanosarcina sp. Strain TM-1

Methanosarcina sp. strain TM-1, an acetotrophic, thermophilic methanogen isolated from an anaerobic sludge digestor, was originally reported to require an anaerobic sludge supernatant for growth. It was found that the sludge supernatant could be replaced with yeast extract (1 g/liter), 6 mM bicarbonate-30% CO₂, and trace metals, with a doubling time on methanol of 14 h. For growth on either methanol or acetate, yeast extract could be replaced with CaCl₂ · 2H₂O (13.6 μM minimum) and the vitamin p-aminobenzoic acid (PABA, ca. 3 nM minimum), with a doubling time on methanol of 8 to 9 h. Filter-sterilized folic acid at 0.3 μM could not replace PABA. The antimetabolite sulfanilamide (20 mM) inhibited growth of and methanogenesis by Methanosarcina sp. strain TM-1, and this inhibition was reversed by the addition of 0.3 μM PABA. When a defined medium buffered with 20 mM N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid was used, it was shown that Methanosarcina sp. strain TM-1 required 6 mM bicarbonate-30% CO₂ for optimal growth and methanogenesis from methanol. Cells growing on acetate were less dependent on bicarbonate-CO₂. When we used a defined medium in which the only organic compounds present were methanol or acetate, nitrilotriacetic acid (0.2 mM), and PABA, it was possible to limit batch cultures of Methanosarcina sp. strain TM-1 for nitrogen at NH₄⁺ concentrations at or below 2.0 mM, in marked contrast with Methanosarcina barkeri 227, which fixes dinitrogen when grown under NH₄⁺ limitation.     

Studies on the mechanism of action of sulfanilamide on mycelial felts of Rhizoctonia solani

Summary Sulfanilamide induced strong inhibitory effects on the growth, respiration and carbohydrate synthesis by mycelial felts of Rhizoctonia solani. The possible reasons for such inhibitory effects are discussed.The inhibitory effects of sulfanilamide were almost completely alleviated by the inclusion of p-aminobenzoic (PABA) acid in the culture medium of the fungal mats. R. solani normally produces certain amounts of PABA and its growth and metabolism is inhibited in presence of excess of it.     

Biosynthesis of folic acid

Summary The influence of various vitamins on the biogenesis of folic acid has been studied in microorganisms requiring these as growth factors. In L. arabinosus, the folic acid synthesised was directly proportional to the availability of both riboflavin and pantothenic acid. The influence of cyanocobalamin on folic synthesis varied radically in different organisms. In case of the B₁₂/methionine auxotroph of E. coli there was an inverse relationship of vitamin B₁₂ to folic acid synthesis, while in Euglena the folic acid elaborated was in proportion to cyanocobalamin supplied. Synthesis of both folic acid and vitamin B₁₂ was depressed when thymine supply was adequate in the nutrition of E. coli 15 T ^-, a thymine auxotroph.     

Metabolic studies with neurospora: Action of 5-bromo- and 5-nitrouracils

Summary There is no growth inhibition of Neurospora crassa even in presence of high levels of 5-bromouracil. Although there is no significant change in the DNA and RNA contents, the analogue is utilized by the organism, a part of it being incorporated in DNA. There is an increase in total folate and citrovorum factor activities with 5-bromouracil.5-Nitrouracil causes a significant growth inhibition at higher concentration which is accompanied by a decrease in pteroylglutamic acid and citrovorum factor levels in the organism.Pteroylglutamic acid and thymidine cause a partial reversal of growth inhibition by 5-nitrouracil and complete reversal when the two metabolites are added simultaneously.     

Relationships between biotin and vitamin B12. Effects of biotin and vitamin B12 on folic acid metabolism

1. The effects of dietary biotin compared with vitamin B₁₂ on the total content and on the distribution of the various folate derivatives in the liver of rats given a biotin-free diet have been studied. The effect of both vitamins on the conversion in vitro of folic acid into citrovorum factor in the same experimental conditions was also examined. 2. In biotin-treated rats as well as in vitamin B₁₂-treated rats the total content of folic acid-active substances measured microbiologically by Pediococcus cerevisiae, Streptococcus faecalis and Lactobacillus casei is significantly higher than that in biotin-deficient rats. The liver distribution of various folate derivatives in the three groups of animals is also markedly modified. 3. The amount of citrovorum factor formed in systems with liver homogenate of rats receiving biotin or vitamin B₁₂ is higher than that with liver homogenates of deficient rats. 4. The results obtained demonstrate the influence of biotin in the metabolism of folic acid, and the similar actions at this level of both biotin and vitamin B₁₂. These results are discussed in relation to the participation of the two vitamins in the metabolism of C₁ units, as a biochemical interpretation of the relationships between vitamin B₁₂ and biotin.     

