Characteristics of dissociation in cultures of Aspergillus brasilense Sp7

Peculiarities of dissociation in the cultures of nitrogen-fixating soil microorganism Azospirillum brasilense have been studied. The possible transfer among colony-morphology variants is established. The relations between variants are described by the following scheme: R in equilibrium with SR----S Electrophoretic analysis of plasmid contents in different variants of Azospirillum brasilense supposes the possible participation of plasmid DNA in the dissociation process in this microorganism.     

Design of Streptomyces nogalater LV65 strains with higher synthesis of nogalamicin using regulatory genes

Influence of cloned regulatory genes on biosynthesis of nogalamicin by Streptomyces nogalater LV65 strains has been studied. Gene snorA from the S. nogalater genome was cloned in multicopy replicative plasmid pSOKA and integrative plasmid pR3A. Introduction of these plasmids into the cells of wild type strain of S. nogalater LV65 resulted in higher synthesis of nogalamicin. A similar effect was observed at heterologous expression of gene ppGpp of synthetase relA cloned in S. coelicolor A3(2). Heterologous expression of genes absA2from S. ghanaensis ATCC14672 and lndyR from genome S. globisporus 1912 decreased synthesis of antibiotic. The study results indicate the presence of homologs of these genes in chromosome of S. nogalater, their possible participation in regulation of nogalamicin biosynthesis, and provide us with a possibility for genetic design of the strains with higher synthesis of this antibiotic.     

Construction of <Emphasis Type="Italic">Streptomyces nogalater</Emphasis> Lv65 strains with enhanced nogalamicin biosynthesis using regulatory genes

Influence of cloned regulatory genes on nogalamycin biosynthesis by Streptomyces nogalater LV65 strain has been studied. Gene snorA from the S. nogalater genome was cloned in multicopy replicative plasmid pSOKA and integrative plasmid pR3A. Introduction of these plasmids into S. nogalater wild type cells resulted in enhanced nogalamicin biosynthesis. A similar effect was observed at heterologous expression of gene (p)ppGpp-synthetase gene relA cloned from Streptomyces coelicolor A3(2). Heterologous expression of genes absA2 from Streptomyces ghanaensis ATCC14672 and lndYR from genome Streptomyces globisporus 1912 decreased synthesis of antibiotic. The study results indicate the presence of homologs of these genes in chromosome of S. nogalater, their possible participation in regulation of nogalamicin biosynthesis, and provide us with a possibility for genetic design of the strains with higher synthesis of this antibiotic.     

农用抗生素产生菌S-10菌的分类鉴定

在筛选防治烟草赤星病农用抗生素过程中,从山东各地分离得到的2604株放线商中筛选得到了一株对烟草赤星病有良好防治效果的链霉菌,编号为S－10。经鉴定,在形态、培养特征和生理生化特性方面与弗氏链霉菌很相似,但有一定的差别,所以定名为弗氏链霉菌山东变种（Streptomucesfradeuarshandongnesisn.var）     

Effect of cloned DNA fragment from Streptomyces chrysomallus No.2 on metabolism in Streptomyces strains

The effects on cloned DNA fragment carrying an actinomycin resistance determinant on physiological processes in streptomyces strains with various potencies in producing this antibiotic, their inactive mutants, and model strain of Streptomyces lividans 66 were studied. This fragment was shown to modulate bacterial resistance to actinomycin and biosynthesis of antibiotics.     

Biosynthesis of the Escherichia coli siderophore enterobactin: sequence of the entF gene, expression and purification of EntF, and analysis of covalent phosphopantetheine.

The sequence of the entF gene which codes for the serine activating enzyme in enterobactin biosynthesis is reported. The gene encodes a protein with a calculated molecular weight of 142,006 and shares homologies with the small subunits of gramicidin S synthetase and tyrocidine synthetase. We have subcloned and overexpressed entF in a multicopy plasmid and attempted to demonstrate L-serine-dependent ATP-[32P]PPi exchange activity and its participation in enterobactin biosynthesis, but the overexpressed enzyme appears to be essentially inactive in crude extract. A partial purification of active EntF from wild-type Escherichia coli, however, has confirmed the expected activities of EntF. In a search for possible causes for the low level of activity of the overexpressed enzyme, we have discovered that EntF contains a covalently bound phosphopantetheine cofactor.     

Plasmid macroevolution in a nosocomial environment: demonstration of a persistent molecular polymorphism and construction of a cladistic phylogeny on the basis of restriction data

Summary Descendants of a gentamicin resistance plasmid first isolated from the Minneapolis Veterans Administration Hospital in 1975 persisted at the hospital until 1983. During this extended period of time several macroevolutionary variants arose and two classes of variants were shown to persist with the initial plasmid. It was possible to construct a cladistic phylogeny on the basis of restriction endonuclease cleavage patterns of the initial and variant plasmids. Additionally, a plasmid polymorphism comprised of members of the plasmid classes was shown to persist at the hospital throughout the endemic period.     

