Bleomycin-induced β-lactamase overexpression in Escherichia coli carrying a bleomycin-resistance gene from Streptomyces verticillus and its application to screen bleomycin analogues

Abstract A bleomycin-resistance gene, designated blmA , has been cloned from bleomycin-producing Streptomyces verticillus by Sugiyama et al. (Gene 151 (1994) 11–16). The present study shows that Escherichia coli harboring the blmA -carrying pUC plasmid overproduced β-lactamase, encoded by an ampicillin-resistance gene on the plasmid, when cultured in the presence of bleomycin, which suggests that bleomycin may act as an inducer (or an activator) for the expression of the specific gene in the presence of blmA . We constructed a vector, designated pMAB50, which senses bleomycin and produces a pigment, using blmA and a Streptomyces tyrosinase gene located under the control of β-lactamase promoter: E. coli harboring pMAB50 produced the melanin pigment in the presence of bleomycin-type antibiotics, suggesting that the transformed E. coli can be employed as a reporter organism to screen bleomycin analogues.     

Production of bleomycin N-acetyltransferase in Escherichia coli and Streptomyces verticillus

Bleomycin-producing Streptomyces verticillus ATCC 15003 has two bleomycin resistance genes, designated blmA and blmB. Bleomycin N-acetyltransferase, encoded by blmB, was overproduced in Escherichia coli as a protein fused to the maltose-binding protein. The protein (fBAT), purified to homogeneity after digestion of the fusion product with blood coagulation factor X_a protease, had an additional 6 N-terminal amino acid residues, but retained its bleomycin-acetylating activity, as did the entire fusion protein. The K_m and V_max values of purified fBAT for the substrate bleomycin were 13.0 μM and 3.4 pmol min⁻¹ ml⁻¹, respectively. The optimal pH for the acetylating activity was 6.0 in 10 mM phosphate buffer. The molecular mass and pI value of fBAT were estimated by polyacrylamide gel electrophoresis to be about 34 500 and 6.13, respectively. An anti-fBAT monoclonal antibody was generated and used to show that bleomycin N-acetyltransferase is expressed simultaneously with bleomycin production in S. verticillus.     

The 1.6-A crystal structure of the copper(II)-bound bleomycin complexed with the bleomycin-binding protein from bleomycin-producing Streptomyces verticillus.

Bleomycin (Bm) in the culture broth of Streptomyces verticillus is complexed with Cu(2+) (Cu(II)). In the present study, we determined the x-ray crystal structures of the Cu(II)-bound and the metal-free types of Bm at a high resolution of 1.6 and 1.8 A, respectively, which are complexed with a Bm resistance determinant from Bm-producing S. verticillus, designated BLMA. In the current model of Cu(II).Bm complexed with BLMA, two Cu(II).Bm molecules bind to the BLMA dimer. The electron density map shows that the copper ion is clearly defined in the metal-binding domain of the Bm molecule. The metal ion is penta-coordinated by a tetragonal monopyramidal cage of nitrogens and binds to the primary amine of the beta-aminoalanine moiety of Bm. The binding experiment between Bm and BLMA showed that each of the two Bm-binding pockets has a different dissociation constant (K(d)(1) and K(d)(2)). The K(d)(1) value of 630 nm for the first Bm binding is larger than the K(d)(2) value of 120 nm, indicating that the first Bm binding gives rise to a cooperative binding of the second Bm to the other pocket.     

Urea-induced conformational changes in cold- and heat-denatured states of a protein, Streptomyces subtilisin inhibitor.

Streptomyces subtilisin inhibitor (SSI) is known to exist in at least two distinct denatured states, cold-denatured (D') and heat-denatured (D) under acidic conditions. In the present work, we investigated the manner how increasing urea concentration from 0 to 8 M changes the polypeptide chain conformation of SSI that exists initially in the D' and D states as well as in the native state (N), in terms of the secondary structure, the tertiary structure, and the chain form, based on the results of the experiments using circular dichroism (CD), small-angle X-ray scattering (SAXS) and 1H-NMR spectroscopy. Our results indicate that the urea-induced conformational transitions of SSI under typical conditions of D' (pH 1.8, 3 degrees C) occur at least in two steps. In the urea concentration range of 0-2 M (step 1), a cooperative destruction of the tertiary structure occurs, resulting in a mildly denatured state (DU), which may still contain a little amount of secondary structures. In the concentration range of 2-4 M urea (step 2), the DU state gradually loses its residual secondary structure, and increases the radius of gyration nearly to a maximum value. At 4 M urea, the polypeptide chain is highly disordered with highly mobile side chains. Increasing the urea concentration up to 8 M probably results in the more highly denatured or alternatively the stiffer chain conformations. The conformational transition starting from the N state proceeds essentially the same way as in the above scheme in which D' is replaced with N. The conformational transition starting from the D state lacks step 1 because the D state contains no tertiary structures and is similar to the DU state. The fact that similar conformations are reached at urea concentrations above 2 M from different conformations of D', D, and N indicates that the effect of urea dominates in determining the polypeptide conformation of SSI in the denatured states rather than the pH and temperature.     