Studies on the vitamin B12 auxotrophy of Rhodocyclus purpureus and two other vitamin B12-requiring purple nonsulfur bacteria

The vitamin B₁₂ requirement of Rhodocyclus purpureus 6770, Rhodospirillum tenue 1/67, and Rhodopseudomonas palustris G 53/2 was determined. A wide variety of biogenetic precursors of the vitamin including cobinamide, cobyric acid, cobinic acid and several partially amidated cobyrinic acids showed growth-promoting activity in all three strains. In R. purpureus vitamin B₁₂ could even be substituted by cobyrinic acid which is the first cobalt-containing precursor of vitamin B₁₂ so far established. Neither methionine, deoxynucleosides, dimethylbenzimidazole nor increased amounts of cobalt could replace vitamin B₁₂ as growth factor.Cupribalamin, which is a strong antimetabolite of vitamin B₁₂ in Escherichia coli 113-3 and Lactobacillus leichmannii ATCC 7830, exhibited only a weak antagonistic effect on growth of R. purpureus and R. tenue. Growth of R. palustris was not inhibited by cupribalamin. The cells of all three strains were shown to contain metal-free corrinoids in addition to cobalt-containing corrinoids. The principal products were identified as 5-deoxyadenosylcobalamin and hydrogenobalamin, the metal free analogue of vitamin B₁₂. The latter does not originate from the vitamin by removal of cobalt but is de novo biosynthesized as could be demonstrated in the case of R. purpureus by a labelling experiment with [¹³C] methyl-l-methionine.     

Induced Resistance to Sulfonamides in Chilomonas paramecium

SUMMARY. Strains of Chilomonas paramecium differing in degree of resistance to sulfanilamide have been established through acclimatization to this sulfonamide at 50, 100 and 200 mg. %. Resistant strains differ from the normal stock in their enhanced sensitivity to p -aminobenzoic acid. In the normal stock, sulfanilamide inhibition is reversed at an SA/PABA ratio of 10,000 but not at 20,000; in the least resistant strain, at a ratio of 400,000 but not at 800,000. In resistant strains inhibition is reversed by folk acid, methionine, adenine, cytosine and thymine; in the normal stock, none of these metabolites produces reversal. In high concentrations of PABA (10–20 mg. %) growth of the normal stock is only retarded, whereas the strain least resistant to sulfanilamide fails to recover from exposure to 20 mg. % PABA. The strain most resistant to sulfanilamide is most susceptible to PABA in high concentrations. The data suggest that resistance to sulfanilamide in C. paramecium may depend mainly upon an accelerated synthesis of PABA.     

Partial purification of a growth factor from orange juice which affects citrus tissue culture and its replacement by citric Acid

Orange (Citrus sinensis L. Osbeck) tissue cultures required a supply of orange juice to the medium for their vigorous growth. The growth-promoting activity of juice seemed to involve both cell division and cell enlargement. Juice had no promotive activity in bioassays for auxins, gibberellins, and cytokinins. The growth promoting activity of juice was mostly transferred into 1-butanol upon partition at pH 2. Gas chromatographic analysis of this acid 1-butanol fraction revealed large amounts of citric acid and negligible amounts of other organic acids. Supply of pure citric acid to the medium, alone or in combination with different concentrations of juice, indicated that citric acid replaces most of the requirement for juice.     

Chemical and biological studies on 1,2-dihydro-S-triazines

Summary A series of 1,2-dihydro-s-triazines has been studied inLactobacillus casei # 7469-pteroylglutamic acid systems. The active derivatives exhibit a competitive inhibition similar to that of 4-aminopteroylglutamic acid, but differ from the latter in that inhibition is not relieved by adenine or guanine, and at appropriate concentrations of inhibitor, is not reversed by excess pteroylglutamic acid. Differences in microbiological activity can be correlated with certain alterations in the structure of the molecule, maximum activity being exhibited by the 2,2-dimethyl-phenyl- and 2,2-dimethyl-m-chlorophenyl derivatives. The inhibitory effect of these compounds is reversed·by appropriate concentrations of dihydropteroylglutamic acid, N¹⁰-formylpteroylglutamic acid, synthetic and natural citrovorum factor, thymine and thymidine. The similarity in inhibition indices obtained vs the various forms of pteroylglutamic acid inLactobacillus casei bioassay systems and the correlation with those vs citrovorum factor inStreptococcus faecalis # 8043 andLeuconostoc citrovorum # 8081 bioassay systems suggests that inhibition ofLactobacillus casei is the result of interference with the utilization of citrovorum factor. Part I: J. Amer. Chem. Soc.74, 855, 1952; Amer. J. Path.28, 599, 1952; II: J. Amer. Chem. Soc. 1955, in press; III: J. Amer. Chem. Soc. 1955, in press; IV: Proc. Soc. Exp. Biol. Med.83, 733, 1953; V: Proc. Soc. Exp. Biol. Med.83, 740, 1953; VI: Proc. Soc. Exp. Biol. Med.83, 742, 1953. The experimental work for this and studies VIII, IX, and X in this series was done in part at the Laboratorium voor de Gezondheidscleer, Universiteit van Amsterdam, Nederland, which is directed by Professor DrA. Charlotte Ruys, and these four reports are based upon a thesis submitted by G.E.F. to the Faculteit der Wis-en Natuurkunde, in partial fulfiment of the requirements for the degree of Doctor of Science.     

