Inhibition of β-galactosidase synthesis inEscherichia coli after infection with different DNA and RNA phages

Infection ofEscherichia coli with T1, T2r⁺, T3 and T4 phages leads to an immediate inhibition of β-galactosidase synthesis. Similar results were obtained with the virulent mutant of phage lambda. The degree of inhibition of β-galactosidase synthesis depends on the time delay between the addition of the inducer and the phage particles, and on the amount of phage DNA, which has penetrated into the host cell. RNA phage MS2 exhibited no inhibitory effect on enzyme synthesis.     

Intergeneric Conjugation Escherichia coli–Streptomyces globisporus1912 Using Integrative Plasmid pSET152 and Its Derivatives

Streptomyces globisporus1912 produces a novel angucycline antitumor antibiotic landomycin E (LE). To study the LE biosynthetic gene cluster in detail, a system for the conjugal transfer of the integrative plasmid pSET152 fromEscherichia coliinto S. globisporus1912 has been developed. It was shown that this plasmid integrates into two sites of the S. globisporuschromosome and is stably inherited under nonselective conditions. pSET152⁺exconjugants of the strain 1912 are characterized by a significant decrease in LE synthesis (by 50–90%). A negative effect of pSET152 integration on antibiotic production was observed even upon the use of the recipient strain with increased LE synthesis, although in this case, the level of LE production in exconjugants was 120–150% of that in the original strain 1912. Based on pSET152, a vector system for gene knockouts in S. globisporuswas developed. The effectivity of this system was shown in the example of disruption of the lndAgene encoding the key enzyme of LE synthesis (-ketoacylsynthase). Inactivation of this gene was shown to lead to the cessation of LE biosynthesis.     

Starch degradation by the mould Trichoderma viride II. Regulation of enzyme synthesis

The synthesis of amylolytic enzymes by the maltose not-utilizing Trichoderma viride strain CBS 354.44 requires the presence of starch or dextrins. Several readily utilizable carbon sources such as glucose and glutamic acid were shown to exert a strong catabolite repression which completely inhibited enzyme induction by starch or dextrins.Enzyme synthesis occurs in the exponential and in the stationary growth phase. In the latter, the ratio between saccharifying and dextrinizing enzyme activity is invariably high. In the exponential growth phase this ratio depends on the nature of the inducing substrate. Growth on starch results in an initially high production of dextrinizing activity, the saccharifying one becoming predominant in the course of exponential growth. The latter activity in dextrin DE 30 cultures is predominant from the very beginning. Thus, the amylolytic enzyme system of T. viride consists of at least two different enzymes, the synthesis of each being controlled specifically. The careful regulation of the synthesis of the dextrinizing enzyme is discussed with special reference to the production of non-utilizable maltose by the latter.     

The Acidocalcisome Vacuolar Transporter Chaperone 4 Catalyzes the Synthesis of Polyphosphate in Insect‐stages of Trypanosoma brucei and T. cruzi

Polyphosphate is a polymer of inorganic phosphate found in both prokaryotes and eukaryotes. Polyphosphate typically accumulates in acidic, calcium‐rich organelles known as acidocalcisomes, and recent research demonstrated that vacuolar transporter chaperone 4 catalyzes its synthesis in yeast. The human pathogens Trypanosoma brucei and T. cruzi possess vacuolar transporter chaperone 4 homologs. We demonstrate that T. cruzi vacuolar transporter chaperone 4 localizes to acidocalcisomes of epimastigotes by immunofluorescence and immuno‐electron microscopy and that the recombinant catalytic region of the T. cruzi enzyme is a polyphosphate kinase. RNA interference of the T. brucei enzyme in procyclic form parasites reduced short chain polyphosphate levels and resulted in accumulation of pyrophosphate. These results suggest that this trypanosome enzyme is an important component of a polyphosphate synthase complex that utilizes ATP to synthesize and translocate polyphosphate to acidocalcisomes in insect stages of these parasites.     

Identification of template strand in heteroduplex DNA directing cell-free enzyme synthesis

Summary Cell-free synthesis of galactose enzymes was directed by heteroduplex DNA carrying an amber mutation in thegal T gene on one DNA strand, the wildtype configuration on the complementary strand. Comparison with the corresponding homoduplex DNA preparations shows that the presence of the wildtype base sequence in the l-strand is necessary and sufficient for the synthesis of active enzyme. Single stranded DNA of either configuration is inactive in this system.     