1H n.m.r. study of geletin gels

Proton spin-lattice and spin-spin relaxation times have been measured in the gelatin-water system during gelatin at various concentrations and quenching temperatures. The spin-spin relaxation time is shown to be a good parameter for following the kinetics of gelation and can be related to the amount of helices measured by polarimetry. The results are analysed within the framework of the Zimmerman-Brittin theory.     

Isolation and characterization of an antibiotic produced by the scab disease-suppressive Streptomyces diastatochromogenes strain PonSSII

An antibiotic produced by the scab disease-suppressive Streptomyces diastatochromogenes strain PonSSII has been isolated and partially characterized. The antibiotic is produced throughout culture growth, with maximum amounts accumulating in the broth when the culture is in the early stationary phase of growth. The activity declines within about 30 h after the culture enters stationary phase. Purification techniques included chromatography on Amberlite XAD-2, DEAE Sephadex and SP Sephadex in addition to C18 HPLC with an average yield of 75%. This antibiotic only inhibits pathogenic strains of S. scabies that cause scab disease on potato and other tuberous vegetables and does not affect S. griseus, S. venezuelae, Actinomyces bovis, Nocardia asteroides, Clostridium perfringens, Bacillus subtilis, Staphylococcus aureus, S. epidermidis, Enterococcus faecalis, Micrococcus luteus, Serratia marcescens and Escherichia coli. The antibiotic has a molecular weight of 500 or less, and is stable for weeks at acidic pH but is very labile at alkaline pH conditions. Received 18 February 1997/ Accepted in revised form 11 August 1997     

Detection of Mobile Proteins by Proton Nuclear Magnetic Resonance Spectroscopy in the Guinea Pig Brain Ex Vivo and Their Partial Purification

Abstract: Proton nuclear magnetic resonance (¹H NMR) spectroscopy was used to study metabolites of the brain cortex ex vivo. The superfused brain cortex preparation was judged to be metabolically viable on the basis of the ³¹P NMR spectrum (intracellular pH of 7.23 ± 0.03 and phosphocreatine/ ATP ratio of 1.21 ± 0.09). Using'H NMR a group of previously unidentified signals was detectable at 0.94, 1.22, and 1.40 ppm with a water-suppressed spin-echo sequence. These signals had shorter spin-spin relaxation times (51-54 ms) than TV-acetylaspartate and lactate (84-93 ms) and also smaller saturation factors, an indication of shorter spin-lattice relaxation times than the latter two low-molecular-weight metabolites. The unidentified signals also displayed homo-nuclear coupling to other spins in the methine region of the spectrum. Acid extraction of the brain slices or cortex from animals that were killed yielded a mixture of proteins that exhibited NMR properties matching the ¹H NMR signals in the brain cortex. The molecular mass of these thermoresistant, "mobile' proteins, which contained proline plus hydroxy-proline (9-16% of all amino acids), ranged between 8 and 40 kDa. These "new' assigμMents of¹H NMR-detectable compounds may influence interpretation of NMR data of some metabolites, as their signals are in the vicinity of the -CH3 ¹H NMR peaks of lactate and alanine.     

产诺加霉素链霉菌中失活snogA基因的研究

诺加霉素是重要的蒽环类抗肿瘤抗生素,由黑胡桃链霉菌ATCC27451发酵产生。本研究从诺加霉素产生茵中克隆得到560bp的氨基甲基化酶（snogA）编码基因片段,并将其插入基因整合型质粒pKCll39的多克隆位点,构建得到基因中断质粒pLMX-3-58。通过接合转移和同源重组,构建得到氨基甲基化酶编码基因被中断的重组菌株删-3-59。基因重组突变株基因型验证结果表明,中断质粒以正确方式整合入基因组,将氨基甲基化酶编码基因中断。发酵验证结果表明,重组茵株发酵产物中不含有诺加霉素。本研究表明snogA基因在诺加霉素生物合成途径中是必需的。这为进一步阐明诺加霉素生物合成途径和组合生物合成改造诺加霉素提供了参考。     

New olivosyl derivatives of methymycin/pikromycin from an engineered strain of Streptomyces venezuelae

