A Protocal For Metabolome Investigation Of Streptomyces Colicolor And Research On Oxytetracycline Efflux Mechanism

Streptomyces are known for their ability to produce a large amount of secondary metabolites, which constitute an important source of small molecule drug leads. The biosynthesis of secondary metabolites is subjected to complex regulation. Elucidation of the regulatory mechanisms could provide clues to improve the production levels of secondary metabolites, and also will help us find the large reservoir of secondary metabolites that are still not exploited.The present work contains two parts, one is the estabilishment of a rapid and accurate metabolites analysis protocol for streptomycetes, another one is the meachnisms of the regulatory protein OtrR and its relationship with oxytetracycline. In the first part, Streptomyces coelicolor was chosen as the object. Several sample prepartion steps including quenching time,cell separation method, conditions for metabolite extraction and metabolite derivatization were optimized. Then, the metabolic profiles of S. coelicolor during different growth stages were analyzed by GC-MS. The optimal sample preparation conditions were as follows:time of low-temperature quenching4min, cell separation by fast filtration, time of freeze-thaw45s/3min and the conditions of metabolite derivatization at40℃for90min. By using this optimized protocol,103metabolites were finally identified from a sample of S. coelicolor, which distribute in central metabolic pathways (glycolysis, pentose phosphate pathway and citrate cycle), amino acid, fatty acid, nucleotide metabolic pathways, etc. By comparing the temporal profiles of these metabolites, the amino acid and fatty acid metabolic pathways were found to stay at a high level during stationary phase, therefore, these pathways may play an important role during the transition between the primary and secondary metabolim. The temporal profiles of metabolites reveal amino acid and fatty acid metabolic pathways may play an important role in the transition from primary to secondary metabolism in S. coelicolor.Many microorganisms could produce molecules with bioactivities as antibiotics to compete with their neighbours for suvival. However, these small moleculars are always toxic to the producers when their concentrations reaching a specific threshold. To address this issue, the efflux pump system in vivo will be activated in time. One very large family of ligand-sensing transcription regulators is exemplified by the TetR-TetA efflux pump system. The regulatory protein TetR controls tetA, a major determinant of resistance to tetracyclines. TetR is a repressor that bind target promoters and prevents transcriptional initiation. When an appropriate small molecular enters a cell, it interacts with the C-terminal domain of TetR, causing it to dissociate from bound DNA, in turn activiating tetA expression and permitting the export of the antibiotic from the cell. Thus in the second part, we mainly focused on the regulatory protein OtrR and the related efflux pump gene otrB involved in the oxytetracycline biosynthetic cluster of Streptomyces rimosus. Similar as ctcS-ctcR in Streptomyces aureus and TetR-tetA in Escherichia coli, OtrR and otrB also transcripted reversely. Additionally, from the same arrangement of the three pairs of genes, we speculate OtrR is the repressor of otrB. OtrR belong to MarR family, members of the MarR family of transcriptional regulators have been described as winged helix proteins that binding directly to the DNA. Most MarR are repressors, and their abilities to binding DNA are abrogated by the binding of small molecule ligands via reactive cysteine residues. At first we purified OtrR as an N-terminal6×His-tag fusion protein, then we carried out gel mobility assay identified His6-OtrR can bind PotrR-otrB and PctcR-ctcs. We speculated that they may have relatively censerved binding sequence in PotrR-otrB and PctcR-ctcs. We obtianed an14bp sequence according to sequence blast. We indentified that the P14bp is the putative operator sites for OtrR by using gel mobility assay and Surface Plasmon Resonance (SPR). We get the equilibrium dissociation constant (KD) of0.14nM. We have identified several molecules that can relieve repression by OtrR, including oxytetracycline, tetracycline, doxycycline and aureomycin.