Clavulanic acid, a β-lactamase inhibitor: biosynthesis and molecular genetics

Clavulanic acid is a secondary metabolite produced by Streptomyces clavuligerus. It possesses a clavam structure and a characteristic 3R,5R stereochemistry essential for action as a β-lactamase inhibitory molecule. It is produced from glyceraldehyde-3-phosphate and arginine in an eight step biosynthetic pathway. The pathway is carried out by unusual enzymes, such as (1) the enzyme condensing both precursors, N ²-(2-carboxyethyl)-arginine (CEA) synthetase, (2) the β-lactam synthetase cyclizing CEA and (3) the clavaminate synthetase, a well-characterized multifunctional enzyme. Genes for biosynthesis of clavulanic acid and other clavams have been cloned and characterized. They offer new possibilities for modification of the pathway and for obtaining new molecules with a clavam structure. The state of the regulatory proteins controlling clavulanic acid biosynthesis, as well as the relationship between the biosynthetic pathway of clavulanic acid and other clavams, is discussed. Received: 9 February 2000 / Received revision: 10 May 2000 / Accepted: 12 May 2000     

The path of unspecific incorporation of selenium in Escherichia coli

The path of unspecific selenium incorporation into proteins was studied in Escherichia coli mutants blocked in the biosynthesis of cysteine and methionine or altered in its regulation. Selenium incorporation required all enzymatic steps of cysteine biosynthesis except sulfite reduction, indicating that intracellular reduction of selenite occurs nonenzymatically. Cysteine (but not methionine) supplementation prevented unspecific incorporation of selenium by repressing cysteine biosynthesis. On the other hand, when the biosynthesis of cysteine was derepressed in regulatory mutants, selenium was incorporated to high levels. These findings and the fact that methionine auxotrophic strains still displayed unspecific incorporation show that selenium incorporation into proteins in E. coli occurs mainly as selenocysteine. These findings also provide information on the labeling conditions for incorporating ⁷⁵Se only and specifically into selenoproteins. Received: 2 May 1997 / Accepted: 23 June 1997     

Effect of Cell Lysis (CLs) Products on Acidophilic Chemolithotrophic Microorganisms and the Role of Acidocella Species

Acidophilic chemolithotrophic microorganisms (CMs) are widely used for bioleaching of mineral resources. However, the growth of bacteria and their leaching activity are often inhibited (restricted) by organic components, e.g. lysates and exudates. The aims of this study were to examine the extent of cell lysis (CLs) inhibition on acidophilic microorganisms and to identify microorganisms that can utilize CLs products and eliminate their inhibition effect on acidophilic microorganisms. Specifically, it was revealed that Acidithiobacillus caldus was severely inhibited at 5% CLs products, whereas A. ferrooxidans and Leptospirillum ferriphilum are severely inhibited at 20%. It has been found that strains RBA and RBB of heterotrophic bacteria, isolated from anaerobic sludge, can biodegrade CLs products and when co-cultured with A. ferrooxidans, they can alleviate the toxic effect of CLs products under low pH (2–3). It has been shown that besides CLs, isolated strains can grow on glucose, glycerol, yeast extract, citric acid, and tryptone soya broth with an optimum temperature of 35°C and a pH of 3. The strains showed the ability to reduce ferric ions to ferrous ions when glycerol was used as a substrate after 2 days under both aerobic and anaerobic conditions. On the basis of morphophysiological and molecular biological studies, the isolated strains RBA and RBB were identified as Acidocella spp.     

Microbial Production of l-Threonine

l-Threonine producing α-amino-β-hydroxyvaleric acid resistant mutants were derived from E. coli K-12 with 3 x 10^-5 frequency. One of mutants, strain β-101, accummulated maximum amount of l-threonine (1. 9 g/liter) in medium. Among isoleucine, methionine and lysine auxotrophs derived from E. coli K-12, only methionine auxotrophs produced l-threonine. In contrast, among isoleucine, methionine and lysine auxotrophs derived from β-101, l-threonine accumulation was generally enhanced in isoleucine auxotrophs. One of isoleucine auxotrophs, strain βI-67, produced maximum amount of l-threonine (4. 7 g/liter). Methionine auxotroph, βM-7, derived from β-101 produced 3.8 g/liter, and βIM-4, methionine auxotroph derived from β1-67, produced 6.1 g/liter, when it was cultured in 3% glucose medium supplemented with 100 μg/ml of l-isoleucine and l-methionine, respectively. These l-threonine productivities of E. coli mutants were discussed with respect to the regulatory mechanisms of threonine biosynthesis. A favourable fermentation medium for l-threonine production by E. coli mutants was established by using strain βM-4.     