On the action of sulfanilamide

Summary Methionine, xanthine and thymine replace para-aminobenzoic acid as a growth factor for the para-aminobenzoic-lessE.coli mutant strain 273.The same substances were able to antagonize non competitively the bacteriostatical activity of 2000 mg/l sulfanilamide completely. It was further shown that the observed differences in sensitivity of the reaction chains leading to methionine, xanthine and thymine are not due to a difference in sensitivity to sulfanilamide but a difference in sensitivity to para-aminobenzoic acid. This seems to indicate that there exists only one reaction which is inhibited by sulfanilamide.XIII: Antonie van Leeuwenhoek15, 136, 1949.     

Growth inhibition of Candida albicans by folate pathway inhibitors. Their potential in the selection of auxotrophs

Growth studies were conducted on C. albicans in a glucose — salts — biotin (GSB) medium in the presence of folate inhibitors. Sulfanilamide inhibited growth which was restored by PABA or tetrahydrofolate (THF). Aminopterin inhibited growth to about the same level as did sulfanilamide, but this inhibition was not reversed with PABA nor THF, singly or in combination. Inhibition by combined sulfanilamide and aminopterin was synergistic, reducing growth by more than 90% for 48 h. The sulfanilamide component of the combined inhibition was reversed by PABA or THF to the level of that of aminopterin alone. Cytochrome synthesis was not affected by the inhibitors, but marked increases occurred in -ketoglutarate, malate, isocitrate, and pyruvate dehydrogenases, especially in the presence of both inhibitors. The pyrimidines in combination with sulfanilamide were as inhibitory as was the combination of aminopterin and sulfanilamide, but they had no effect when added alone or in combination with aminopterin. Unlike the pyrimidines, the purines stimulated about a 50% recovery from inhibition by either of the inhibitors. Growth inhibition by combined sulfanilamide and aminopterin was overcome by about 50% by the addition of the THF-mediated end-produits: deoxythymidylate, adenine, histidine and methionine. The use of GSB medium containing adenine, histidine, methionine and the folate inhibitors but without deoxythymidylate resulted in thymineless death of prototrophic cells providing a method for the selection of auxotrophic mutants.     

Role of Listeria monocytogenes σB in Survival of Lethal Acidic Conditions and in the Acquired Acid Tolerance Response

The food-borne pathogen Listeria monocytogenes can acquire enhanced resistance to lethal acid conditions through multiple mechanisms. We investigated contributions of the stress-responsive alternative sigma factor, σ^B, which is encoded by sigB, to growth phase-dependent acid resistance (AR) and to the adaptive acid tolerance response in L. monocytogenes. At various points throughout growth, we compared the relative survival of L. monocytogenes wild-type and ΔsigB strains that had been exposed to either brain heart infusion (pH 2.5) or synthetic gastric fluid (pH 2.5) with and without prior acid adaptation. Under these conditions, survival of the ΔsigB strain was consistently lower than that of the wild-type strain throughout all phases of growth, ranging from 4 orders of magnitude less in mid-log phase to 2 orders of magnitude less in stationary phase. Survival of both ΔsigB and wild-type L. monocytogenes strains increased by 6 orders of magnitude upon entry into stationary phase, demonstrating that the L. monocytogenes growth phase-dependent AR mechanism is σ^B independent. σ^B-mediated contributions to acquired acid tolerance appear to be greatest in early logarithmic growth. Loss of a functional σ^B reduced the survival of L. monocytogenes at pH 2.5 to a greater extent in the presence of organic acid (100 mM acetic acid) than in the presence of inorganic acid alone (HCl), suggesting that L. monocytogenes protection against organic and inorganic acid may be mediated through different mechanisms. σ^B does not appear to contribute to pH_i homeostasis through regulation of net proton movement across the cell membrane or by regulation of pH_i buffering by the GAD system under the conditions examined in this study. In summary, a functional σ^B protein is necessary for full resistance of L. monocytogenes to lethal acid treatments.     

