NovG, a DNA-binding protein acting as a positive regulator of novobiocin biosynthesis

Alessandra S. Eustáquio1, Shu-Ming Li1 and Lutz Heide1

1 Pharmazeutische Biologie, Pharmazeutisches Institut, Eberhard-Karls-Universität Tübingen, Auf der Morgenstelle 8, 72076 Tübingen, Germany

Correspondence
Lutz Heide
heide{at}uni-tuebingen.de


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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 {Delta}novG 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 novGnovH and the cloGcloY 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.


Abbreviations: DIG, HTH, helix–turn–helix; PHD, Profile network prediction Heidelberg; SARP, Streptomyces antibiotic regulatory protein

The GenBank/EMBL/DDBJ accession numbers for the genes and DNA regions used in this study are AF170880 (novobiocin cluster) and AF329398 (clorobiocin cluster).


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Streptomycetes produce two-thirds of the clinically useful antibiotics of natural origin (Kieser et al., 2000). Therefore, considerable efforts are directed towards understanding how these filamentous soil bacteria synthesize antibiotics, in order to manipulate the biosynthetic gene clusters and to produce novel compounds with improved properties. In addition, the identification and characterization of regulatory genes is crucial for the rational engineering of strains with enhanced antibiotic production.

Biosynthesis of secondary metabolites by Streptomyces species is a complex process involving several levels of regulation. In this respect, two phylogenetically distant species have received the most extensive attention so far: Streptomyces coelicolor A3(2) and Streptomyces griseus. The former because of its early development as an excellent genetic system, which allowed the analysis of morphological differentiation and secondary metabolite formation, and the latter because it provided the first well-studied bacterial example of extracellular signalling by a hormone-like acylated lactone (the {gamma}-butyrolactone A-factor) (Chater & Horinouchi, 2003). In both cases, pleiotropic regulatory genes affect antibiotic biosynthesis by influencing the expression of pathway-specific regulatory genes, which are usually clustered with the structural biosynthetic genes (Chater & Bibb, 1997).

In S. griseus, StrR has been characterized as a pathway-specific regulatory protein. It activates the expression of streptomycin biosynthetic genes by binding to DNA loci which are characterized by an inverted repeat with the consensus sequence GTTCGActG(N)11CagTcGAAc, and located upstream of their respective promoter regions (Retzlaff & Distler, 1995). StrR has a putative helix–turn–helix (HTH) motif in the central region of its primary structure (Retzlaff & Distler, 1995), which is typical for a family of bacterial and phage DNA-binding proteins (Pabo & Sauer, 1992).

Analysis of ActII-ORF4 from S. coelicolor A3(2) (Arias et al., 1999; Wietzorrek & Bibb, 1997) and of DnrI from Streptomyces peucetius (Sheldon et al., 2002; Wietzorrek & Bibb, 1997) revealed another family of Streptomyces antibiotic regulatory proteins (SARPs). They show in their predicted secondary structure an OmpR-like DNA-binding domain with a different structure than the typical HTH motif (Sheldon et al., 2002; Wietzorrek & Bibb, 1997). These proteins activate transcription of target genes by binding to DNA loci characterized by direct (rather than inverted) heptameric repeats with the consensus sequence TCGAGCG/C located close to the transcriptional start sites (Arias et al., 1999; Sheldon et al., 2002; Wietzorrek & Bibb, 1997).

Furthermore, other types of pathway-specific regulatory genes have been identified from different Streptomyces strains, such as srmR of the spiramycin cluster in Streptomyces ambofaciens, the predicted product of which shows no significant sequence similarity to any other known regulatory protein; mmyR of the methylenomycin cluster in S. coelicolor A3(2), representing the first identified negative pathway-specific regulator of antibiotic production; and response regulator genes of two-component systems like dnrN of the daunorubicin cluster in S. peucetius and redZ of the undecylprodigiosin cluster in S. coelicolor A3(2) (Chater & Bibb, 1997).

