S.AMBOFACIENS PRODUCES SPIRAMYCIN (A MACROLIDE ANTIBIOTIC). IT IS SPECIALLY ACTIVE AGAINST GRAM-POSITIVE BACTERIA, AND IS CONSIDERED AS VERY IMPORTANT TO ANTIBIOTIC THERAPY (HUMAN AND ANIMAL).
THE ISOLATION AND CHARACTERIZATION OF RESISTANCE DETERMINANTS TO SPIRAMYCIN WILL ALLOW A BETTER KNOWLEDGE OF HOW THE STRAIN PROTECTS ITSELF AGAINST THE MACROLIDES IT PRODUCES. IN ADDITION THIS COULD ENABLE UNDERSTANDING OF HOW THE RESISTANCE TO AND THE ANTIBIOTIC PRODUCTION ARE COORDINATELY REGULATED. PLASMID PSAM2 FROM S. AMBOFACIENS CAN INTERACT WITH THE CHROMOSOME, UNDERGO ALTERATIONS AND WILL BE USED FOR THE CONSTRUCTION OF A VECTOR WHICH COULD BEHAVE EITHER AS AN INTEGRATIVE OR A REPLICATIVE ONE. S. AMBOFACIENS LIKE OTHERS STREPTOMYCES AND ESPECIALLY MACROLIDE PRODUCERS DISPLAYS GENETIC INSTABILITY AND POSSESSES AMPLIFIABLE DNA SEQUENCES. THEREFORE IT IS POSSIBLE TO STUDY IN THIS INDUSTRIAL STRAIN MANY PROBLEMS COMMON TO STREPTOMYCES AND THIS COULD HELP IN THE CONSTRUCTION OF MORE PERFORMANT STRAINS.
Streptomyces produces macrolide antibiotics. Macrolides are widely used in human and animal therapeutics and inhibit protein synthesis by a mechanism involving the binding of the antibiotic to the large subunit of the ribosome.
The integrative element pSAM2 from S ambofaciens was characterized using genetic and molecular biology.The genes involved in the site specific integration and excision in the transfer, kill-kor and in replecation were identified. pSAM2 integrate site specifically in the genome of many Streptomyces and some actimonomycetes through an attachment site present both on the element (attP) and on the chromosome (attB). attB overlaps the 3' ned of a tRNA gene.
Integrative vectors derived from pSAM2 have been constructed. They do not need any selective pressure to be maintained and they have a wide host range amongst actinomycetes. The gene pra (pSAM2 replication activation) involved in the positive regulation of replication during ttransfer have been characterized.
Two genes conferring resistance by ribosomal modification (23S rRna methylation) have been characterized. A gene whose product inactivates some macrolides by glycosylation and a gene supposed to be involved in antibiotic export are also present in S ambofaciens. A fifth resistance gene has not been characterized yet.
Two genes have been identified in S lividans: arepressor of the LacI type having pleiotropic effects on the expression of amylases, chitinases and xylanases, and a gene showing sequences homologies with inositol mono-phosphatases.
THE LABORATORY OF DR GUERINEAU IS WORKING WITH STREPTOMYCES AMBOFACIENS, THE SPIRAMYCIN PRODUCER, AND IS MAINLY INTERESTED IN THE FOLLOWING POINTS :
- MACROLIDE RESISTANCE IN THE PRODUCING ORGANISM.
- BIOLOGY OF THE PLASMID, PSAM2 AND CONSTRUCTION OF CLONING VECTORS USING THIS PLASMID.
- GENETIC INSTABILITY AND AMPLIFICATION.
DR GUERINEAU WILL ESTABLISH A GENETIC MAP OF S. AMBOFACIENS AND WILL STUDY THE INSTABILITY OF SOME GENETIC MARKERS. THE AMPLIFIED UNITS FROM S. AMBOFACIENS WILL BE COMPARED WITH THOSE OF OTHER MACROLIDE PRODUCERS AND WILL BE USED TO STUDY THEIR TRANSCRIPTION AND POSSIBLE TRANSPOSITION.
THE INTERACTION WITH THE CHROMOSOME OF SOME PLASMIDS PRESENT IN S. AMBOFACIENS, PROVOKES REARRANGEMENTS. INTEGRATION AND EXCISION WILL BE INVESTIGATED IN ORDER TO ELUCIDATE THE INVOLVED MECHANISMS.
S. AMBOFACIENS EXHIBIT AN INDUCIBLE RESISTANCE AGAINST MACROLITES, LINCOSAMINES AND STREPTOGRAMINES (MLS ANTIBIOTICS). GENES CODING FOR THE MLS RESISTANCE WILL BE CHARACTERIZED AND ITS REGULATION WILL BE ANALYZED IN DETAIL. GENETIC STUDIES WILL BE PERFORMED TO LOCALIZE THIS DETERMINANT.
Funding SchemeCSC - Cost-sharing contracts