LcpRVH2 – regulating the expression of latex-clearing proteins in Gordonia polyisoprenivorans VH2
| Autor: | Anna Coenen, Rense Jongsma, Sylvia Oetermann, Alexander Steinbüchel, Jeanne Keller |
|---|---|
| Rok vydání: | 2019 |
| Předmět: |
Latex
Inverted repeat medicine.disease_cause Microbiology 03 medical and health sciences Hemiterpenes Bacterial Proteins Escherichia coli medicine Native state Electrophoretic mobility shift assay Binding site Gordonia Bacterium Gene 030304 developmental biology 0303 health sciences Binding Sites 030306 microbiology Chemistry Gordonia polyisoprenivorans DNase-I Footprinting Gene Expression Regulation Bacterial Molecular biology Recombinant Proteins Molecular Weight Biodegradation Environmental Trans-Activators Protein Multimerization |
| Zdroj: | Microbiology. 165:343-354 |
| ISSN: | 1465-2080 1350-0872 |
| Popis: | Gordonia polyisoprenivorans VH2 harbours two latex clearing proteins, which are responsible for the cleavage of poly(cis-1,4-isoprene) into oligoisoprenes, thereby allowing growth in presence of, e.g. natural rubber. A gene coding for a putative regulator of the TetR-family (lcpR VH2) is located 131 bp upstream of lcp1 VH2. We heterologously expressed lcpR VH2 in Escherichia coli , and purified and characterized the protein with respect to its ability to bind to the operator region of lcp1 VH2. LcpRVH2 forms a dimer in its native state. The size of the dimer was determined to be 52.7 kDa by size exclusion chromatography, whereas the calculated size of a monomer was 24.1 kDa. Electrophoretic mobility shift assays (EMSAs) with the purified protein revealed a shift upon binding to the intergenic region between lcpR VH2 and lcp1 VH2. Within this region, an inverted repeat was identified in silico, probably being the binding site of LcpRVH2. This binding sequence was confirmed by a DNase I footprinting assay. A shift also occurred in EMSAs with this 44 bp sequence only. Interestingly, no regulator was detected upstream of the second lcp (lcp2 VH2). Therefore, we performed EMSA studies with LcpRVH2 and the putative operator region upstream of lcp2 VH2, and discovered by DNase I footprinting another binding sequence upstream of lcp2 VH2. Hence, we concluded that LcpRVH2 binds the operator region of both lcps and, most likely, regulates their expression in G. polyisoprenivorans VH2. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|