A mutant strain of Streptomyces venezuelae was engineered by deletion of the entire gene cluster related to biosynthesis of the endogenous deoxysugar (TDP-D-desosamine) and replacement with genes required for biosynthesis of an intermediate sugar (TDP-4-keto-6-deoxy-D-glucose) or an exogenous sugar (TDP-D-olivose), from the oleandomycin and urdamycin deoxysugar pathways. The 'sugar-flexible' glycosyltransferase (DesVII) was able to attach the intermediate sugar and the new sugar to both 12- and 14-membered macrolactones thus producing quinovose or olivose glycosylated 10-deoxymethynolide and narbonolide, respectively. In addition, hydroxylated analogs of the new metabolites were detected. These results demonstrate a successful attempt of engineering the deoxysugar pathway for generation of novel hybrid macrolide antibiotics.     

In vivo manipulation of the bleomycin biosynthetic gene cluster in Streptomyces verticillus ATCC15003 revealing new insights into its biosynthetic pathway

Bleomycin (BLM), an important clinically used antitumor compound, and its analogs are challenging to prepare by chemical synthesis. Genetic engineering of the biosynthetic pathway in the producer strain would provide an efficient and convenient method of generating new derivatives of this complex molecule in vivo. However, the BLM producing Streptomyces verticillus ATCC15003 has been refractory to all means of introducing plasmid DNA into its cells for nearly two decades. Several years after cloning and identification of the bleomycin biosynthetic gene cluster, this study demonstrates, for the first time, genetic accessibility of this pharmaceutically relevant producer strain by intergeneric Escherichia coli-Streptomyces conjugation. Gene replacement and in-frame deletion mutants were created by lambdaRED-mediated PCR targeting mutagenesis, and the secondary metabolite profile of the resultant mutants confirmed the identity of the BLM biosynthetic gene cluster and established its boundaries. Ultimately, the in-frame blmD deletion mutant strain S. verticillus SB5 resulted in the production of a bleomycin intermediate. The structure of this compound, decarbamoyl-BLM, was elucidated, and its DNA cleavage activity was compared with the parent compounds.     

High-Resolution Proton Magnetic Resonance Spectroscopy of Rabbit Brain: Regional Metabolite Levels and Postmortem Changes

The changes in 16 cerebral metabolites produced by cardiac arrest and subsequent room temperature autolysis were studied using high-resolution proton nuclear magnetic resonance spectroscopy. Biopsies of rabbit cerebral cortex, cerebral white matter, and cerebellum were quantitatively analyzed for acetate, alanine, gamma-aminobutyric acid, creatine, glutamate, glycine, inositol, lactate, N-acetylaspartate, phosphocreatine, succinate, taurine, and threonine. Of these, N-acetylaspartate and the total creatine pool are the best candidates for use as concentration reference standards linking in vitro to in vivo 1H nuclear magnetic resonance measurements. Both changed little immediately after death, and they varied in a distinctive way among cortex, white matter, and cerebellum.     

High-yield actinorhodin production in fed-batch culture by a Streptomyces lividans strain overexpressing the pathway-specific activator gene actll-ORF4

Streptomyces lividans 1326 usually does not produce the red/blue colored polyketide actinorhodin in liquid culture even though it carries the entire actinorhodin biosynthesis gene cluster. The bacterium can be forced to produce this secondary metabolite by introducing actII-ORF4, the actinorhodin pathway-specific activator gene from Streptomyces coelicolor, on a multicopy plasmid. The production of actinorhodin by such a strain has been optimized by medium and process manipulations in fed-batch cultures. With high-yield cultivation conditions, 5 g actinorhodin/l are produced during 7 days of cultivation; or approximately 0.1 g actinorhodin/g dry weight (DW)/day in the production phase. The yield in this phase is 0.15 Cmol actinorhodin/Cmol glucose, which is in the range of 25% to 40% of the maximum theoretical yield. This high-level production mineral medium is phosphate limited. In contrast, nitrogen limitation resulted in low-level production of actinorhodin and high production of α-ketoglutaric acid. Ammonium as nitrogen source was superior to nitrate supporting an almost three times higher actinorhodin yield as well as a two times higher specific production rate. The wild-type strain lacking the multicopy plasmid did not produce actinorhodin when cultivated under any of these conditions. This work examines the actinorhodin-producing potential of the strain, as well as the necessity to improve the culture conditions to fully utilize this potential. The overexpression of biosynthetic pathway-specific activator genes seems to be a rational first step in the design of secondary metabolite overproducing strains prior to alteration of primary metabolic pathways for redirection of metabolic fluxes. Journal of Industrial Microbiology & Biotechnology (2002) 28, 103–111 DOI: 10.1038/sj/jim/7000219 Received 04 April 2001/ Accepted in revised form 30 October 2001     