The methods of protoplast formation and transformation of Streptomyces strains

Factors influencing formation, regeneration and transformation of protoplasts in streptomyces are described. Conditions for formation and regeneration of protoplasts in 4 industrial strains producing the macrolide antibiotic tylosin were studied. It was demonstrated possible to apply the method for transformation of the S. lividans type culture to 3 industrial strains of S. griseus producing grisin, an antibiotic used as a feed additive. Potential increasing of the efficiency of protoplast transformation and transfection in various actinomycetous strains including industrial ones is discussed. The stimulating effect of lyposomes on transformation of protoplasts in S. lividans 66 with DNA of plasmids pVG101 and pIJ350 as well as transfection with DNA of phages SH10 and KS404 was shown. The tylosin resistance genes in S. fradiae strain B45 were cloned which enabled isolating the cluster of the genes participating in tylosin biosynthesis.     

一株产天然蓝色素海洋链霉菌的筛选鉴定

从胶州湾附近海域水样中分离筛选到一株可产生天然蓝色素的链霉菌。采用通用引物16F27/16R1492扩增该菌株的16SrDNA,对测序结果进行序列分析,采用Neighbor-Joining(N-J)法构建系统发育进化树,同时结合链霉菌的传统形态学和生理生化特性对菌株进行鉴定。16SrDNA序列分析表明菌株与Streptomyces violaceolatus KCTC 9772相近,相似率为99%。在Neighbour-Joining法构建的系统发育进化树中,该菌株与Streptomyces violaceolatus聚类在同一分枝上。因此将该菌株鉴定为紫边链霉菌Streptomyces violaceolatus。     

Preparation and analysis of mutants of rhizospheric pseudomonads, resistant to toxic analogs of tryptophan and phenylalanine]

The absence of plasmids in strains of fluorescent pseudomonads characterized by high level of synthesis of phytohormone indole-3-acetic acid (IAA) as well as invariability of this feature in plasmid and non-plasmid variants of strain BSP8 suggests chromosomal control of IAA synthesis by the rhizosphere bacteria tested. Using toxic analogues of aromatic amino acids -5-fluorine-tryptophan and 5-methyl-tryptophan variants were obtained which synthesized and secreted only anthranilic acid. Mutants with resistance to p-fluorine-phenylalanine and capable of secreting tryptophan and/or phenylalanine were found. Testing of the secreting variants failed to reveal any differences between the levels of IAA biosynthesis in comparison with the wild-type strains.     

Effect of heat shock on biosynthesis of antibiotics by three strains of Streptomyces]

Effects of heat shock on the biosynthesis of antibiotics, actinomycin C (in cultures of Streptomyces sp. 26-115 and S. chrysomallus 23209) and antibiotics of the nonactin group (in the culture of S. werraensis 1365) were studied. After heat shock, the formation of antibiotics of the nonactin group and actinomycin C were shown to increase by 30% and 27%, respectively, in comparison to control values. Thus, heat shock stimulates the biosynthesis of antibiotics in all three strains of streptomyces studied.     

Polyketide-nonribosomal peptide epothilone antitumor agents: the EpoA,B, C subunits

The epothilones are a family of macrolactone natural products from the myxobacterial species Sorangium cellulosum. Similar to taxol, they are of current clinical interest as anticancer agents. Sequence analysis of the epothilone gene cluster allowed the identification of polyketide synthase and nonribosomal peptide synthetase modules involved in catalyzing epothilone biosynthesis. Given this information, it has been possible to test the predicted functions of several modules to date. EpoA ACP, EpoB, and EpoC have been overproduced in Escherichia coli, allowing in vitro reconstitution of the EpoA/B/C interface and production of the expected epothilone precursor. Further experiments probed the tolerance of EpoB and EpoC for unnatural substrates. These studies of the first three modules of the epothilone biosynthetic cluster suggest that combinatorial biosynthesis may lead to the production of a variety of epothilone analogs that incorporate diversity into the heterocycle starter unit. Additional efforts with the remaining modules, coupled with increased understanding of the macrocyclizing thioesterase domain, may lead to the production of epothilone variants with improved clinical properties.     

