The Natural Product Domain Seeker NaPDoS: A Phylogeny Based Bioinformatic Tool to Classify Secondary Metabolite Gene Diversity

New bioinformatic tools are needed to analyze the growing volume of DNA sequence data. This is especially true in the case of secondary metabolite biosynthesis, where the highly repetitive nature of the associated genes creates major challenges for accurate sequence assembly and analysis. Here we introduce the web tool Natural Product Domain Seeker (NaPDoS), which provides an automated method to assess the secondary metabolite biosynthetic gene diversity and novelty of strains or environments. NaPDoS analyses are based on the phylogenetic relationships of sequence tags derived from polyketide synthase (PKS) and non-ribosomal peptide synthetase (NRPS) genes, respectively. The sequence tags correspond to PKS-derived ketosynthase domains and NRPS-derived condensation domains and are compared to an internal database of experimentally characterized biosynthetic genes. NaPDoS provides a rapid mechanism to extract and classify ketosynthase and condensation domains from PCR products, genomes, and metagenomic datasets. Close database matches provide a mechanism to infer the generalized structures of secondary metabolites while new phylogenetic lineages provide targets for the discovery of new enzyme architectures or mechanisms of secondary metabolite assembly. Here we outline the main features of NaPDoS and test it on four draft genome sequences and two metagenomic datasets. The results provide a rapid method to assess secondary metabolite biosynthetic gene diversity and richness in organisms or environments and a mechanism to identify genes that may be associated with uncharacterized biochemistry

Stimulation of polyketide metabolism in Streptomyces fradiae by tylosin and its glycosylated precursors

Three glycosyltransferases are involved in tylosin biosynthesis in Streptomyces fradiae. The first sugar to be added to the polyketide aglycone (tylactone) is mycaminose and the gene encoding mycaminosyltransferase is orf2* (tylM2). However, targeted disruption of orf2* did not lead to the accumulation of tylactone under conditions that normally favour tylosin production; instead, the synthesis of tylactone was virtually abolished. This may, in part, have resulted from a polar effect on the expression of genes downstream of orf2*, particularly orf4* (ccr) which encodes crotonyl-CoA reductase, an enzyme that supplies 4-carbon extender units for polyketide metabolism However, that cannot be the entire explanation, since tylosin production was restored at about 10% of the wild-type level when orf2* was re-introduced into the disrupted strain. When glycosylated precursors of tylosin were fed to the disrupted strain, they were converted to tylosin, confirming that two of the three glycosyltransferase activities associated with tylosin biosynthesis were still intact. Interestingly, however, tyladone also accumulated under such conditions and, to a much lesser extent, when tylosin was added to similar fermentations. It is concluded that glycosylated macrolides exert a pronounced positive effect on polyketide metabolism in S. fradiae

Cloning and characterization of gentamicin-resistance genes from Micromonospora purpurea and Micromonospora rosea

Aminoglycoside-resistance genes (grm) were cloned from a gentamicin producer Micromonospora purpurea and a sisomicin producer Micromonospora rosea. The nucleotide (nt) sequences of both genes were determined and the similarity between them was very high (90.4% identity). In either case, the transcription start point was localised to about 11 nt upstream from the likely translation start codons of grm, which is expressed as a polycistronic transcript. In studies to be reported elsewhere, it has been established that the M. purpurea grm gene encodes a ribosomal RNA methyltransferase. Here, we confirmed that the similarity of the two genes exists not only at the structural but also at the functional level