一株生防圆突起链霉菌的分类鉴定

菌株SCY311是从河南省凤凰山土壤样品中分离到的对多种植物病原真菌具有拮抗活性的一株放线菌。为了明确其分类地位, 在形态特征、培养特征、生理生化特征、细胞壁组分测定等传统分类学方法的基础上, 测定和分析了菌株的16S rRNA基因序列。结果表明, 菌株SCY311在高氏一号培养基上生长良好, 基内菌丝呈褐色; 气生菌丝灰色至鼠灰色, 不产生可溶性色素, 无吸水现象; 孢子链卷曲, 末端形成闭合或开放螺旋; 孢子椭圆或圆柱状, 表面形成结节状突起; 生理生化特征和在国际链霉菌计划(ISP)培养基上的培养特     

链霉菌育种进展

链霉菌是放线菌中最重要的一属,它产生绝大部分抗生素和其它众多活性物质。随着分子生物学的发展,链霉菌育种技术也日益多样。本文从基因突变与育种,遗传重组与育种、基因表达调控与代谢调节育种、基因工程与定向育种等四个方面综述链霉菌育种的进展。     

链霉菌发酵麦草产木聚糖酶的试验研究

通过正交设计试验 ,找出利用链霉菌和麦草基质发酵生产木聚糖酶的试验条件。培养基 (g/L) :麦草粉 ,4 5 ;(NH4 ) 2 SO4 ,7.5 ;酵母膏 ,8;K2 HPO4 ·3H2 O ,1;MgSO4 ·7H2 O ,0 .5 ;NaCl,0 .3。接种量为 5 .0× 10 8个孢子 / 5 0mL培养基 ,振荡培养 (12 0r/min) 5d     

A bleaching herbicidal activity of methoxyhygromycin (MHM) produced by an actinomycete strain Streptomyces sp. 8E-12

AIMS: To screen bioherbicidal isolates and evaluate herbicidal activity of methoxyhygromycin (MHM) produced by Streptomyces sp. 8E-12. METHODS AND RESULTS: Streptomyces sp. 8E-12 with herbicidal activity was selected through seed germination bioassay. An active metabolite, MHM was isolated from culture broth by carbon absorption, butanol extraction, silica gel, Sephadex LH-20 and G-10 chromatography, and preparative HPLC. The metabolite was identified by electrospray ionization mass spectra (ESI-MS) and 1H- and 13C-NMR spectral data analyses. In vivo herbicidal activity was examined against weeds and crops grown on pots. CONCLUSIONS: Streptomyces sp. 8E-12 produced a selective herbicidal metabolite which was identified as MHM. The metabolite showed stronger in vivo activity against monocotyledonous plants than dicotyledonous plants, and caused a bleaching (albino symptom) on some weeds including Digitaria sanguinalis and Echinochloa crus-galli. SIGNIFICANCE AND IMPACT OF THE STUDY: These results showed that Streptomyces sp. 8E-12 produced a bioherbicidal metabolite, MHM and can be developed as a biocontrol agent (BCA) for weed control.     

角蛋白酶基因gm2886在密旋链霉菌ACT12中的表达及鉴定

通过生物信息学分析,在本实验室分离得到的1株羽毛高效降解菌微白黄链霉菌Fea-10基因组中发现基因gm2886(GenBank Accession Number:KY368946)可能编码一新的角蛋白酶,通过在该基因5'端和3'端分别连接红霉素抗性基因启动子(PermE)和组氨酸标签编码序列并构建在大肠杆菌-链霉菌穿梭质粒pSET152上,接合转入密旋链霉菌Streptomyces pactum ACT12,从而实现了异源表达,蛋白纯化后对其酶学性质进行了研究。实验结果表明,带有组氨酸标签编码序列的gm2886在密旋链霉菌ACT12中可以表达分泌得到1个大小约为36 kDa的蛋白。多种底物检测表明异源表达得到的重组蛋白GM2886-His6具有蛋白酶活性,可以降解水不溶性的天青角蛋白和羽毛粉;其最适温度和pH分别为50℃和pH 10.0。PMSF可抑制GM2886-His6的酶活,而EDTA不能,说明该酶为丝氨酸蛋白酶。本研究为从分子水平上解析羽毛高效降解菌Fea-10的活性机理,从而进一步开发其应用潜力提供了基础,同时可为该类蛋白酶的研究提供借鉴。