Cloning and characterization of the pyrrolomycin biosynthetic gene clusters from Actinosporangium vitaminophilum ATCC 31673 and Streptomyces sp. strain UC 11065.
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.
Zhang
Xiujun
X
Rice University, Department of Chemistry MS60, 6100 Main Street, Houston, TX 77005, USA.
Parry
Ronald J
RJ
D000900:Anti-Bacterial Agents / Q000096:biosynthesis*; D015139:Blotting, Southern; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D003360:Cosmids / Q000235:genetics; D004269:DNA, Bacterial / Q000096:biosynthesis / Q000235:genetics; D005285:Fermentation; D015723:Gene Library; D008969:Molecular Sequence Data; D005810:Multigene Family; D010088:Oxidoreductases / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013302:Streptomyces / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism*
D000900:Anti-Bacterial Agents; D004269:DNA, Bacterial; D010088:Oxidoreductases; C407364:monodechloroaminopyrrolnitrin halogenase