<Emphasis Type="Italic">In vitro</Emphasis> inhibitory effect of cranberry (<Emphasis Type="Italic">Vaccinium macrocarpum</Emphasis> Ait.) juice on pathogenic microorganisms

The purpose of this study was to determine the inhibitory effects of cranberry juice on pathogenic microorganisms. The microorganisms analyzed were Escherichia coli from patients with urinary infections, Salmonella spp., Listeria monocytogenes, Pseudomona aeruginosa, and Staphylococcus aureus. The disc method was used to determine the sensitivity of bacteria to cranberry juice (CJ, both concentrated and diluted). A lawn of 10⁶ cfu/ml was grown on agar surfaces in Petri dishes and on Whatman discs that had been previously saturated with CJ and CJ : water. 1 : 1 to 1 : 50 juice solutions had been placed on the discs, which were cultured and incubated. The results indicated that S. aureus was more susceptible to cranberry juice inhibition than the other microorganisms. L. monocytogenes was the most resistant to the inhibitory action of cranberry juice, showing a significant difference from the inhibition of P. aeruginosa, uropathogenic E. coli, Salmonella spp., and S. aureus. This study also demonstrated that the inhibitory activity of cranberry juice for E. coli took place up to a dilution of 1 : 20. Published in Russian in Prikladnaya Biokhimiya i Mikrobiologiya, 2008, Vol. 44, No. 3, pp. 333–336. The text was submitted by the authors in English.     

Synthetic Analogues of the Termite Trail-following Substance

The effect of tripropyltin chloride (TPT) on some functional reactions in E. coli was investigated. The inhibition on respiration and protein, DNA and RNA syntheses were examined in vivo. Oxygen consumption by E. coli cells was scarcely inhibited even at the concentration of 30 µg/ml TPT. The incorporations of ¹⁴C-labeled amino acids into protein fraction were inhibited. Especially, in the case of l-leucine, it was inhibited 60% even at 10 µg/ml TPT. Both incorporations of ¹⁴C-adenine into DNA and RNA fraction were inhibited 50–60% at 20 µg/ml TPT. RNA polymerase was prepared from E. coli cells and the effect of organotin compounds on the enzyme activity was examined. Organotin compounds inhibited the enzyme activity only at high concentrations (5-10mm). and dialkyltin chlorides which possess no antimicrobial action showed the inhibition more intensely than trialkyltin chlorides. The effect on membrane-bound ATPase was also examined in vitro. We found that TPT has high inhibitory action on membrane-bound ATPase. However, it slightly inhibited the activity of ATPase separated from membrane.     

A quantitative assessment of the antimicrobial activity of garlic (Allium sativum)

An aqueous extract of freeze-dried garlic (Allium sativum), when incorporated into growth media, inhibited many representative bacteria, yeasts, fungi and a virus. All microorganisms tested were susceptible to garlic. Quantitative assessment of the minimum inhibitory concentrations for bacteria and yeasts showed values ranging from 0.8 to 40.0 mg garlic ml^-1. Fungal radial colony growth was inhibited by at least 25% at concentrations as low as 2.0 mg garlic ml^-1. The 50% endpoint neutralization titre for rotavirus was 2.4 to 2.8 g ml^-1. Lactic acid bacteria were the least sensitive microorganisms to the inhibitory effects of garlic. In mixed culture studies of Lactobacillus acidophilus and Escherichia coli, garlic prevented the establishment of E. coli, although the final outcome of competition was not affected.L.P. Rees, S.F. Minney and N.T. Plummer are with Interprise Ltd, Baglan Bay Industrial Park, Port Talbot SA 12 7DJ, UK. J.H. Slater and D.A. Skyrme are with the School of Pure and Applied Biology, University of Wales, Cardiff, P.O. Box 915, Cardiff CF1 3TL, UK.     