The aminocoumarin antibiotics novobiocin, clorobiocin and coumermycin A1 are very potent inhibitors of DNA gyrase produced by different Streptomyces strains (Maxwell, 1993). The biosynthetic gene clusters for these antibiotics have been cloned and sequenced (Pojer et al., 2002; Steffensky et al., 2000; Wang et al., 2000), which allowed detailed investigations of the biosynthetic pathways (reviewed by Li & Heide, 2004) as well as the generation of novel antibiotics by metabolic engineering (Eustáquio et al., 2003a, 2004), chemo-enzymic synthesis (Xu et al., 2004) and precursor-directed biosynthesis (Galm et al., 2004).

In contrast, little is known about how aminocoumarin antibiotic production is regulated. novE is likely to have a positive regulatory function in novobiocin biosynthesis, as shown by an inactivation experiment (Eustáquio et al., 2003b). The predicted gene product of novE shows sequence similarity only to LmbU, which may be involved in the regulation of lincomycin biosynthesis (Peschke et al., 1995). The present study concentrated on another putative regulatory gene of novobiocin biosynthesis, i.e. novG. NovG shows sequence similarity to StrR, the well-established positive regulator of streptomycin biosynthesis (Retzlaff & Distler, 1995).

The aims of the present study were to provide functional proof, by genetic and biochemical approaches, for the role of novG as a regulator of novobiocin biosynthesis, and to investigate whether overexpression of novG can be used to upregulate novobiocin production.


   METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacterial strains, plasmids, cosmids and culture conditions.
Bacterial strains, plasmids and cosmids used in this study are listed in Table 1. The REDIRECT technology kit for PCR targeting (Gust et al., 2003) was obtained from Plant Bioscience (Norwich, UK).


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Table 1. Bacterial strains, plasmids and cosmids

 
S. coelicolor M512 strains were routinely cultured in baffled Erlenmeyer flasks containing a stainless steel spring. YMG medium containing 1 % malt extract, 0·4 % yeast extract and 0·4 % glucose (pH 7·3) was used routinely for cultivation, carried out at 30 °C and 200 r.p.m. for 2 days. For preparation of protoplasts or isolation of genomic DNA, strains were grown in YEME medium (Kieser et al., 2000). For analysis of secondary metabolites, cells from a YMG pre-culture (1 ml) were inoculated in 50 ml CDM production medium (Kominek, 1972) and cultured at 30 °C and 200 r.p.m. for 7 days. The cultivation in production medium was carried out without addition of antibiotics. Escherichia coli strains were cultured in LB medium as described by Sambrook & Russell (2001).

Kanamycin (15 µg ml–1 in liquid medium and 50 µg ml–1 in solid medium for Streptomyces species; 50 µg ml–1 for E. coli), chloramphenicol (25–50 µg ml–1), apramycin (50 µg ml–1), carbenicillin (50–100 µg ml–1) and thiostrepton (15 µg ml–1 in liquid medium and 50 µg ml–1 in solid medium) were used for selection of recombinant strains.

Before transformation of S. coelicolor M512 strains, the recombinant plasmids and cosmids were amplified in E. coli ET12567 to avoid methyl-sensing restriction (MacNeil et al., 1992).

Plasmid construction.
All the plasmid constructions are summarized in Table 1. For the construction of the NovG expression plasmid pAE-G5, novG was amplified by PCR using cosmid nov-BG1 as template and the primer pair PnovG_f (5'-TGG GGA TCC CAT GAC CAA CAG-3') and PnovG_r (5'-GAT TCA AGC TTT TGA ACG TCA GG-3'); bold letters represent mutations inserted in comparison to the original sequence to give the underlined restriction sites BamHI and HindIII, respectively. The PCR reaction was carried out in 50 µl volume with 100 ng template, 0·2 mM dNTPs, 50 pmol each primer, 5 % (v/v) DMSO, using the Expand High Fidelity PCR System (Roche Molecular Biochemicals): denaturation at 94 °C for 2 min, then 30 cycles with denaturation at 94 °C for 45 s, annealing at 55 °C for 45 s, extension at 72 °C for 90 s, and a final elongation step at 72 °C for 5 min. After purification, the PCR product was ligated into pGEM-T to give pAE-G4. The insert of pAE-G4 was checked by nucleotide sequencing, and the BamHI–HindIII fragment was ligated into the same sites of pRSET B to give pAE-G5.

