几种诱变因子对龟裂链霉菌原生质体的影响

作者应用通电、ＵＶ、ＮＴＧ和亚硫酸氢钠等诱变因子对土霉素产生菌——龟裂链霉菌１３８—ｌ原生质体进行处理。结果表明,各种诱变因子在对原生质体致死率５０％左右时具有较好的诱变效应。经亚硫酸氢钠＋ＬｉＣｌ处理获得的Ｃｌ１８菌株通过１６５００１发酵罐试验,最高发酵水平达３３３３０ｕ／ｍｌ,平均发酵水平为３０５４２．５ｕ／ｍｌ,比出发菌株１３８—１提高２０．６％。     

Genetic Recombination in Crosses Between Streptomyces aureofaciens and Streptomyces rimosus

Polsinelli, M. (University of Pavia, Pavia, Italy), and Maria Beretta. Genetic recombination in crosses between Streptomyces aureofaciens and Streptomyces rimosus. J. Bacteriol. 91:63-68. 1966.-Biochemical mutants were obtained from Streptomyces rimosus and S. aureofaciens by ultraviolet irradiation. Crosses were performed between auxotrophic strains of S. rimosus and S. aureofaciens with positive results. Data are reported which indicate that the interaction observed in some crosses is due to gene recombination.     

高产番茄红素链霉菌选育及摇瓶发酵研究

番茄红素的抗氧化能力目前在类胡萝卜素中最强,是近年来国际上功能食品成分研究的热点。在国内首次利用龟裂链霉菌（Streptomyces rimosus）发酵生产番茄红素,建立了分光光度计法和HPLC法等番茄红素测定方法;以一株龟裂链霉菌Fc作为出发菌株,进行紫外诱变,筛选到一株突变高产菌株Fc’,其番茄红素产量较出发菌株提高2.5倍;通过摇瓶发酵实验优化培养条件,使菌株Fc’的番茄红素产量达到230 mg/L,并且在不添加任何阻断剂的情况下,利用链霉菌发酵可获得纯度较高的番茄红素。该结果为今后利用链霉菌工业化生产番茄红素奠定了良好基础。     

工程改造龟裂链霉菌提高土霉素产量

土霉素是由龟裂链霉菌合成的一类广谱性抗生素,前期研究工作证明其生物合成受其自身途径特异性调控蛋白OtcR的直接调节,OtcR能够激活和促进土霉素合成基因簇的转录表达。在龟裂链霉菌M4018宿主内利用强启动子单独过表达OtcR蛋白,使土霉素的产量提高到原来产量的4倍;为了进一步提高土霉素产量,在M4108宿主内表达乙酰辅酶A羧化酶基因,提高其胞内土霉素合成的前体物丙二酸单酰辅酶A的含量。对出发菌株M4018进行工程改造,同时过表达途径特异性调控蛋白OtcR和乙酰辅酶A羧化酶,发酵检测改造后的重组工程菌株土霉素的产量由1.37g/L提高到9.09g/L,该研究策略对工程改造龟裂链霉菌提高土霉素的产量具有重要的指导意义。     

链霉菌11371原生质体的制备与再生

为选育链霉菌11371的高产Tetramycin菌株,摸索该菌株的原生质体的制备与再生。结果表明,链霉菌11371原生质体制备和再生的最佳条件为菌丝生长培养液中甘氨酸浓度为0．7％,培养温度为28℃,培养时间为42h,溶菌酶浓度为3mg／mL,酶解温度为37℃,酶解时间为90min。最佳再生培养基为R2YE培养基。     

链霉菌702产孢子固体培养基和培养条件的筛选

通过单因素实验、均匀设计和正交实验对链霉菌702产抱子培养基和培养条件进行筛选,筛选到的最佳培养基组成为：马铃薯2009／L,葡萄糖25g／L．最佳培养条件为培养温度37℃、培养时间5天和培养基初始pH值8．实验结果表明,采用优化后的培养基和培养条件,使链霉菌702产抱子数达到4．07亿,比原来培养基（高氏一号培养基）产抱予量提高了8．7倍,培养时间却从原来的7天缩短到现在的5天．     

Isolation and characterization of a linear plasmid from Streptomyces rimosus

Abstract A linear plasmid was isolated from a strain of Streptomyces rimosus . This plasmid was separated by agarose gel electrophoresis and its size, about 43 kb, determined both by this method and by electron microscopy. The cleavage pattern of the linear plasmid with 5 restriction endonucleases is given. A protein, which is removed by proteinase K, is probably associated to this plasmid. By ethidium bromides or acridine orange treatment we obtained mutants which had lost their aerial mycelium and their linear plasmid.     

