LcpRVH2 - regulating the expression of latex-clearing proteins in Gordonia polyisoprenivorans VH2.

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). LcpR(VH2) 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 LcpR(VH2). 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 LcpR(VH2) 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 LcpR(VH2) binds the operator region of both lcps and, most likely, regulates their expression in G. polyisoprenivorans VH2.