DNA isolation, manipulation and cloning.
Standard procedures for DNA isolation and manipulation were performed as described by Sambrook & Russell (2001) and Kieser et al. (2000). Isolation of DNA fragments from agarose gel and purification of PCR products were carried out with the NucleoSpin 2 in 1 Extract Kit (Macherey–Nagel). Isolation of cosmids and plasmids from E. coli was carried out with ion-exchange columns (Nucleobond AX kits, Macherey–Nagel) according to the manufacturer's protocol. Isolation of plasmid DNA from Streptomyces strains was carried out by alkaline lysis and potassium acetate precipitation, procedure D (Kieser et al., 2000). If required, the plasmid DNA isolated from Streptomyces species was amplified in E. coli XL-1 Blue MRF' before restriction analysis.

Genomic DNA was isolated from S. coelicolor M512 strains using the Kirby mix procedure (Kieser et al., 2000). Southern blot analysis was performed on Hybond-N nylon membrane (Amersham Biosciences) with a digoxigenin-labelled probe by using the DIG high prime DNA labelling and detection starter kit II (Roche Molecular Biochemicals).

Heterologous expression of the novobiocin biosynthetic gene cluster.
In cosmid 10-9C, containing the complete novobiocin biosynthetic gene cluster, the ampicillin resistance gene bla of the SuperCos1 backbone was replaced, using {lambda}-Red-mediated recombination, with a cassette from pIJ787 containing the integrase gene, int, and attachment site, attP, of phage {pi}C31, as well as a tetracycline resistance gene, tet, giving nov-BG1. Cosmid nov-BG1, still carrying the kanamycin resistance gene neo, was then introduced into S. coelicolor M512 via PEG-mediated protoplast transformation (Kieser et al., 2000). Kanamycin-resistant clones were checked for specific genomic integration of the cosmid into the {pi}C31 attachment site by Southern blot analysis. For details, see Eustáquio et al. (2004, 2005).

Inactivation of novG in cosmid nov-BG1, and heterologous expression of the {Delta}novG cosmid.
In cosmid nov-BG1, novG was replaced, via {lambda}-Red-mediated recombination (Gust et al., 2003), by an apramycin resistance (aac(3)IV) cassette from pUG019 (Eustáquio et al., 2004, 2005), which is flanked by XbaI and SpeI recognition sites. The cassette for replacement of novG was generated by PCR using the primer pair P1-novG (5'-GAT CCG GAC CAG ACC ATT AAG TCC TAT GGG GGT TAC ATG ATT CCG GGG ATC TCT AGA TC-3') and P2-novG (5'-CAA CCG AAT GAT TCG AGC AGT TGA ACG TCA GGC GGT GTC ACT AGT CTG GAG CTG CTT C-3'). Underlined letters represent 39 nt homologous extensions to the DNA regions immediately upstream and downstream of novG, including the putative translational start and stop codons of novG, respectively; the XbaI and SpeI restriction sites are presented in bold letters. The PCR reaction was carried out in 50 µl volume with 100 ng template (pUG019 digested with EcoRI, HindIII and DraI), 0·2 mM dNTPs, 50 pmol each primer, 5 % (v/v) DMSO, using the Expand High Fidelity PCR System (Roche Molecular Biochemicals): denaturation at 94 °C for 2 min; 10 cycles with denaturation at 94 °C for 45 s, annealing at 45 °C for 45 s and extension at 72 °C for 90 s; 15 cycles with annealing at 48 °C, and a final elongation step at 72 °C for 5 min.

