SVIBOR - Project code: 1-08-021

MINISTRY OF SCIENCE AND TECHNOLOGY

Strossmayerov trg 4, HR - 10000 ZAGREB
tel.: +385 1 459 44 44, fax: +385 1 459 44 69
E-mail: ured@znanost.hr

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Project code: 1-08-021


STRUCTURE, COMPLEXITY AND STABILITY OF Streptomyces GENOME


Main researcher: HRANUELI, DASLAV (16520)


Assistants
Type of research: basic
Duration from: 01/01/91. to 12/15/95.
Papers on project (total): 35
Papers on project quoted in Current Contents: 7
Institution name: Prehrambeno-biotehnološki fakultet, Zagreb (58)
Department/Institute: Faculty of Food Technology and Biotechnology Department of Biochemical Engineering Laboratory for Biology and Microbial Genetics
Address: Pierottijeva 6
City: 10000 - Zagreb, Croatia
Communication
Phone: +385 (0)1 41-70-44
Phone: +385 (0)1 18-19-59
Fax: +385 (0)1 41-14-36
Fax: +385 (0)1 18-16-06
Summary: The Streptomyces rimosus strain R6 shows a pattern of genetic instability, typical for strain degeneration with the loss of one or more of five phenotypic properties (sporulation, pigmentation, colony morphology, oxytetracycline production, oxytetracycline resistance). In this proposal, the frequency of segregation of different variants will be investigated in detail and the collection of representative variants will be assembled. The variants will be studied for evidence of DNA amplification or large deletions. A cosmid gene bank of S. rimosus will be constructed. This will be used to clone the parental type sequences in regions subjected to DNA rearrangements (in particular, amplifications and deletions) to characterize the nature of the rearrangements. Methods will be developed to transfer marker genes into the chromosome of S. rimosus. These will be based on the methods used successfully with S. lividans. Loss of the marker genes will be used to monitor deletion frequencies; in addition, it will be investigated whether selection for marker genes in deletogenic regions can prevent accumulation of deletions and lead to strain stabilization. The S. rimosus R6 contains a giant linear plasmid and two prophages, RP2 and RP3. The molecular biology of S. rimosus R6 will be investigated further to prepare a physical map of the chromosome (and an endogenous linear plasmid), to characterize two prophages of this strain and to combine this new information to develop stable cloning vectors with utility in S. rimosus. IS117, the 2.6 kb mini-circle of S. coelicolor A3(2), is a transposable element previously shown to be integrated into two distant sites in the chromosome. Upon the introduction into S. lividans, it is expected that IS117 will integrate into one preferred chromosomal site, but that many secondary sites also exist. Nucleotide sequences of eventual secondary integration sites will be revealed and compared with the preferred site. Possible chromosomal rearrangements mediated by the integration of IS117 into eventual secondary sites will be studied.

Keywords: Streptomyces, Genetic instability, Bacterial chromosome, Linear plasmid, Prophages, Transposable element

Research goals: Streptomycetes make 75 % of all commercial antibiotics as well asmany other useful therapeutic agents (e.g. anti-tumour agents,anti-parasitic agents, enzyme inhibitors and immunosuppressants).For more than 40 years industry has been selecting strains forbetter productivity, but improved strains are often geneticallyunstable. The broad objective of the proposed research is: better understanding of the mechanisms that underlie genetic instability in hyper-producing strains and the use of this information to generate stable derivatives with enhancedproduction potential. Specific objectives are as follows: (a) To analyze genetic rearrangements (e.g. deletions, duplications and reiterations) that occur in unstable derivativesof S. rimosus R6. (b) To characterize accessory genetic elements (giant linear plasmid and prophages), evaluate their roles in antibiotic production and in genetic instability, and to construct stable vectors. (c) To construct the physical map of S. rimosus R6 chromosome and to assign genetic loci to the physical map. (d) To analyze preferred and secondary chromosomal sites for the integration of the S. coelicolor A3(2) transposable element IS117 and to determine degrees of similarity between them. The genomes of some prokaryotes are very stable, whereas others, like those of streptomycetes, show remarkable plasticity. The proposed research, by revealing details of the mechanisms that underlie genetic instability in streptomycetes will contribute toour understanding of this enigma. Secondly, it will provide the information necessary for the generation of stable production strains for the fermentation industry. The fundamental knowledge/tools obtained will be applied immediately to the other aspects of this proposal, to help in achieving the overall objectives.

COOPERATION - PROJECTS


  1. Name of project: 1-08-058 Ekspresija gena u micelijskim mikroorganizmima
    Name of institution: PLIVA Istraživački institut, Biosinteza i biotehnologija
    City: 10000 - Zagreb, Croatia

  2. Name of project: CI1*/0527-C(MB) Molecular mechanisms of genetic instability and strain degeneration in streptomycetes
    Name of institution: Universit„t Kaiserslautern, LB Genetik
    City: D-67653 - Kaiserslautern, FRG

  3. Name of project: The study of actinophages RP2 and RP3 as the accessory genetic elements of Streptomyces rimosus genome
    Name of institution: Universit„t Mnchen, Institut fr Genetik und Mikrobiologie
    City: D-80638 - Mnchen, FRG

COOPERATION - INSTITUTIONS


  1. Name of institution: PLIVA Istraživački institut, Biosinteza i biotehnologija
    Type of institution: Non-profit
    Type of cooperation: Joint project
    City: 10000 - Zagreb, Croatia
Other information about the project.
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