Cloning and heterologous expression in Streptomyces lividans of Streptomyces rimosus genes involved in oxytetracycline biosynthesis.

The anhydrotetracycline (ATC) oxygenase enzyme which carries out the conversion of ATC to dehydrotetracycline was purified and the N-terminal amino acid sequence was determined. The sequence displays a significant similarity to that of the p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens. This is consistent with the activity of the oxygenase, i.e., addition of a hydroxyl moiety to an aromatic ring structure. Oligonucleotide probes were designed and used to clone the corresponding fragment of chromosomal DNA from Streptomyces rimosus. This DNA fragment was used to screen a cosmid library, allowing the isolation of flanking DNA sequences. Surprisingly, the gene was located within the previously cloned cluster of genes involved in the synthesis of the biosynthetic intermediate ATC and not as had been expected (P. M. Rhodes, N. Winskill, E. J. Friend, and M. Warren, J. Gen. Microbiol. 124:329-338, 1981) at a separate locus on the other side of the chromosome. Subcloning of an appropriate DNA fragment from one of the cosmid clones onto pIJ916 produced Streptomyces lividans transformants which synthesized oxytetracycline.     

In vivo and enzymatic conversion of toyocamycin to sangivamycin by Streptomyces rimosus

The pyrrolopyrimidine nucleosides, toyocamycin, sangivamycin, and tubercidin are isolated from the culture filtrates of 14 species of the Streptomyces. Although earlier experiments showed that the biosynthesis of the pyrrolopyrimidine nucleosides require GTP as the common precursor, there was no experimental evidence to demonstrate the interconversion of these naturally occurring nucleoside analogs. The data presented here describe two types of experiments to prove that toyocamycin is the precursor for sangivamycin. First, in vivo experiments show that radioactive toyocamycin is converted to sangivamycin. Second, the enzyme, toyocamycin nitrile hydrolase, that catalyzes the conversion of toyocamycin to sangivamycin has been isolated and partially purified from the soluble fraction of S. rimosus. The nitrile hydrolase is not present in cell-free extracts of the Streptomyces that synthesize tubercidin or toyocamycin. Activity can be assayed by measuring the formation of radioactive sangivamycin from toyocamycin. The enzyme has been purified 24-fold with an over-all yield of 5%. The pH optimum is 6.5 and the K_m is 0.5 mm. Most nitriles tested are competitive inhibitors but they are not substrates. The activity of the hydrolase is limited to the conversion of the nitrile group to the carboxamide group. Hydrolase activity is observed in cell-frre estracts of S. rimosus before toyocamycin production begins. The in vivo and in vitro studies demonstrate that toyocamycin is not a precursor for tubercidin. The experimental evidence strongly suggests that there must be a branch point in the biosynthesis of the pyrrolopyrimidine nucleoside antibiotics.     

Genetic interactions in Streptomyces rimosus mediated by conjugation and by protoplast fusion

The development of a protoplast fusion technique for oxytetracycline-producing Streptomyces rimosus strains, and its evaluation for the application for a breeding programme, has been described. Treatment of S. rimosus protoplasts with 40% (w/v) PEG 1550 for 30 min gave optimal numbers of recombinants ranging from 1 to 10% of the total progeny. Therefore, by comparison with conjugation, protoplast fusion increased the frequency of recombination by two to three orders of magnitude. The proportion of multiple crossover classes amongst recombinants was higher, by a factor of ten, after protoplast fusion (13.3%) than after conjugation (1.5%). Participation of less frequent complementary genotype doubled from 9.0% in conjugation to 17.9% in protoplast fusion. Overall, this suggested that the opportunities for crossing over in a fusion of S. rimosus protoplasts were spatially and/or temporally extended leading to a loosening of linkage with a near-random assortment of genotypes in a cross. However, by minimizing the multiple crossover classes and calculating allele frequency gradients, it was shown that the protoplast fusion technique allows arrangement of genetic markers on the S. rimosus chromosome. These are ideal characteristics for the recombination of divergent lines in a strain improvement programme.     

