RT Journal Article SR Electronic(1) A1 Eustáquio, Alessandra S. A1 Li, Shu-Ming A1 Heide, LutzYR 2005 T1 NovG, a DNA-binding protein acting as a positive regulator of novobiocin biosynthesis JF Microbiology, VO 151 IS 6 SP 1949 OP 1961 DO https://doi.org/10.1099/mic.0.27669-0 PB Microbiology Society, SN 1465-2080, AB The biosynthetic gene cluster of the aminocoumarin antibiotic novobiocin contains two putative regulatory genes, i.e. novE and novG. The predicted gene product of novG shows a putative helix–turn–helix DNA-binding motif and shares sequence similarity with StrR, a well-studied pathway-specific transcriptional activator of streptomycin biosynthesis. Here functional proof is provided, by genetic and biochemical approaches, for the role of NovG as a positive regulator of novobiocin biosynthesis. The entire novobiocin cluster of the producer organism Streptomyces spheroides was expressed in the heterologous host Streptomyces coelicolor M512, and additional strains were produced which lacked the novG gene within the heterologously expressed cluster. These ΔnovG strains produced only 2 % of the novobiocin formed by the S. coelicolor M512 strains carrying the intact novobiocin cluster. The production could be restored by introducing an intact copy of novG into the mutant. The presence of novG on a multicopy plasmid in the strain containing the intact cluster led to almost threefold overproduction of the antibiotic, suggesting that novobiocin biosynthesis is limited by the availability of NovG protein. Furthermore, purified N-terminal His6-tagged NovG showed specific DNA-binding activity for the novG–novH and the cloG–cloY intergenic regions of the novobiocin and clorobiocin biosynthetic gene clusters, respectively. By comparing the DNA sequences of the fragments binding NovG, conserved inverted repeats were identified in both fragments, similar to those identified as the binding sites for StrR. The consensus sequence for the StrR and the putative NovG binding sites was GTTCRACTG(N)11CRGTYGAAC. Therefore, NovG and StrR apparently belong to the same family of DNA-binding regulatory proteins., UL https://www.microbiologyresearch.org/content/journal/micro/10.1099/mic.0.27669-0