Molecular detection of type II polyketide synthase genes in Cuban soils
Molecular detection methods were developed to study the distribution of type II polyketide synthase (PKS) genes in Cuban soils. A PCR based detection method targeting the α and β ketosynthase genes was applied to a number of different total community DNA samples. These genes were detected in 43% of samples tested from a number of different locations. A botanical garden site located in Havana, Cuba, was found to show the greatest distribution of type II PKS genes across the sites tested. It was not possible to amplify type II PKS genes from a pristine island site off the coast of Cuba. Further investigation revealed that actinornycetes containing type II PKS were present in the soil community at a level above the detection limit of the PCR protocol. Further total community DNA cleanup steps failed to allow the detection of type II PKS genes within the DNA samples suggesting PCR inhibition was responsible for negative results. The molecular detection of type II PKS genes in total community DNA was compared to the detection of type II PKS genes in actinomycete isolates. A lack of correlation between these two approaches was observed with the molecular detection limit unable to amplify type II PKS genes in <50% of crop soils tested. Actinomycetes containing type II PKS genes could be isolated from all crop soils tested. No difference was seen in the detection of type II PKS genes between rhizosphere and bulk soil samples. Actinomycetes were isolated using a selective isolation procedure at a level of approximately 10(7) cfu g-1 soil compared to 10(8) cfu g-1 for total bacterial counts. Actinomycetes were isolated from Cuban crop soils and screened for the presence of type II PKS genes. Out of 100 isolates 26 were found to contain the genes of interest. Phylogenetic analysis of these isolates based on 16S rDNA and recA sequence data showed them to be closely grouped within the streptomycetes. Sequence data based on KSα genes from Cuban isolates showed them to be representative of both spore pigment and antibiotic polyketide genes. A representative clone library was constructed containing type II PKS genes amplified from total community DNA. Rhizosphere and bulk soil samples were compared from the same site. Sequences obtained from rhizosphere total community DNA appeared to be widely distributed when compared to published sequences and included examples of both spore pigment and antibiotic polyketide genes. A molecular method was developed to amplify near full length α and β KS genes from type II PKS gene clusters. Expression vectors were constructed to allow these genes to be expressed along with an ACP to give a functional minimal PKS for polyketide chain production. This method was used on total community DNA in an attempt to extract diverse genes from as yet uncultured organisms.