Studies on the central metabolism of Thermus aquaticus,an extreme thermophilic bacterium: Anaplerotic reactions and their regulation

The physiology of Thermus aquaticus strain Z05 was investigated. Substantial evidence for gene and enzyme regulation in the central metabolism of this extreme thermophile was found.Two anaplerotic pathways were detected: (1) phosphoenolpyruvate carboxylase; (2) a glyoxylate shunt which proved to be essential for growth on pyruvate as well as acetate. The synthesis of isocitrate lyase and malate synthase were found to depend on a common control mechanism. Pronounced regulatory effects were observed on the activity of malic enzyme, pyruvate kinase and phosphoenolpyruvate carboxylase. The data could be fitted together into a picture of the metabolism during glycolysis and gluconeogenesis which shows how variations of enzyme levels and activities correlate with the apparent needs of the cell.Our results call attention to a peculiar metabolic analogy between T. aquaticus and Acinetobacter Abbreviations ace acetate nonutilizing - Acetyl-CoA acetyl-coenzyme A - I.U. international unit - PEP phosphoenolpyruvate - T Thermus     

Biological control of southern corn leaf blight by Trichoderma atroviride SG3403

Trichoderma atroviride SG3403 showed high biocontrol activity against southern corn leaf blight (SCLB; pathogen: Cochliobolus heterostrophus). T. atroviride SG3403 could cause death of C. heterostrophus race O hypha on plates. Spraying T. atroviride SG3403 conidia suspension over maize seedling leaves protected the corn from SCLB infection. Biocontrol effect lasted for 30 days in the field. Trichoderma strain was able to induce resistance response in corn leaves against pathogen infection. In corn leaves treated with T. atroviride SG3403, the enzyme activities of phenylalanine ammonia lyase (PAL) and superoxide dismutase (SOD) reached the highest at 24 h, enzyme activity of catalase (CAT) reached the highest at 36 h after inoculation of pathogen C. heterostrophus race O. RNA expression levels of Pal, Sod and Cat (which synthesis enzyme PAL, SOD and CAT) were also upregulated and corresponded to the enzyme activity at the same time point. Enzyme activities and corresponding genes expression induced by Trichoderma SG3403 was more obvious than that induced by pathogen only, which implies that T. atroviride SG3403 induced corn defense gene expression against pathogen infection. Thus, induced resistance mechanism was possibly involved in the biocontrol of SCLB by T. atroviride SG3403.     

Evaluation of β-1,3-glucanase and β-1,4-glucanase enzymes production in some Trichoderma species

Trichoderma species have become the important means of biological control for fungal diseases. This research was carried on to access the high β-1,3-glucanase and β-1,4-glucanase enzyme producer of Trichoderma species isolates using two different carbon sources for finding a method to obtain more concentrate culture filtrates. Therefore, 14 Trichoderma isolates belonging to species: Trichoderma ceramicum, T. virens, T. pseudokoningii, T. koningii, T. koningiosis, T. atroviridae, T. viridescens, T. asperellum, T. harzianum1, T. orientalis, T. harzianum2, T. brevicompactum, T. viride and T. spirale were cultured in Wiendling’s liquid medium plus 0.5% glycerol or 0.5% Phytophthora sojae-hyphe as the carbon source in shaking and non-shaking (stagnant) statuses. Enzyme activity rate and total protein were evaluated in raw, acetony and lyophilized concentrated culture filtrates and the specific enzyme activity of β-1,3-glucanase and β-1,4-glucanase were measured by milligramme glucose equivalent released per minute per milligramme total protein in culture filtrates. The results showed that using Phytophthora – hyphe in medium increased the enzyme activities as compared to glycerol at all Trichoderma species which suggested that these substrates can also act as inducer for synthesis of lytic enzymes, in addition the most enzymes activity was observed in the lyophilised concentrated culture filtrate. The most successful species in β-1,3-glucanase and β-1,4-glucanase enzymes activities were T. brevicompactum and T. virens and these species can be used for mass production of these enzymes which are supposed to be used in commercial formulation and also will be able to control P. sojae directly.     

Molecular interactions involving Escherichia coli nucleoside diphosphate kinase

Nucleoside diphosphate kinase plays a distinctive metabolic role as the enzyme poised between the last reaction of deoxyribonucleoside triphosphate (dNTP) biosynthesis and the DNA polymerization apparatus. In bacteriophage T4 infection, NDP kinase is one of very few enzymes of host cell origin to participate in either dNTP synthesis or DNA replication. Yet NDP kinase forms specific contacts with phage-coded proteins of dNTP and DNA synthesis. This article summarizes work from our laboratory that identifies and characterizes these interactions. Despite these specific interactions, the enzyme appears to be dispensable, both for T4 replication and for growth of the host, Escherichia coli, because site-specific disruption of ndk, the structural gene for NDP kinase, does not interfere with growth of the host cell and only partly inhibits phage replication. However, ndk disruption unbalances the dNTP pools and stimulates mutagenesis. We discuss our attempts to understand the basis for this enhanced mutagenesis.     