After isolation from the non-methylating E. coli ET12567, cosmid DNA was digested with XbaI and SpeI and ligated overnight at 4 °C. E. coli XL-1 Blue MRF' cells were transformed with 100 ng DNA. Apramycin-sensitive, kanamycin-resistant clones were analysed using restriction enzymes and gel electrophoresis. The generated novG cosmid nov-AE10, carrying the kanamycin resistance gene neo, was introduced into S. coelicolor M512 by PEG-mediated protoplast transformation (Kieser et al., 2000). Kanamycin-resistant clones were checked for specific genomic integration of cosmid nov-AE10 into {pi}C31 attB by Southern blot analysis.

Complementation with plasmids pAE-G2_1, pAE-G2_2, pAE8 or pWHM3.
Introduction of plasmids pAE-G2_1, pAE-G2_2, pAE8 or pWHM3 into the {Delta}novG mutant [S. coelicolor(nov-AE10)], or into S. coelicolor(nov-BG1) was carried out by PEG-mediated protoplast transformation (Kieser et al., 2000).

Production and analysis of secondary metabolites.
Integration mutants, transformants and the parental strains of S. coelicolor M512 were cultured as described above, and assayed for novobiocin production by HPLC as described by Eustáquio et al. (2003b).

Negative-ion FAB mass spectra were recorded on a TSQ70 spectrometer (Finnigan), using diethanolamine as matrix. The substance isolated from the {Delta}novG mutants gave the following signals: m/z (relative intensity in %): 611 (4, [M–H]), 568 (2, [M–CONH]), 416 (7), 394 (14), 255 (100), 209 (74), corresponding to novobiocin (C31H36N2O11; molecular mass, 612).

Expression and purification of His6-tagged NovG.
Five litres of LB medium (Sambrook & Russell, 2001) containing 50 µg carbenicillin ml–1 and 35 µg chloramphenicol ml–1 was inoculated with E. coli BL21(DE3)pLysS(pAE-G5) cells; sufficient inoculum was used to reach an initial OD600 of 0·1. Cells were then cultured at 30 °C. When the OD600 reached 0·6, IPTG was added to a final concentration of 0·5 mM. After further growth for 1·5 h at 30 °C, cells were harvested by centrifugation and frozen at –70 °C. All subsequent steps were carried out at 4 °C. After thawing on ice, cells (16 g) were suspended in 20 ml lysis buffer (50 mM NaH2PO4, pH 8·0, 300 mM NaCl, 10 mM imidazole, 2 mg lysozyme ml–1) and incubated on ice for 30 min. The cell suspension was sonicated for 7x30 s with 30 s intervals between each treatment (Branson Sonifier 250). Cellular debris was removed by centrifugation (17 500 g for 30 min). Three millilitres of Ni-NTA-agarose slurry [50 % (w/v) nickel-nitrilotriacetic acid agarose resin suspension in 30 % (v/v) ethanol, precharged with Ni2+) (Qiagen)] was added to 3 ml lysis buffer and stirred gently for about 15 min. The supernatant was added to the Ni-NTA-agarose mixture described above and stirred gently for 60 min. Ten millilitres of washing buffer (50 mM NaH2PO4, pH 8·0, 300 mM NaCl, 20 mM imidazole) were added and the protein-Ni-NTA-agarose mixture was harvested by centrifugation (5000 g for 10 min at 4 °C) and washed twice with 30 ml washing buffer (5000 g for 10 min at 4 °C) to remove unbound proteins. The pellet was suspended in washing buffer and loaded into a column. After further washing with 3x4 ml washing buffer containing 50 mM imidazole, unspecifically bound proteins were eluted using a stepwise imidazole gradient: 2x1 ml 100 mM, 1x1 ml 150 mM and 1x1 ml 200 mM imidazole in washing buffer. The NovG fusion protein was subsequently eluted with 4 ml elution buffer (50 mM NaH2PO4, pH 8·0, 300 mM NaCl, 250 mM imidazole). Aliquots (10 µl) were analysed by SDS-PAGE, carried out according to the method of Laemmli (1970), and protein bands were stained with Coomassie brilliant blue R-250. Eluate fractions (1 ml) containing the NovG fusion protein (38·1 kDa) were applied onto a NAP 10 column (Amersham Biosciences) and eluted with 1·2 ml storage buffer [25 mM Tris/HCl (pH 7·5), 10 % (w/v) glycerol, 2 mM DTT]. Aliquots of NovG fusion protein in storage buffer were either frozen in liquid nitrogen and stored at –70 °C, or used immediately in DNA-binding assays.

