Identification of LcpRBA3(2), a novel regulator of lcp expression in Streptomyces coelicolor A3(2)
2019
Coenen, Anna | Oetermann, Sylvia | Steinbüchel, A.
Streptomyces coelicolor A3(2) is a rubber-degrading actinomycete that harbors one gene coding for a latex clearing protein (lcpA₃₍₂₎). Within the genome of S. coelicolor A3(2), we identified a gene coding for a novel protein of the TetR family (LcpRBA₃₍₂₎) downstream of lcpA₃₍₂₎ and demonstrated its binding upstream of lcpA₃₍₂₎. This indicates a role of LcpRBA₃₍₂₎ in the regulation of lcp expression. LcpRBA₃₍₂₎ shows no homology to LcpRVH₂, a putative regulator of lcp expression in Gordonia polyisoprenivorans VH2. Additionally, LcpRVH₂ homologs did not occur in the genome of S. coelicolor A3(2). Reverse transcriptase (RT) experiments showed that the expression of lcpA₃₍₂₎ and lcpRBA₃₍₂₎ is induced with poly(cis-1,4-isoprene) as sole carbon source. For further experiments, we heterologously expressed lcpRBA₃₍₂₎ in Escherichia coli, purified the protein, and subsequently verified a binding of LcpRBA₃₍₂₎ upstream of lcpA₃₍₂₎. The operator site was examined by a DNase I footprinting assay: it comprises 31 bp and exhibits an inverted repeat of nine bases for the putative binding region. Interestingly, two N-terminal DNA-binding HTH domains of the TetR-type (PF00440) were identified within the sequence of LcpRBA₃₍₂₎. The native molecular weight of LcpRBA₃₍₂₎ was determined as 44 kDa by size exclusion chromatography which correlates to the molecular weight of a monomer. Normally, proteins of the TetR family occur as dimers so that the monomeric state is a novelty. Furthermore, LcpRBA₃₍₂₎ homologs were identified in silico in several Lcp-containing actinomycetes, suspecting a conserved regulation mechanism. Apparently, the expression of lcps is regulated either by an LcpRB or by an LcpR.
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