Molecular Evolution of Aromatic Polyketides and Comparative Sequence Analysis of Polyketide Ketosynthase and 16S Ribosomal DNA Genes from Various Streptomyces Species

Abstract

A 613-bp fragment of an essential ketosynthase gene from the biosynthetic pathway of aromatic polyketide antibiotics was sequenced from 99 actinomycetes isolated from soil. Phylogenetic analysis showed that the isolates clustered into clades that correspond to the various classes of aromatic polyketides. Additionally, sequencing of a 120-bp fragment from the ␥-variable region of 16S ribosomal DNA (rDNA) and subsequent comparative sequence analysis revealed incongruity between the ketosynthase and 16S rDNA phylogenetic trees, which strongly suggests that there has been horizontal transfer of aromatic polyketide biosynthesis genes. The results show that the ketosynthase tree could be used for DNA fingerprinting of secondary metabolites and for screening interesting aromatic polyketide biosynthesis genes. Furthermore, the movement of the ketosynthase genes suggests that traditional marker molecules like 16S rDNA give misleading information about the biosynthesis potential of aromatic polyketides, and thus only molecules that are directly involved in the biosynthesis of secondary metabolites can be used to gain information about the biodiversity of antibiotic production in different actinomycetes.Soil actinomycetes, especially those that belong to the genus Streptomyces, have been the focus of intensive research for the past several decades. The interest in Streptomyces arose from the finding that this group of bacteria seems to have the ability to produce a large variety of different bioactive compounds that have a wide spectrum of activity. From the 1950s to the mid-1970s numerous new bioactive molecules were discovered through large screening programs, and these molecules subsequently found their way into various clinical uses ranging from control of infections to cancer treatment (21).In more recent years, modern high-throughput screening methods have exponentially increased the number of strains screened annually, but the number of novel compounds discovered has not increased in the same proportion. One of the many reasons, presumably the most important one, for this problem is that old molecules (and strains) are being rediscovered with the screening procedures that are in use today (21).In a previous paper (18) Metsä-Ketelä et al. reported a method that could be used for preliminary classification of strains on the basis of their genetic abilities to produce various compounds belonging to the aromatic polyketide group. This method is based on PCR amplification of a gene fragment that is essential in the biosynthesis pathway of aromatic polyketides and on analysis of the amplified regions by phylogenetic methods. The degenerate primers designed for this purpose amplify a portion of a ketosynthase gene (KS ␣ ), which in collaboration with KS ␤ and an acyl carrier protein condenses small carboxylic acids in a stepwise manner to form a long polyketide chain that is subsequently folded into a range of different aromatic compounds by various ketoreductases, cyclases, and aromatases. Later, the molecule formed is of...

Isolation and characterization of culturable endophytic actinobacteria associated with Artemisia annua L.

“…It is equally important to explore useful natural products from these valuable isolates. It is accepted that the approach of detection biosynthetic gene sequences is helpful for obtaining information about the production of active metabolites in different actinomycetes (Metsa-Ketela et al 2002). In this study, the results of biosynthetic gene sequence amplification showed that PKS-I, PKS-II and NRPS biosynthetic systems were widespread in the tested strains.…”

“…Three sets of PCR primers were used: A3F (5 0 -GCS TAC SYS ATS TAC ACS TCS GG-3 0 ) and A7R (5 0 -SAS GTC VCC SGT SCG GTA S-3 0 ) targeting NRPS sequences (Gonzalez et al 2005); K1F (5 0 -TSA AGT CSA ACA TCG GBC A-3 0 ) and M6R (5 0 -CGC AGG TTS CSG TAC CAG TA-3 0 ) targeting PKS-I sequences (Gonzalez et al 2005); KSaF (5 0 -TSG CST GCT TGG AYG CSA TC-3 0 ) and KSaR (5 0 -TGG AAN CCG CCG AAB CCG CT-3 0 ) targeting KSa genes (Metsa-Ketela et al 2002). PCR amplifications were performed in a Biometra thermal cycler in a final volume of 25 ll containing 0.2 lmol l -1 of each primer, 0.1 mmol l -1 of each of the four dNTPs (TaKaRa), 2.5 ll of extracted DNA, 0.5 unit of Taq DNA polymerase (with its recommended reaction buffer) and 10% of DMSO.…”

Emergent gene order in a model of modular polyketide synthases

“…Because PKS protein domains from different organisms retain a high degree of sequence identity, homology-preferring recombinant processes are expected to drive the shuffling of PKS genes between DNA strands. This expectation is supported by the evidence from comparative genomics (1,20,21) including several examples of HGT events that have been inferred from sudden transitions in sequence identity along PKS gene clusters (1). If one accepts the hypothesis that HGT and homologous recombination are the main drivers of the search for novel polyketides, it becomes interesting to consider how natural selection might augment the modular features of the system that facilitate its adaptation to changing circumstances, or, in other words, make it more "evolvable" (22).…”

Diversity of nonribosomal peptide synthetase and polyketide synthase genes in the genus Actinoplanes found in Mongolia

“…For phylogenetic analysis, the 16S rDNA sequences were aligned with genus Actinoplanes reference sequences using the CLUSTAL_X software and a phylogenetic tree constructed using the neighbor-joining method. 12 Adenylation (A) domain regions in NRPS genes, ketosynthase (KS) domain regions in type-I PKS genes and KSa genes in type-II PKS genes were amplified using specific primer sets described by AyusoSacido et al, 8 Schermer et al 13 and Metsa-Ketela et al, 14 respectively. The PCR products were cloned, sequenced and searched by BLASTX on the NCBI website and phylogenetic trees were constructed.…”

“…The PCR products were cloned, sequenced and searched by BLASTX on the NCBI website and phylogenetic trees were constructed. 10,14 The substrate-specificity-conferring residues, comprising the aminoacid (aa)-binding pocket 15 and the substrate aa of A domains in the NRPS genes were predicted using the PKS/NRPS Analysis Web-site (http://nrps.igs.umaryland.edu/nrps/).…”