Expression of the pyranose 2-oxidase from Trametes pubescens in Escherichia coli and characterization of the recombinant enzyme

cDNA-encoding pyranose 2-oxidase (P2O) from Trametes pubescens was sequenced and cloned into Escherichia coli strain BL21/DE3 on a multicopy plasmid under the control of trc promoter. The synthesis of P2O was studied in a batch culture in M9-based mineral medium: the enzyme was synthesized constitutively at 28 °C in amount corresponding to 8% of the cell soluble protein (0.6 U mg⁻¹). Only small portion of P2O (11%) was in the form of non-active inclusion bodies. Purified recombinant enzyme has similar physico-chemical and kinetic parameters with other P2Os. When compared to the expression of p2o of Trametes ochracea, a ratio of the mature enzyme to inclusion bodies found in the same E. coli host at 28 °C is as much as nine times higher. The finding makes the enzyme from T. pubescens preferable for the large-scale production by recombinant bacteria. The difference in amino acid sequences of the P2O from T. ochracea and T. pubescens may explain the favourable trait of the latter enzyme regarding protein folding.     

Fatty acid synthesis inTrichophyton rubrum

Analysis of the fatty acid content ofT. rubrum showed that the organism is capable of synthesizing a variety of saturated and unsaturated long-chain fatty acids (Kostiw, Vicher &Lyon, 1966). The present study was undertaken to determine the mechanism of synthesis of these fatty acids. Experimental data point to the presence of two mechanisms of fatty acid synthesis inT. rubrum:de novo synthesis and chain elongation. The presence of both mechanisms is suggested by the nature of the enzyme preparation, by the cofactor requirements for either pathway, and by the reaction products.University of Illinois at the Medical Center, and Chicago Medical School, Chicago, Illinois. Presented in part at the International Society of Mycology Meeting, New Orleans, Louisiana, August 1967.     

Identification of the "sense" -strand of T7 DNA through heteroduplex directed in vitro enzyme synthesis

Summary Enzyme synthesisin vitro directed by T7 heteroduplex DNA is observed when the H strand [the strand assuming the higher density in combination with poly(G,U)], carries the wild allele. This is true for enzyme synthesis mediated byE. coli RNA polymerase or by T7 phage RNA polymerase. Experiments of Wetekam (accompanying paper) using heteroduplexes with markers in thegal operon ofE. coli gave analogous results.     

Cloning and expression in Escherichia coli of a Thermoanaerobacter cellulolyticus gene coding for heat-stable β-glucanase

Summary A 4.8 kb HindIII fragment of Thermoanaerobacter cellulolyticus DNA cloned in Escherichia coli was shown to direct the synthesis of -glucanase. The enzyme produced by the transformant was extremely heat-stable and the optimum temperature for the enzyme reaction was 80°C. The cloned enzyme could hydrolyse carboxymethyl cellulose and lichenan, but could not digest laminarin, xylan and cellobiose. Although T. cellulolyticus secreted cellulase(s) into the medium, most of the cloned enzyme activity was detected only in cytoplasm in the recombinant clone.     

In vitro andin vivo nitrogenase activity ofRhizobium mutants and their symbiotic effectivity

A slow growing nitrogen-fixing strain ofVigna radiata var.aureus (mung bean)Rhizobium which expressed nitrogenase activity in a synthetic medium was isolated from its native population. Mutants with decreased and increased nitrogenase activity were derived from this strain by treatment with acridine orange and ethidium bromide. These mutants were tested for symbiotic effectivity invivo. The effectivity of mutants with decreased nitrogenase activity in the culture medium was lower than the parent strain; however, the effectivity of mutants with higher nitrogenase activity did not increase above that of the parent. This suggests that the plant is perhaps a limiting factor in the full expression of rhizobial nitrogenase in the nodules.     

Enhanced esterification activity through interfacial activation and cross‐linked immobilization mechanism of Rhizopus oryzae lipase in a nonaqueous medium

Interfacial activation via surfactant (Tween 80, Triton X‐100) treatment was conducted to improve the esterification activity of Rhizopus oryzae lipase that had undergone immobilization through cross‐linked enzyme aggregates (CLEA®) technique. Surfactant pretreated immobilized enzymes exhibited better esterification activity compared to free and non‐pretreated immobilized enzyme (Control CLEAs) since higher conversion rates were obtained within shorter times. The superiority of surfactant pretreated CLEAs, especially Tween 80 pretreated CLEAs (T 80 PT CLEAs), were clearly pronounced when longer alcohols were used as substrates. Conversion values exceeded 90% for octyl octanoate, oleyl octanoate and oleyl oleate synthesis with T 80 PT CLEAs whereas Control CLEAs and free enzyme showed no activity. Maximum conversions were achieved in the case equal molars of the substrates or in the case excess of the alcohol to acid in cyclohexane. In solvent free medium containing equal molars of substrates the conversion rates were 85% and 87% with T 80 PT CLEAs respectively for octyl octanoate and oleyl oleate within 2 hours. T 80 PT CLEAs showed 59% of its original activity after 7 consecutive usage for oleyl oleate synthesis. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:899–904, 2016     

