Cloning and Characterization of the Pyrrolomycin Biosynthetic Gene Clusters from Actinosporangium vitaminophilum ATCC 31673 and Streptomyces sp. Strain UC 11065

Abstract

The pyrrolomycins are a family of polyketide antibiotics, some of which contain a nitro group. To gain insight into the nitration mechanism associated with the formation of these antibiotics, the pyrrolomycin biosynthetic gene cluster from Actinosporangium vitaminophilum was cloned. Sequencing of ca. 56 kb of A. vitaminophilum DNA revealed 35 open reading frames (ORFs). Sequence analysis revealed a clear relationship between some of these ORFs and the biosynthetic gene cluster for pyoluteorin, a structurally related antibiotic. Since a gene transfer system could not be devised for A. vitaminophilum, additional proof for the identity of the cloned gene cluster was sought by cloning the pyrrolomycin gene cluster from Streptomyces sp. strain UC 11065, a transformable pyrrolomycin producer. Sequencing of ca. 26 kb of UC 11065 DNA revealed the presence of 17 ORFs, 15 of which exhibit strong similarity to ORFs in the A. vitaminophilum cluster as well as a nearly identical organization. Single-crossover disruption of two genes in the UC 11065 cluster abolished pyrrolomycin production in both cases. These results confirm that the genetic locus cloned from UC 11065 is essential for pyrrolomycin production, and they also confirm that the highly similar locus in A. vitaminophilum encodes pyrrolomycin biosynthetic genes. Sequence analysis revealed that both clusters contain genes encoding the two components of an assimilatory nitrate reductase. This finding suggests that nitrite is required for the formation of the nitrated pyrrolomycins. However, sequence analysis did not provide additional insights into the nitration process, suggesting the operation of a novel nitration mechanism.

Characterization of the Biosynthesis Gene Cluster for the Pyrrole Polyether Antibiotic Calcimycin (A23187) in Streptomyces chartreusis NRRL 3882

“…(i) PCR amplification of the proline adenyltransferase gene from S. chartreusis NRRL 3882. Fifteen sets of degenerate primers were designed on the basis of the conserved domains of bacterial proline adenytransferase genes found in pyolu-teorin, coumermycin A 1 , prodiginines, clorobiocin, and pyrrolomycin biosynthetic gene clusters (5,23,24,34,37). PCR amplification was run for 30 cycles.…”

Prodigiosin biosynthesis gene cluster in the roseophilin producer Streptomyces griseoviridis

“…10 PCR was carried out using several primers designed from the conserved amino acid sequences of L-prolyl-AMP ligase and L-prolyl-PCP dehydrogenase genes in the coumermycin A 1 producer, Streptomyces rishiriensis DSM 40489 (proB and proA), 11 in the pyrrolomycin producers, Streptomyces vitaminophilum ATCC 31673 (pyr8 and pyr7) and Streptomyces sp. Strain UC 11065 (dox8 and dox7) 12 and in the prodigiosin producer, S. coelicolor A3(2) (redM and red W). DNA sequencing of the PCR products identified single genes encoding L-prolyl-AMP ligase and L-prolyl-PCP dehydrogenase in S. griseoviridis.…”

Insights into the Evolution of Macrolactam Biosynthesis through Cloning and Comparative Analysis of the Biosynthetic Gene Cluster for a Novel Macrocyclic Lactam, ML-449

“…A probe for the ML-449 biosynthetic gene cluster was obtained by PCR amplification of conserved KS domain-coding regions by using degenerate primers. Cloning and sequencing of these DNA fragments and subsequent BLAST analysis of the translated sequences revealed KS domains with high similarity to PKS sequences from the pyrrolomycin biosynthetic gene cluster (44) and to VinP1 from the vicenistatin biosynthetic gene cluster (30). As a DNA fragment encoding a KS domain with high similarity to VinP1 was successfully used as a probe for the BE-14106 biosynthetic gene cluster (19), the latter was chosen as a probe for identification of the ML-449 gene cluster.…”