Artificial chromosomes for antibiotic-producing actinomycetes

Abstract

Bacteria belonging to the order Actinomycetales produce most microbial metabolites thus far described, several of which have found applications in medicine and agriculture. However, most strains were discovered by their ability to produce a given molecule and are, therefore, poorly characterized physiologically and genetically. Thus, methodologies for genetic manipulation of actinomycetes are not available and efficient tools have been developed for just a few strains. This constitutes a serious limitation to applying molecular genetics approaches to strain development and structural manipulation of microbial metabolites. To overcome this hurdle, we have developed bacterial artificial chromosomes (BAC) that can be shuttled among Escherichia coli, where they replicate autonomously, and a suitable Streptomyces host, where they integrate site-specifically into the chromosome. The existence of gene clusters and of genetically amenable host strains, such as Streptomyces coelicolor or Streptomyces lividans, makes this a sensible approach. We report here that 100 kb segments of actinomycete DNA can be cloned into these vectors and introduced into genetically accessible S. lividans, where they are stably maintained in integrated form in its chromosome.

Genetically Modified Bacterial Strains and Novel Bacterial Artificial Chromosome Shuttle Vectors for Constructing Environmental Libraries and Detecting Heterologous Natural Products in Multiple Expression Hosts

“…To our knowledge, this is the first example of conjugative transfer of such high-molecular-weight plasmids from E. coli to Streptomyces. Sosio et al (32) have constructed E. coli-Streptomyces shuttle BACs that also use C31-mediated site-specific recombination to integrate in the Streptomyces chromosome, and they showed that inserts up to 120 kb can be introduced and maintained in S. lividans. Their vectors, however, do not contain the oriT sequence and thus have to be transferred into Streptomyces by protoplast transformation, which is not amenable to high throughput.…”

Characterization of the biosynthetic gene cluster (ata) for the A201A aminonucleoside antibiotic from Saccharothrix mutabilis subsp. capreolus

“…18 The library was screened by colony hybridization on nitrocellulose Hybond-N membranes (Amersham) using fragments from the ends of the cosmid inserts of pCAR13 and pCAR23 as DNA probes described, 16,19 isolating plasmid pIES. This plasmid contained an insert of~60 kb that included the A201A biosynthetic (ata) gene cluster from S. mutabilis subsp.…”

Bacterial genomics: the use of DNA microarrays and bacterial artificial chromosomes

“…Gene clusters encoding antibiotic synthesis could be cloned into a BAC vector and expressed in an appropriate host. As previously mentioned, E. coli -Streptomyces artificial chromosomes (ESAC) have been developed in which genes from non-culturable actinomycete strains have been integrated into a foreign genome and expressed in a culturable host (Sosio et al, 2000). Classical genetic, molecular and biochemical tech-niques can be employed to optimize expression of the recombinant genes.…”

“…ESACs will permit the analysis of previously genetically uncharacterizable actinomycetes. Optimization of expression of integrated genes can potentially lead to the synthesis of production hosts for antibiotic and other industrially and medically important actinomycete metabolites (Sosio et al, 2000).…”

“…This genomic technique allowed rapid identification of functional loci in B. cereus, a microorganism not easily studied by traditional molecular techniques. Sosio et al (2000) created BACs harbouring actinomycete genomic clusters that could replicate autonomously in E. coli host or stably integrate into a Streptomyces genome by site-specific recombination. The E. coli -Streptomyces artificial chromosomes (ESAC) were made using the previously described approach or by homologous replication of overlapping clones in E. coli.…”