Mechanistic studies of DepR in regulating FK228 biosynthesis in Chromobacterium violaceum no. 968

Autor: Tiantian Tong, Dongqing Zhu, Wenjing Lin, Yi-Qiang Cheng, Jiao Xue, Yongjian Qiao, Zixin Deng
Jazyk: angličtina
Rok vydání: 2018
Předmět:
0301 basic medicine
Operon
Gene Expression
lcsh:Medicine
Electrophoretic Mobility Shift Assay
Artificial Gene Amplification and Extension
Restriction Fragment Mapping
Pathology and Laboratory Medicine
Biochemistry
Polymerase Chain Reaction
Genes
Reporter
Transcription (biology)
Chromobacterium
Nucleic Acids
Depsipeptides
Gene Order
Gene cluster
Medicine and Health Sciences
Promoter Regions
Genetic
lcsh:Science
Multidisciplinary
biology
Chemistry
Transcriptional Control
Bacterial Pathogens
Complementation
Medical Microbiology
Pathogens
Research Article
Protein Binding
030106 microbiology
Research and Analysis Methods
Biosynthesis
Microbiology
Models
Biological
03 medical and health sciences
Bacterial Proteins
Genetics
Gene Regulation
Electrophoretic mobility shift assay
Nucleotide Motifs
Molecular Biology Techniques
Operons
Molecular Biology
Microbial Pathogens
Binding Sites
Point mutation
Gene Mapping
lcsh:R
Biology and Life Sciences
DNA
Gene Expression Regulation
Bacterial
biology.organism_classification
030104 developmental biology
lcsh:Q
Chromobacterium Violaceum
Chromobacterium violaceum
Transcription Factors
Zdroj: PLoS ONE, Vol 13, Iss 4, p e0196173 (2018)
PLoS ONE
ISSN: 1932-6203
Popis: DepR, a LysR-type transcriptional regulator encoded by the last gene of the putative min operon (orf21-20-19-depR) located at the downstream region of the anticancer agent FK228 biosynthetic gene cluster in Chromobacterium violaceum No. 968, positively regulates the biosynthesis of FK228. In this work, the mechanism underlining this positive regulation was probed by multiple approaches. Electrophoretic mobility shift assay (EMSA) and DNase I footprinting assay (DIFA) identified a conserved 35-nt DNA segment in the orf21-orf22 intergenic region where the purified recombinant DepR binds to. Quantitative reverse transcription PCR (RT-qPCR) and green fluorescent protein (GFP) promoter probe assays established that transcription of phasin gene orf22 increases in the depR deletion mutant of C. violaceum (CvΔdepR) compared to the wild-type strain. FK228 production in the orf22-overexpressed strain C. violaceum was reduced compared with the wild-type strain. DepR has two conserved cysteine residues C199 and C208 presumed to form a disulfide bridge upon sensing oxidative stress. C199X point mutations that locked DepR in a reduced conformation decreased the DNA-binding affinity of DepR; T232A or R278A mutation also had a negative impact on DNA binding of DepR. Complementation of CvΔdepR with any of those versions of depR carrying a single codon mutation was not able to restore FK228 production to the level of wild-type strain. All evidences collectively suggested that DepR positively regulates the biosynthesis of FK228 through indirect metabolic networking.
Databáze: OpenAIRE
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