Identification of unique type II polyketide synthase genes in soil

Abstract

Many bacteria, particularly actinomycetes, are known to produce secondary metabolites synthesized by polyketide synthases (PKS). Bacterial polyketides are a particularly rich source of bioactive molecules, many of which are of potential pharmaceutical relevance. To directly access PKS gene diversity from soil, we developed degenerate PCR primers for actinomycete type II KS(alpha) (ketosynthase) genes. Twenty-one soil samples were collected from diverse sources in New Jersey, and their bacterial communities were compared by terminal restriction fragment length polymorphism (TRFLP) analysis of PCR products generated using bacterial 16S rRNA gene primers (27F and 1525R) as well as an actinomycete-specific forward primer. The distribution of actinomycetes was highly variable but correlated with the overall bacterial species composition as determined by TRFLP. Two samples were identified to contain a particularly rich and unique actinomycete community based on their TRFLP patterns. The same samples also contained the greatest diversity of KS(alpha) genes as determined by TRFLP analysis of KS(alpha) PCR products. KS(alpha) PCR products from these and three additional samples with interesting TRFLP pattern were cloned, and seven novel clades of KS(alpha) genes were identified. Greatest sequence diversity was observed in a sample containing a moderate number of peaks in its KS(alpha) TRFLP. The nucleotide sequences were between 74 and 81% identical to known sequences in GenBank. One cluster of sequences was most similar to the KS(alpha) involved in ardacin (glycopeptide antibiotic) production by Kibdelosporangium aridum. The remaining sequences showed greatest similarity to the KS(alpha) genes in pathways producing the angucycline-derived antibiotics simocyclinone, pradimicin, and jasomycin.

FIG. 1.

TRFLP dendrograms. Labels indicate the identity of soil samples (NSX) where X is the sample number and NS (1S, 2S, 3S, and 4S) indicates the sampling collection site (see Materials and Methods). (A) Bacterial 16S rRNA genes (27F/1525R). (B) Actinomycete 16S rRNA genes (243F/1401R). A distance of 10% (scale bar) indicates a dissimilarity of 10% between two samples as calculated by the Sorenson index.

FIG. 2.

Phylogenetic analysis of DNA sequences obtained by cloning of KS_α PCR products from samples 2S1 and 2S5, which were chosen because they contained a large number of peaks in their actinomycete 16S TRFLPs. Species names indicate sequences obtained from GenBank. A subset of GenBank sequences used to design the PCR primer set as well as GenBank sequences most similar to cloned sequences (Actinomadura hibisca for 2S5 and Streptomyces antibioticus for 2S1 clones) was used for analysis. Numbers indicate bootstrap percentages. Only bootstraps >65% are shown. A star (*) after a sequence identifier indicates that this sequence and its identity to other sequences are shown in Table 2.

FIG. 3.

Phylogenetic analysis of DNA sequences obtained by cloning of KS_α PCR products from samples 1S2, 3S6, and 4S4, which were chosen because they contained a large number of peaks in their KS_α TRFLPs. Only the most divergent subset (n = 35) of the sequences we obtained (n = 97) for these three samples are shown. Also shown is a subset of sequences from the 2S1 and 2S5 samples. Species names indicate sequences obtained from GenBank. A subset of GenBank sequences used to design the PCR primer set as well as GenBank sequences most similar to cloned sequences (Actinomadura hibisca for 3S6 and 4S4, Streptomyces venezuelae for 1S2 A, and Kibdelosporangium ardium for 1S2 B and C clones) was used for analysis. Numbers indicate bootstrap percentages. Only bootstraps >65% are shown. A star (*) after a sequence identifier indicates that this sequence and its % identity to other sequences are shown in Table 2.