Deficiency in methionine adenosyltransferase resulting in limited repressibility of methionine biosynthetic enzymes in Aspergillus nidulans

Summary In Aspergillus nidulans methionine can be metabolized to cysteine. Mutants blocked in this pathway were selected and divided into three groups representing three separate loci: mecA, mecB and mecC. mecC13 mutant possesses a low level of methionine adenosyltransferase and shows a limited extent of methionine-caused repression of three enzymes of the methionine biosynthetic pathway: sulfate permease, sulfite reductase and 0-acetylhomoserine sulfhydrylase. Intracellular pools of methionine do not differ markedly in the mutant and in wild type, while the S-adenosylmethionine (SAM) pool is decreased in the mutant. Methionine adenosyltransferase was found to be inducible by methionine, SAM is postulated to be involved in regulation of methionine biosynthetic enzymes in A. nidulans. Differences in regulation of methionine biosynthesis in A. nidulans, Escherichia coli and Saccharomyces cerevisiae are discussed.     

Ophiocordin,an antifungal antibiotic of Cordyceps ophioglossoides

An unknown antibiotic, ophiocordin, C₂₁H₂₂N₂O₈, MW: 430, was isolated from submerged cultures of Cordyceps ophioglossoides, strain TÜ 276, grown in a glycerol soybean meal medium at 27°C. The antibiotic was extracted from acidified culture fluids with n-butanol and purified by subsequent column chromatography on DEAE-Sephadex and cellulose. Studies including nuclear magnetic resonance and mass spectrometry resulted in proposals of partial structures of the molecule. Inhibition by ophiocordin could be demonstrated for a small number of fungi belonging to different taxonomic groups. Bacteria were not inhibited. The antifungal effect was antagonized by ammonia and nitrate ions and by certain amino acids.Metabolic products of microorganisms, 161st Communication. 160. Mitteilung: J. M. Müller, H. Fuhrer, J. Gruner, and W. Voser: Conocandin, ein fungistatisches Antibiotikum aus Hormococcus conorum (Sacc. et Roum.) Roback. Helv. chim. Acta 59, 2506–2514 (1976)     

Glutamate kinase from Thermotoga maritima: characterization of a thermophilic enzyme for proline biosynthesis

Glutamate kinase (GK), an enzyme involved in osmoprotection in plants and microorganisms, catalyses the first and controlling step of proline biosynthesis. The proB gene encoding GK was cloned from the hyperthermophilic bacterium Thermotoga maritima and overexpressed in Escherichia coli, and the resulting protein was purified to homogeneity in three simple steps. T. maritima GK behaved as a tetramer, showing maximal activity at 83°C, and was inhibited by ADP and proline. Although T. maritima GK exhibited high amino acid similarity to the mesophilic E. coli GK, it was less dependent of Mg ions and was not aggregated in the presence of proline. Moreover, it displayed a greater thermostability and higher catalytic efficiency than its mesophilic counterpart at elevated temperatures.     

Stoffwechselprodukte von Mikroorganismen

Zusammenfassung Der Streptomycet Tü 901, Streptomyces tendae, bildet ein antifungisch wirkendes Nukleosid-Antibioticum, Nikkomycin. Als Angriffsort kommt die Chitinsynthese in Frage.Mit Hilfe der Massenspektrometrie und des chemischen Abbaus konnten Uracil, eine Aminohexuronsäure und eine neue, einen Pyridinring enthaltende Aminosäure nachgewiesen werden.

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Metabolic products of microorganisms154. Nikkomycin, a new inhibitor of fungal chitin synthesis

From the fermentation broth of Streptomyces tendae Tü 901 a substance was isolated, which inhibits the growth of several fungi. The new antibiotic affects the chitchin biosynthesis. Its structure was identified by mass spectrometry of the products obtained after chemical degradation. Nikkomycin is a nucleoside-peptide antibiotic consisting of uracil, an amino hexuronic acid and a new amino acid containing a pyridin ring.

153. Mitteilung: G. Wolf, J. Wörth, H. Achenbach: Untersuchungen der Pigmente aus Streptomyces michiganesis. Arch. Microbiol. 106, 245–249 (1975)     

Metabolic products of microorganisms. 185. The anthraquinones of the Aspergillus glaucus group. I. Occurrence,isolation, identification and antimicrobial activity