Non-competitive antagonists for derivatives of p-aminobenzoic acid

Summary Shive's method of “inhibition analysis” was applied to the growth inhibition ofE. coli by some derivatives of p-aminobenzoic acid. In accordance with the findings ofWinkler andde Haan methionine, xanthine, serine, thymine, and valine were shown to be non-competitive antagonists of sulfanilamide forE. coli strain Bray β. It is highly probable that for this bacterium glycine should be added to this series. Methionine is the only non-competitive antagonist of the 2-chloro, 2-bromo, and 2-amino derivatives of P.A.B., provided their action is investigated at no higher concentrations than can be antagonised by P.A.B. The same complete series of non-competitive antagonists of sulfanilamide was shown to be active against the bacteriostatic action of the 2-methyl, 3-methyl, and 3-amino derivatives of P.A.B. The hypothesis is proposed that these derivatives are not competing directly with P.A.B. for an essential enzyme but that they are used by the bacterium in the formation of derivatives of pteroylglutamic acid; these may be competitive antagonists of co-enzymes, related to pteroylglutamic acid. There is some evidence in favour of this supposition. Further investigations are needed, however, before any judgment regarding its value can be given.     

Fatty acids of plant vacuolar membrane lipids

Fatty acid (FA) composition of vacuolar membrane lipids from storage tissues of umbelliferous plants, viz., parsnip (Pastinaca sativa L.), parsley (Petroselinium crispum L.), and carrot (Daucus carota L.) is studied by gas-liquid chromatography and the FA biosynthetic pathways are considered. Vacuolar membrane lipids are characterized by high (78% of the total FA pool) content of unsaturated FA among which linoleic acid is predominant. Its content in vacuolar lipids of parsnip, parsley and carrot is 53.5, 55.1, and 54.9%, respectively. Parsnip and parsley vacuolar lipids contain large amounts of hexadecadienoic C16:2ω6 acid (8.0 and 4.6%, respectively). The content of α-linolenic acid in vacuolar lipids of tested plants varies from 4.8 to 7.3%. Palmitic acid (18.0–20.7%) predominates among saturated FA. High content of linoleic and hexadecadienoic acid in parsnip and parsley vacuolar lipids is suggestive of a crucial role of the microsomal ω6 fatty-acid desaturase fad2 gene in resistance and acclimation of plants to low temperatures.     

Characterization of a novel plant growth-promoting bacteria strain Delftia tsuruhatensis HR4 both as a diazotroph and a potential biocontrol agent against various plant pathogens

A novel, plant growth-promoting bacterium Delftia tsuruhatensis, strain HR4, was isolated from the rhizoplane of rice (Oryza sativa L., cv. Yueguang) in North China. In vitro antagonistic assay showed this strain could suppress the growth of various plant pathogens effectively, especially the three main rice pathogens (Xanthomonas oryzae pv. oryzae, Rhizoctonia solani and Pyricularia oryzae Cavara). Treated with strain HR4 culture, rice blast, rice bacterial blight and rice sheath blight for cv. Yuefu and cv. Nonghu 6 were evidently controlled in the greenhouse. Strain HR4 also showed a high nitrogen-fixing activity in N-free Döbereiner culture medium. The acetylene reduction activity and ¹⁵N₂-fixing activity (N₂FA) were 13.06 C₂H₄ nmol ml⁻¹ h⁻¹ and 2.052 ¹⁵Na.e.%, respectively. The nif gene was located in the chromosome of this strain. Based on phenotypic, physiological, biochemical and phylogenetic studies, strain HR4 could be classified as a member of D. tsuruhatensis. However, comparisons of characteristics with other known species of the genus Delftia suggested that strain HR4 was a novel dizotrophic PGPB strain.     

Hpa1 harpin needs nitroxyl terminus to promote vegetative growth and leaf photosynthesis in Arabidopsis

Hpa1 is a harpin protein produced by Xanthomonas oryzae, an important bacterial pathogen of rice, and has the growth-promoting activity in plants. To understand the molecular basis for the function of Hpa1, we generated an inactive variant protein, Hpa1ΔNT, by deleting the nitroxyl-terminal region of the Hpa1 sequence and compared Hpa1ΔNT with the full-length protein in terms of the effects on vegetative growth and related physiological responses in Arabidopsis. When Hpa1 was applied to plants, it acted to enhance the vegetative growth but did not affect the floral development. Enhanced plant growth was accompanied by induced expression of growth-promoting genes in plant leaves. The growth-promoting activity of Hpa1 was further correlated with a physiological consequence shown as promoted leaf photosynthesis as a result of facilitated CO₂ conduction through leaf stomata and mesophyll cells. On the contrary, plant growth, growth-promoting gene expression, and the physiological consequence changed little in response to the Hpa1ΔNT treatment. These analyses suggest that Hpa1 requires the nitroxyl-terminus to facilitate CO₂ transport inside leaf cells and promote leaf photosynthesis and vegetative growth of the plant.