Enzymic synthesis ofL-Lysine fromDL-α-Amino-ε-caprolactam by new microbial strains

The production of L-lysine fromDL-α-amino-ε-caprolactam (DL-ACL) by new strains producingL-α-amino-ε-caprolactamase and aminocaprolactam racemase is described. Optimal conditions for hydrolysis ofL-ACL byCryptococcus sp. and for racemization of ACL by cells of a strain isolated in nature and identified asPseudomonas sp. were determined. Synthesis ofL-α-amino-ε-caprolactamase is induced byDL-ACL orL-lysine with the same effectivity. A positive effect of phosphates (potassium salts) on reduction of the induction lag was detected, the synthesis of this enzyme was found to be repressed by glucose and some possibilities of the reversion of this repressive effect were demonstrated. Under conditions optimal for the production of both enzymes a quantitative theoretical conversion of 10 % aqueousDL-ACL toL-lysine by a mixture of native cells in a mass ratio of 1: 2 (producer of ACL-hydrolase to producer of ACL-racemase) occurred in 8 h at 40 °C and pH 8.0     

Degradation of catechin and purification and partial characterization of catechin oxygenase fromChaetomium cupreum

An enzyme capable of cleaving catechin was present in the mycelium ofCheatomium cupreum. Maximum synthesis of the enzyme occurred after 15 days growth. Sucrose and maltose increased enzyme synthesis among the carbon sources tested. Catechol, protocatechuic acid and phloroglucinol carboxylic acid were the intermediates of catechin degradation.Cheatomium cupreum containedmeta-cleaving enzymes for catechol and protocatechuic acid metabolism. Pyruvate was identified as an end-product. Catechin oxygenase from the mycelium ofC. cupreum was purified to homogeneity. It was optimum at pH 7.0 and 50°C and was highly specific for catechin, with a K_m of 4 m. Its molecular size was 40 kDa, as determined by gel filtration and gel electrophoresis, and it had a pI of 9.1.p-Chloromercuric benzoate, iodoacetate, N-ethylmaleimide, 2,2-dipyridyl and EDTA markedly inhibited the enzyme activity. It was a glycoprotein.T. Sambandam was and A. Mahedevan is with the Center for Advanced Studies in Botany, University of Madras, Guindy Campus, Madras-600025, India. T. Sambandam is now with the Department of Medicine, University of Alabama at Birmingham, Birmingham, AL 35294, USA.     

Increased DNA polymerase and ATP-dependent deoxyribonuclease activities following DNA damage in Mycobacterium smegmatis

Summary Treatment of growing cultures of Mycobacterium smegmatis with alkylating agents (methyl methaneusulphonate, ethyl methanesulphonate, nitrogen mustard, or mitomycin C) or with ultraviolet light resulted in enhanced specific activities of a DNA polymerase and of an ATP-dependent deoxyribonuclease. Similar results had previously been obtained with hydroxyurea and with iron limitation. The three of these treatments which were tested (methyl methane-sulphonate, mitomycin C and hydroxyurea) produced strand breaks or alkali-labile regions in the DNA of this organism. The increased enzyme activities could be prevented by simultaneous treatment with inhibitors of protein synthesis.In contrast, treatment of the cultures with intercalating agents (ethidium bromide, acridine orange, or proflavine), 5-fluorouracil, caffeine, or nalidixic acid, inhibited DNA synthesis without increasing the enzyme activities. These treatments did not produce strand breaks in the DNA of this organism.The results support the hypothesis that, in M. smegmatis, damage to DNA induces increased synthesis of enzymes associated with DNA repair.     

In vitro studies on the role of phage T7 gene 4 product in DNA replication

Summary A soluble enzyme fraction prepared from T7-infected E. coli is able to initiate DNA synthesis on circular single-stranded phage DNA. The product synthesized in vitro is a full-length linear complementary strand as judged by alkaline sucrose gradient analysis. DNA synthesis requires the products of the phage genes 4 and 5, Mg⁺⁺, dNTPs and rNTPs; however, ATP by itself can almost completely satisfy the rNTP requirement. The gene 4 product is essential for DNA chain initiation on unprimed single-stranded DNA, but is dispensable for the replication of a X174 DNA-RNA hybrid. The enzyme system from T7-infected cells does not discriminate between the DNA templates from phages X174, M13 or fd and is also capable of replicating native T7 DNA. However, a striking difference with regard to the template DNA is revealed by complementation analysis. Extracts of T7 mutant-infected cells complement each other only with T7 DNA but not with X174 DNA as template.Abbreviations rNTP ribonucleoside triphosphate - dNTP deoxyribonucleoside triphosphate - BSA bovine serum albumin