The occurrence of emodin, erythroglaucin, physcion, physcion-9-anthrone, questin, catenarin, and catenarin-8-methyl ether in different species of the Aspergillus glaucus group (genus Eurotium) was investigated. So far catenarin-8-methyl ether (1, 4, 6-trihydroxy-8-methoxy-3-methylanthraquinone) has not been described as a natural product; it was therefore given the name rubrocristin. The chemical and physical properties of rubrocristin are reported. In addition a new violet pigment (C₁₆H₁₂O₅) was isolated and characterized by its MS-, IR- and UV-spectra.The antimicrobial properties of all substances were examined in the agar diffusion assay. Gram-positive bacteria were the most sensitive organisms and catenarin was the most active naturally occurring substance. Synthetically obtained 1, 4, 6, 8-tetrahydroxy-anthraquinone was slightly more active than catenarin, whereas rubrocristin showed no antibacterial activity.Abbreviations MIC Minimal inhibitory concentration - TLC Thin layer chromatography - PTLC Preparative thin layer chromatography Metabolic products of microorganisms. 184. H. Anke: On the mode of action of cladosporin. J. Antibiotics 32, 952–958 (1979)     

Biosynthesis of L-5-methyltetrahydrofolate by genetically engineered Escherichia coli

L-5-Methyltetrahydrofolate (L-5-MTHF) is the only biologically active form of folate in the human body. Production of L-5-MTHF by using microbes is an emerging consideration for green synthesis. However, microbes naturally produce only a small amount of L-5-MTHF. Here, Escherichia coli BL21(DE3) was engineered to increase the production of L-5-MTHF by overexpressing the intrinsic genes of dihydrofolate reductase and methylenetetrahydrofolate (methylene-THF) reductase, introducing the genes encoding formate-THF ligase, formyl-THF cyclohydrolase and methylene-THF dehydrogenase from the one-carbon metabolic pathway of Methylobacterium extorquens or Clostridium autoethanogenum and disrupting the gene of methionine synthase involved in the consumption and synthesis inhibition of the target product. Thus, upon its native pathway, an additional pathway for L-5-MTHF synthesis was developed in E. coli, which was further analysed and confirmed by qRT-PCR, enzyme assays and metabolite determination. After optimizing the conditions of induction time, temperature, cell density and concentration of IPTG and supplementing exogenous substances (folic acid, sodium formate and glucose) to the culture, the highest yield of 527.84 μg g⁻¹ of dry cell weight for L-5-MTHF was obtained, which was about 11.8 folds of that of the original strain. This study paves the way for further metabolic engineering to improve the biosynthesis of L-5-MTHF in E. coli.     

Inhibition of ethylene production by rhizobitoxine

Rhizobitoxine, an inhibitor of methionine biosynthesis in Salmonella typhimurium, inhibited ethylene production about 75% in light-grown sorghum seedlings and in senescent apple tissue. Ethylene production stimulated by indoleacetic acid and kinetin in sorghum was similarly inhibited. With both apple and sorghum, the inhibition could only be partially relieved by additions of methionine. A methionine analogue, α-keto-γ-methylthiobutyric acid, which has been suggested as an intermediate between methionine and ethylene, had no effect on the inhibition.     

Cystathionine as a precursor of methionine in Escherichia coli and Aerobacter aerogenes

Balish, Edward (Argonne National Laboratory, Argonne, Ill.), and Stanley K. Shapiro. Cystathionine as a precursor of methionine in Escherichai coli and Aerobacter aerogenes. J. Bacteriol. 92:1331-1336. 1966.-Cystathionine has been shown to be a precursor of methionine biosynthesis in Escherichia coli and Aerobacter aerogenes. A double enzyme assay was developed to show the formation of homocysteine from cystathionine. The results obtained support the concept that cystathionine serves as a precursor of methionine via the intermediate formation of homocysteine. The latter compound is methylated by the homocysteine methyltransferase of these microorganisms. Sulfhydryl and keto acid assays were used to demonstrate cystathionase activity. Methionine represses both homocysteine methyltransferase formation and cystathionase formation. However, the presence of methionine in reaction mixtures resulted in product inhibition of homocysteine methyltransferase activity, but not of cystathionase activity.     

Porphyrin metabolism in lead and mercury treated bajra (Pennisetum typhoideum) seedlings

Lead and mercury inhibited porphyrin biosynthesis significantly in the germinating seeds of bajra (Pennisetum typhoideum). Both 5-aminolevulinic acid dehydratase and porphobilinogen deaminase activities were inhibited by these metals. A comparative study of the inhibition of these two enzymes under invivo andin vitro conditions showed that 5-aminolevulinic acid dehydratase is the major site of action of heavy metals in porphyrin biosynthesis. Further, over-all production of porphyrinsviz., protoporphyrin-IX, Mg-protoporphyrin (ester) and protochlorophyllide was repressed by lead and mercury in both light and dark grown seedlings. Similarly, chlorophylla and chlorophyllb and total chlorophyll contents in dark-grown seedlings were also significantly decreased, suggesting the impairment of chlorophyll biosynthesis by lead and mercury in germinating seedlings.     

