Rapid identification of teleocidins in fermentation broth using HPLC photodiode array and LC/MS methodology

Summary During the course of screening for inhibitors of phorbol ester binding to protein kinase C, several actinomycete cultures were discovered that produce active metabolites. HPLC coupled to photodiode array and LC/MS techniques were applied to broth extracts to identify the presence of indolactams belonging to the teleocidin family. Various members of this family were rapidly identified from crude broth extracts using a combination of these spectroanalytical procedures. An analytical HPLC system was developed to optimize separation of teleocidin, A and B analogues directly from ethyl acetate extracts of whole broth cultures. This technique allowed us to identify a novel homologue of pendolmycin and demonstrated the utility of photodiode array HPLC coupled with LC/MS as an intial analytical tool in the analyses of these secondary metabolites produced by soil microorganisms.     

Purification and characterization of a thermostable pullulanase fromThermoactinomyces thalpophilus

Summary Thermoactinomyces thalpophilus No. 15 produced an extracellular pullulanase in an aerobic fermentation with soluble starch, salts, and complex nitrogen sources. Acetone fractionation, ion-exchange chromatography, and gel filtration purified the enzyme from cell-free broth 16-fold to an electrophoretically homogeneous state (specific activity, 1352 U/mg protein; yield, 4%). The purified enzyme (estimated MW 79 000) was optimally active at pH 7.0 and 70°C and retained 90% relative activity at 80°C (30 min) in the absence of substrate. The enzyme was activated by Co²⁺, inhibited by Hg²⁺, and exhibited enhanced stability in the presence of Ca²⁺. The enzyme hydrolyzed pullulan (K _m 0.32%, w/v) forming maltotriose, and hydrolyzed amylopectin (K _m 0.36%, w/v), amylopectin beta-limit dextrin (K _m 0.45%, w/v) and glycogen beta-limit dextrin (K _m 1.11%, w/v) forming maltotriose and maltose.     

rRNA operon restriction derived taxa for Streptomyces (RiDiTS)

Abstract A method for grouping Streptomyces strains by fingerprints of their rRNA operons is described. In polyacrylamide gels, multicopy rRNA operon fragments in Streptomyces genomic Mse I fingerprints produced intense bands which are well resolved from the less conspicuous low copy fragments interspersed between them. The high intensity multicopy rRNA bands are easily distinguished from the low intensity bands, eliminating the need for Southern blot hybridization to visualize the rRNA fragments. Direct evidence that the high-intensity bands in these polyacrylamide gels originated from rRNA operons was provided by a 'differential' Southern blot technique. We have used this method to assign 98 strains to 11 rRNA fingerprint type groups. This clustering method may be applicable to any prokaryote with a high G + C content genome.     

DTPA soil extractable and plant heavy metal concentrations with soil-added Cd treatments

Summary The numbers, weights, and ages of root nodules were determined in a homogeneous 6- to 7-year-old stand ofHippophaë rhamnoides in a coastal sand-dune area. Large variations in the number of nodules, which ranges from 30 to 281 per m² were found. Most of the nodules occurred between 10 and 60 cm below the soil surface. In the surface layer and lower than 60 cm only small numbers of nodules were present. It is concluded that relatively large samples have to be investigated to obtain an impression of the nodulation of a given stand and to permit a comparison of the contribution of dinitrogen fixation in different stands.Close correlation was found between nodule numbers and the age of the roots, young roots having the highest numbers. However, the wide variation in the number of nodules per square metre could not be explained solely by the age distribution of the roots. The possible role of some other factors is discussed. No relationship was found between the nodule weight and the age of the nodule. The mean dry weight of living nodule material was 5.3 g per m². Most nodules reached an age of up to 3 years. The significance of nodule decay for the distribution of the endophyte in the soil and in the process of nodule formation is discussed.     

Identification of a lipoarabinomannan-like lipoglycan in the actinomycete Gordonia bronchialis

The cell envelopes of actinomycetes contain lipidated macroamphiphiles, of which the most extensively characterised are the lipoarabinomannans of mycobacteria and related bacteria. We have investigated the mycolic acid-containing actinomycete Gordonia bronchialis and identified the presence of a lipoarabinomannan-like lipoglycan. The extraction and purification procedures recovered a second amphiphilic fraction with properties suggesting a phosphatidylinositol mannoside, consistent with studies of other Gordonia species.Dedicated to the memory of our former colleague Dr. David Bendell.     

A new cytochrome P450 belonging to the 107L subfamily is responsible for the efficient hydroxylation of the drug terfenadine by Streptomyces platensis

Fexofenadine, an antihistamine drug used in allergic rhinitis treatment, can be produced by oxidative biotransformation of terfenadine by Streptomyces platensis, which involves three consecutive oxidation reactions. We report here the purification and identification of the enzyme responsible for the first step, a cytochrome P450 (P450)-dependent monooxygenase. The corresponding P450, designated P450_terf, was found to catalyze the hydroxylation of the t-butyl group of terfenadine and exhibited UV–Vis characteristics of a P450. Its interaction with terfenadine led to a shift of its Soret peak from 418 to 390 nm, as expected for the formation of a P450–substrate complex. In combination with spinach ferredoxin:NADP(+) oxidoreductase and ferredoxin, and in the presence of NADPH, it catalyzed the hydroxylation of terfenadine and some of its analogues, such as terfenadone and ebastine, with k_m values at the μM level, and k_cat values around 30 min⁻¹. Sequencing of the p450_terf gene led to a 1206 bp sequence, encoding for a 402 aminoacid polypeptide exhibiting 56–65% identity with the P450s from the 107L family. These results confirmed that P450s from Streptomyces species are interesting tools for the biotechnological production of secondary metabolites, such as antibiotics or antitumor compounds, and in the oxidative biotransformation of xenobiotics, such as drugs.     

Production of the glycopeptide antibiotic A40926 by<Emphasis Type="Italic"> Nonomuraea</Emphasis> sp. ATCC 39727: influence of medium composition in batch fermentation

Nonomuraea sp. ATCC 39727 is a novel actinomycete species and the producer of A40926, a glycopeptide antibiotic structurally similar to teichoplanin. In the present study, a defined minimal medium was designed for Nonomuraea fermentation. The influence of initial phosphate, glucose and ammonium concentrations on antibiotic productivity was investigated in batch fermentation and the effect of glucose limitation was studied in fed-batch fermentation. It was found that low initial concentrations of phosphate and ammonium are beneficial for A40926 production and that productivity is not enhanced during glucose limitation. Furthermore, the initiation of A40926 production was not governed by residual ammonium and phosphate concentrations, although the level of these nutrients strongly influenced A40926 production rates and final titers. Electronic Publication     

Proteome analysis of <Emphasis Type="Italic">Streptomyces coelicolor</Emphasis> mutants affected in the proteasome system reveals changes in stress-responsive proteins

Prokaryotic 20S proteasomes are confined to archaebacteria and actinomycetes. Bacterial targets of this compartmentalized multi-subunit protease have not yet been identified and its physiological function in prokaryotes remains unknown. In this study, intracellular and extracellular proteomes of Streptomyces coelicolor A3(2) mutants affected in the structural genes of the 20S proteasome, in the gene encoding the presumed proteasome-accessory AAA ATPase ARC, or in two putative proteasome-associated actinomycete-specific genes (sco1646, sco1647) were analysed, revealing modified patterns of stress-responsive proteins. In addition, the extracellular protease profile of the sco1647 mutant was significantly altered. The most prominent change, common to the four mutants, was a strongly increased level of the non-heme chloroperoxidase SCO0465, coinciding with an increased resistance to cumene hydroperoxide. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.