Fig 1.
Novobiocin production of rokB deletion and overexpression mutants.
Novobiocin production of S. coelicolor M512(nov-BG1)/pUWL-apra-oriT 1–6, S. coelicolor M512(nov-BG1)/pPBB12 1–5 (+rokB) and S. coelicolor M512(nov-BG1)/ΔrokB 1–3 compared relatively to S. coelicolor M512(nov-BG1) novobiocin production. Error bars indicate standard deviations between individual clones.
Fig 2.
SPR experiments with RokB and PnovH.
a Scheme of the promoter of novH with localisation of the tested DNA fragments (oligo 1–18) on PnovH and binding curves of RokB to the tested oligonucleotides. b Binding site of RokB on PnovH. Boundaries determined by SPR spectroscopy are indicated by vertical lines. Start codon of novH in grey (TTG). c SPR binding curves of RokB to 42 bp starting sequence shortened by 2 bp each for determination of the left-hand and right hand boundary of the RokB binding site on PnovH.
Fig 3.
SPR binding curves with RokB and PnovH-BS, ProkB/sco6114-BS1 40 bp and ProkB/sco6114-BS2 40 bp.
Fig 4.
a Binding site of RokB on ProkB/sco6114-BS1. Boundaries are indicated by vertical lines. SPR binding curves of RokB to 33 bp starting sequence shortened by 2 bp each for determination of the left and right-hand boundary of the binding site. b Binding site of RokB on ProkB/sco6114-BS2. Boundaries are indicated by vertical lines. SPR binding curves of RokB to 34 bp starting sequence shortened by 2 bp each for determination of the left and right-hand boundary of the binding site.
Fig 5.
RokB binding sites on the genome of S. coelicolor.
a SPR binding curves with RokB and ProkB/sco6114-BS1, ProkB/sco6114-BS2, Psco3215/3216-BS, Psco6108-BS, Psco0938-BS. b Binding motif of RokB on ProkB/sco6114-BS1, ProkB/sco6114-BS2, Psco3215/3216-BS, Psco6108-BS, Psco0938-BS, calculated by MEME and binding site of RokB on PnovH. Inverted repeat is underlined, TTG Startcodon of novH depicted in grey.
Fig 6.
Binding sites of RokB in the Genome of S. coelicolor.
a Location of RokB binding sites (marked by vertical bars) in the promoter regions of the respective operons. b Sequences of the 5 identified genomic RokB Binding sites. c Annotations of genes belonging to a possible RokB regulon.
Fig 7.
Novobiocin production after feeding with L-tyrosine and casamino acids.
a Novobiocin production [mg g-1 dry weight] of S. coelicolor M512(nov-BG1) unfed, after feeding of 1mM L-tyrosine once and twice and after feeding of 1g L-1 casamino acids once and twice. b S. coelicolor M512(nov-BG1)/ pPBB12 (+rokB) 1–5 unfed, after feeding of 1mM L-tyrosine once and twice and after feeding of 1g L-1 casamino acids once and twice. c S. coelicolor M512(nov-BG1) ΔrokB 1–3 unfed, after feeding of 1mM L-tyrosine once and twice and after feeding of 1g L-1 casamino acids once and twice. Error bars indicate standard deviation between triplicated measurements.