Ethylene formation by cultures of Escherichia coli

Growth of Escherichia coli strain B SPAO on a medium containing glucose, NH₄Cl and methionine resulted in production of ethylene into the culture headspace. When methionine was excluded from the medium there was little formation of ethylene. Ethylene formation in methionine-containing medium occurred for a brief period at the end of exponential growth. Ethylene formation was stimulated by increasing the medium concentration of Fe³⁺ when it was chelated to EDTA. Lowering the medium phosphate concentration also appeared to stimulate ethylene formation. Ethylene formation was inhibited in cultures where NH₄Cl remained in the stationary phase. Synthesis of the ethylene-forming enzyme system was determined by harvesting bacteria at various stages of growth and assaying the capacity of the bacteria to form ethylene from methionine. Ethylene forming capacity was greatest in cultures harvested immediately before and during the period of optimal ethylene formation. It is concluded that ethylene production by E. coli exhibits the typical properties of secondary metabolism.Abbreviations HMBA 2-Hydroxy-4-methylthiobutyric acid (methionine hydroxy analogue) - KMBA 2-keto-4-methylthiobutyric acid - MOPS 3-[N-morpholino] propanesulphonic acid     

Evolved Cobalamin-Independent Methionine Synthase (MetE) Improves the Acetate and Thermal Tolerance of Escherichia coli

Acetate-mediated growth inhibition of Escherichia coli has been found to be a consequence of the accumulation of homocysteine, the substrate of the cobalamin-independent methionine synthase (MetE) that catalyzes the final step of methionine biosynthesis. To improve the acetate resistance of E. coli, we randomly mutated the MetE enzyme and isolated a mutant enzyme, designated MetE-214 (V39A, R46C, T106I, and K713E), that conferred accelerated growth in the E. coli K-12 WE strain in the presence of acetate. Additionally, replacement of cysteine 645, which is a unique site of oxidation in the MetE protein, with alanine improved acetate tolerance, and introduction of the C645A mutation into the MetE-214 mutant enzyme resulted in the highest growth rate in acetate-treated E. coli cells among three mutant MetE proteins. E. coli WE strains harboring acetate-tolerant MetE mutants were less inhibited by homocysteine in l-isoleucine-enriched medium. Furthermore, the acetate-tolerant MetE mutants stimulated the growth of the host strain at elevated temperatures (44 and 45°C). Unexpectedly, the mutant MetE enzymes displayed a reduced melting temperature (T_m) but an enhanced in vivo stability. Thus, we demonstrate improved E. coli growth in the presence of acetate or at elevated temperatures solely due to mutations in the MetE enzyme. Furthermore, when an E. coli WE strain carrying the MetE mutant was combined with a previously found MetA (homoserine o-succinyltransferase) mutant enzyme, the MetA/MetE strain was found to grow at 45°C, a nonpermissive growth temperature for E. coli in defined medium, with a similar growth rate as if it were supplemented by l-methionine.     

Antibacterial and membrane‐damaging activities of β‐bungarotoxin B chain

This study investigates whether the B chain of β‐bungarotoxin exerted antibacterial activity against Escherichia coli (Gram‐negative bacteria) and Staphylococcus aureus (Gram‐positive bacteria) via its membrane‐damaging activity. The B chain exhibited a growth inhibition effect on E. coli but did not show a bactericidal effect on S. aureus. The B‐chain bactericidal action on E. coli positively correlated with an increase in membrane permeability in the bacterial cells. Lipopolysaccharide (LPS) layer destabilization and lipoteichoic acid (LTA) biosynthesis inhibition in the cell wall increased the B‐chain bactericidal effect on E. coli and S. aureus. The B chain induced leakage and fusion in E. coli and S. aureus membrane‐mimicking liposomes. Compared with LPS, LTA notably suppressed the membrane‐damaging activity and fusogenicity of the B chain. The B chain showed similar binding affinity with LPS and LTA, whereas LPS and LTA binding differently induced B‐chain conformational change as evidenced by the circular dichroism spectra. Taken together, our data indicate that the antibacterial action of the B chain is related to its ability to induce membrane permeability and suggest that the LPS‐induced and LTA‐induced B‐chain conformational change differently affects the bactericidal action of the B chain. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.