ABSTRACT
OBJECTIVES: The aim of this study was to obtain Streptomyces xinghaiensis (fradiae) ATCC 19609 mutants resistant to oligomycin A and its derivatives and to identify the underlying mechanism of resistance. This study was based on the premise that S. xinghaiensis ATCC 19609 contains several oligomycin A biological targets, explaining why the strain remains supersensitive to oligomycin A despite all efforts to obtain resistant mutants using standard genetic methods. METHODS: The method to obtain oligomycin A-resistant mutants was performed in two steps: first, mutants slightly resistant to an oligomycin A derivative with an attenuated effect were obtained; and second, oligomycin A-resistant mutants were obtained from those mutants obtained earlier. The genomes of the mutants were then sequenced and a bioinformatics analysis of the detected mutations was conducted. RESULTS: Mutants with seven mutations were required to obtain oligomycin A-resistant mutant strains of S. xinghaiensis characterised by a level of resistance comparable with that of the model organism Streptomyces lividans. Five of these mutations caused amino acid substitutions in the well-known oligomycin A biological target, namely the F0F1-ATP synthase A subunit, and the others caused amino acid substitutions in unexplored biological targets, including RecB-like recombinase, type IV helicase, DNA ligase and single-domain response regulator. CONCLUSION: A new oligomycin resistance mechanism involving a pathway that repairs double-strand breaks in DNA known as non-homologous end joining (NHEJ) was discovered.
Subject(s)
Computational Biology , Drug Resistance, Bacterial/genetics , Oligomycins , Streptomyces/genetics , Mutation , Oligomycins/pharmacology , Streptomyces/drug effectsABSTRACT
We report a draft genome sequence of Streptomyces xinghaiensis (fradiae) OlgR, which is resistant to oligomycin A. This mutant strain is derived from S. xinghaiensis OlgR2.100, which is resistant to (33S)-azido-33-deoxyoligomycin A. We have identified single nucleotide polymorphisms (SNPs) in 7 genes, which may lead to oligomycin A resistance.
ABSTRACT
We describe Streptomyces fradiae mechanisms of sensitivity to nitrone-oligomycin A, a derivative of oligomycin A. We obtained S. fradiae-nitR+ bld, a nitrone-oligomycin A resistant mutant with a «bald¼ phenotype. Comparative genomic analysis of the wild-type S. fradiae ATCC19609 and S. fradiae-nitR+ bld revealed a mutation in padR - a gene encoding a multifunction transcription regulator, which resulted in the amino acid replacement in a highly conserved DNA-binding domain. Bioinformatics genome analysis of S. fradiae ATCC19609 discovered a PadR binding site 13 bp upstream the start codon of the marR transcription factor gene. Induction of S. fradiaenitR+ bld and w.t. strains with nitrone-oligomycin A lead to a significant increase in expression level of the marR gene in the w.t. strain, but no change observed in mutant strain. We identified differences between DNA-protein interactions of the mutant and native PadR proteins with its putative binding site in S. fradiae ATCC19609. This allowed us to suggest that the padR gene, that harbored a single nucleotide mutation in the S. fradiaenitR+ bld strain, might be involved in the mechanism of resistance to nitrone-oligomycin A. We assume the participation of the transcriptional factorpadR in the formation of the bald phenotype.
Subject(s)
Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Drug Resistance, Bacterial/genetics , Nitrogen Oxides/pharmacology , Oligomycins/pharmacology , Streptomyces/drug effects , Streptomyces/genetics , Anti-Bacterial Agents/pharmacology , Binding Sites/genetics , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Drug Resistance, Bacterial/physiology , Gene Expression Regulation, Bacterial , Genome, Bacterial/genetics , Mutation , Protein Binding , Streptomyces/metabolism , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
We report a draft genome sequence of Streptomyces fradiae olg1-1, a mutant strain derived from the model object S. fradiae ATCC 19609, which is resistant to nitrone-oligomycin and has a mutation in the DNA-binding domain of a transcriptional regulator PadR.
ABSTRACT
A data set consisting of twenty-two sertindole analogues and ten structurally diverse inhibitors, spanning a wide range in potency, was analyzed using CoMSiA. A homology model of HERG was constructed from the crystal structure of the open MthK potassium channel. A complementary relationship between our CoMSiA and homology models is apparent when the long inhibitor axis is oriented parallel to the longitudinal axis of the pore, with the tail region pointed toward the selectivity filter. The key elements of the pharmacophore, the CoMSiA and the homology model are: (1) The hydrophobic feature optimally consists of an aromatic group that is capable of engaging in pi-stacking with a Phe656 side chain. Optionally, a second aromatic or hydrophobic group present in some inhibitors may contact an additional Phe656 side chain. (2) The basic nitrogen appears to undergo a pi-cation interaction with Tyr652. (3) The pore diameter (12A+), and depth of the selectivity loop relative to the intracellular opening, act as constraints on the conformation-dependent inhibitor dimensions.