Biosynthesis of chloramphenicol

The current knowledge concerning the biosynthesis of chloramphenicol is discussed. Cultures of Streptomyces sp. 3022a fed ¹⁴C-shikimie acid incorporated the label to the same extent into phenylalanine, tyrosine, and chloramphenicol. Of possible precursors of the phenylpropanoid nucleus of this antibiotic only p-aminophenylalanine and DL-threo-p-amino phenylserine specifically labeled chloramphenicol. On the basis of these results a pathway for the biosynthesis of chloramphenicol is presented. The lack of specific incorporation of ¹⁵N-nitrogen from a competitive feeding experiment in which both ^l5N-nitrate and ¹⁴N-DL-serine were fed to growing cultures suggests that both the amido- and the nitro-nitrogen atom present in this antibiotic are derived from a common pool. Studies on the enzyme, DAHP synthetase, show that in streptomyces sp. 3022a it is not subject to feed back inhibition by either phenylalanine, tyrosine, or chloramphenicol.     

In vivo selection of functional variations in essential sites of ribosomal RNA

The technique of "in vivo selection of functional ribosomes" is a genetic approach to dissecting the link between the structure and function of critical sites of rRNA. This method proceeds through selection of functional variants among cells that express ribosomes from a pool of rRNA-containing randomized sites. The selection of bacterial clones with functional ribosomes is based on the use of a plasmid carrying a rRNA operon in which a site of interest has been randomized and a point mutation conferring an antibiotic resistance has been introduced. Cells expressing functional ribosomes are then selected on medium containing the antibiotic. With this approach one can isolate at once all the possible variations at a given rRNA site that are able to sustain normal ribosome function. The identification of covariations in between several nucleotides that maintain wild-type ribosome activity can thus help demonstrate the function of specific interactions in rRNA.     

Mutation detection in plasmid-based biopharmaceuticals

As the number of applications involving therapeutic plasmid DNA (pDNA) increases worldwide, there is a growing concern over maintaining rigorous quality control through a panel of high-quality assays. For this reason, efficient, cost-effective and sensitive technologies enabling the identification of genetic variants and unwanted side products are needed to successfully establish the identity and stability of a plasmid-based biopharmaceutical. This review highlights several bioinformatic tools for ab initio detection of potentially unstable DNA regions, as well as techniques used for mutation detection in nucleic acids, with particular emphasis on pDNA.     

Ethyl methanesulphonate mutagenesis with L5178Y mouse lymphoma cells: a comparison of ouabain, thioguanine and excess thymidine resistance.

Complete inhibition of growth of sensitive L5178Y mouse lymphoma cells in culture was obtained with 10(-3)M ouabain, 1.65 X 10(-3)M thymidine, 1.8 X 10(-4)M thioguanine and 10(-6)M cytosine arabinoside. The toxicity of methotrexate was dependent upon cell density and this compound was excluded from further study. The expression time before addition of the selective agent was important for detecting EMS induced resistant variants. Ouabain-resistant variants appeared immediately after treatment and were present over a broad time span. No excess thymidine- or thioguanine-resistant variants were seen initially; a peak in variant numbers was seen for excess thymidine resistance at 48-96 h and for thioguanine resistance at 144-192 h. Using induced mutation frequencies at optimum expression times, equal EMS treatments yielded substantially more variants resistant to thioguanine than to ouabain. It is suggested that this difference may have origin in possible constraints in the classes of mutants which are permissible in a vital function, maintenance of the Na+/K+ balance, when compared with a non-vital function, salvage purine biosynthesis. Some data are presented on the stability in culture of resistant variants. A limited number of observations were made following treatment in the peritoneal cavity of the mouse which were in broad agreement with the above results.     

Multiplex amplification test system for the identification and differentiation of Bacillus anthracis

The multiplex amplification test system for the identification of Bacillus anthracis with primers to plasmid cya (pX01), capC (pX02) genes and chromosomal sap gene were developed. The primers to sap gene were selected by the authors and, after being tested on 72 microbial strains of the genus Bacillus, proposed as more specific in comparison with the known primers to chromosomal locus Ba 813. The proposed test system permitted the simultaneous identification of B. anthracis of all plasmid variants, the evaluation of their potential virulence and the differentiation of B. anthracis nonplasmid strains from bacilli of the group Bacillus cereus.     

The human steroid hydroxylases CYP1B1 and CYP11B2

Major advances have been made during the last decade in our understanding of adrenal steroid hormone biosynthesis. Two key players in these pathways are the human mitochondrial cytochrome P450 enzymes CYP11B1 and CYP11B2, which catalyze the final steps in the biosynthesis of cortisol and aldosterone. Using data from mutations found in patients suffering from steroid hormone-related diseases, from mutagenesis studies and from the construction of three-dimensional models of these enzymes, structural information could be deduced that provide a clue to the stereo- and regiospecific steroid hydroxylation reactions carried out by these enzymes. In this review, we summarize the current knowledge on the physiological function and the biochemistry of these enzymes. Furthermore, the pharmacological and toxicological importance of these steroid hydroxylases, the means for the identification of their potential inhibitors and possible biotechnological applications are discussed.