Antagonism and Action Mechanism of Antifungal Metabolites from Streptomyces rimosus MY02

The genus of Streptomyces , a saprophytic Gram-positive bacterium, has properties, which make them useful as pharmaceutical and biocontrol agents. A streptomyces strain MY02 from soil samples showed significant antagonism against 14 plant pathogenic fungi including Fusarium oxysporum f. sp. cucumarinum . Antifungal metabolite(s) SN06 from the culture of the strain MY02 were extracted with n -butanol and purified by silica gel column chromatography. The minimum concentration of SN06 inhibiting any visible fungal growth of F. oxysporum f. sp. cucumarinum is 12.5 μg/ml by twofold serial dilutions method. The mycelia of F. oxysporum f. sp. cucumarinum treated with SN06 were observed under the normal optics microscope. The results showed that some cells of hyphae began to dilate and formed some strings of beads. The cytoplasm oozed out of the cells with the culture time and so most of the cells became empty. The hyphae broke into many segments and then collapsed after 48 h. After inoculated in potato dextrose medium for 48 h, the filtrate of mycelia treated with 1% NaCl containing 12.5 μg/ml SN06 was scanned using ultraviolet spectrophotometer and absorption peak at 260 nm showed that the mycelia cell membrane of F. oxysporum f. sp. cucumarinum was broken and that nucleic acid oozed out of the cell.     

Streptomyces rimosus extracellular proteases 3. Isolation and characterization of leucine aminopeptidase

Summary A leucine aminopeptidase was purified to homogeneity fromStreptomyces rimosus culture filtrates, which are waste broth of oxytetracycline bioproduction process. Purification procedure includes ultrafiltration and chromatography on CM-Sephadex, AH-Sepharose and FPLC Mono S column. The enzyme is a monomer with molecular weight of 27,500 Daltons and pI of 7.3, stable in broad pH range and up to 70°C. It is a metallo enzyme dependent on Ca²⁺ ions for its full activity. By its specificity it is a true aminopeptidase active on amino acid amide, arylamide, peptide and ester bonds. The hydrolysing activity shows preference for leucine at the N-terminal position of substrates, also acts on aromatic acids and methionine, but does not release glycine, proline, acidic amino acids orD-amino acid residues.     

Development of an intergeneric conjugal transfer system for rimocidin‐producing Streptomyces rimosus

Aims: To develop an intergeneric conjugation system for rimocidin‐producing Streptomyces rimosus. Methods and Results: High efficiencies of conjugation [10^?2–10^?3 transconjugants/recipient colony forming units (CFU)] were obtained when spores of S. rimosus were heat treated at 40°C for 10 min prior to mixing with E. coli ET12567(pUZ8002/pIJ8600) as donor. Mycelium from liquid grown cultures of S. rimosus could also be used as recipient instead of spores, with 24‐h cultures giving optimal results. TSA (Oxoid) medium containing 10 m mol l^?1 MgCl₂ was the preferred medium for conjugation. Southern hybridization was used to confirm that transconjugants of S. rimosus contained a single copy of pIJ8600 integrated at a unique chromosomal attachment site (attB). The transconjugants exhibited a high stability of plasmid integration and showed strong expression of green fluorescent protein when using pIJ8655 as the conjugative vector. Conclusion: Intergeneric conjugation between E. coli and S. rimosus was achieved at high efficiency using both spores and mycelium. Significance and Impact of the Study: The conjugation system developed provides a convenient gene expression system for S. rimosus R7 and will enable the genetic manipulation of the rimocidin gene cluster.     

Characterization and persistence of actinophage RP2 isolated from Streptomyces rimosus ATCC 10970

While searching for true lysogens among oxytetracycline-producing Streptomyces rimosus strains, free phage particles were detected and isolated from a liquid culture of S. rimosus ATCC 10970 (R7). The actinophage, designated RP2, appears to be a typical temperate DNA phage producing turbid plaques on the sensitive strain S. rimosus R6. Electron microscopic examination of RP2 lysates showed that it belongs to group B of Bradley's morphological classification. The rate of RP2 adsoprption at 28 degrees C appeared to be low. The length of the latent period was about 6 h and the average burst size about 120 phage particles. The lysogenic nature of the host-virus system described was established on the basis of the following characteristics: spontaneous lysis frequency of 2 X 10(-6) per cell, resistance to curing with phage-specific antiserum, spontaneous curing frequency of less than 0.05% and immunity to superinfection with the homologous phage. Clear-plaque mutants of RP2, which failed to lysogenize sensitive cultures, arose at a frequency of 10(-5).