Preparation of 3'-end DIG-labelled DNA fragments.
The following DNA fragments were used in gel mobility-shift assays: (1) the 325 bp AvaI fragment obtained from pMS32 (position 4463–4787 in AF170880, i.e. upstream of novE); (2) the 341 bp PvuI–SalI fragment obtained from pMS32 (position 4231–4571 in AF170880, i.e. upstream of novE); (3) the 272 bp PCR product obtained using pMS63 as template and the primer pair Pnov$F_f (5'-AGG ACC ACT GGC TCG ATT TCG-3') and Pnov$F_r (5'-GTC ACG CGC GAA GCC GTG AG-3') (position 5238–5509 in AF170880, i.e. upstream of novF); (4) the 265 bp PCR product obtained using pMS33 as template and the primer pair Pnov$G_f (5'-GAG CTG GCC CGC CTC TTC GA-3') and Pnov$G_r (5'-ACT TAA TGG TCT GGT CCG GAT CG-3') (position 6451–6715 in AF170880, i.e. upstream of novG); (5) the 180 bp Van91I–HindIII fragment obtained from pMS63 (position 7725–7904 in AF170880, i.e. upstream of novH); (6) 209 bp SalI–EcoRI fragment obtained from pMS33 (position 7613–7821 in AF170880, i.e. upstream of novH); (7) the 244 bp BamHI fragment obtained from pMS61 (position 17 264–17 507 in AF170880, i.e. upstream of novO); (8) the 252 bp PCR product obtained using pMS61 as template and the primer pair Pnov$O_f (5'-TGT ACG AGC TGC TCA CCC ACG-3') and Pnov$O_r (5'-TGA ATT GAG CCT ACA CGG ACA C-3') (position 17 101–17 352 in AF170880, i.e. upstream of novO); (9) the 231 bp PCR product obtained using pMS62 as template and the primer pair Pgyr$_f (5'-GCG CAG AGG TGC TCT CGT TCC-3') and Pgyr$_r (5'-TGC GGG TGT CGT AAG AAG TCA C-3') (position 168–398 in AF205854, i.e. upstream of gyrBR); (10) the 277 bp SmaI fragment obtained from pMS62 (position 25 608–25 617 in AF170880 and 4–270 in AF205854, i.e. upstream of gyrBR); (11) the 235 bp NotI–MluI fragment obtained from pAE9 (Table 1) (position 12 654–12 888 in AF329398, i.e. upstream of cloY); (12) the 255 bp PCR product obtained using cosmid K1F2 (Table 1) as template and the primer pair Pclo$H_1f (5'-GAA CGG CTC CTA TCT GGT CC-3') and Pclo$H_1r (5'-CGG CCT TCG AAC AAC CTT CG-3') (position 12 946–13 200 in AF329398, i.e. upstream of cloH); and (13) the 260 bp PCR product obtained using cosmid K1F2 as template and the primer pair Pclo$H_2f (5'-TGG GTG GCG AGT AGC ATC TG-3') and Pclo$H_2r (5'-CTT AAG TCT CCA TGC CAT TGG-3') (position 13 122–13 381 in AF329398, i.e. upstream of cloH). PCR products were obtained using the Expand High Fidelity PCR System (Roche Molecular Biochemicals) as described above (‘Plasmid construction’). pMS plasmids are subclones of cosmid 10-9C or 9-6G (Steffensky et al., 2000).

After purification by NuSieve GTG-agarose (FMC BioProducts) gel electrophoresis, DNA fragments were 3'-end-labelled with DIG-11-ddUTP using the DIG Gel Shift Kit, 2nd Generation (Roche Molecular Biochemicals) according to the manufacturer's instructions.

Gel mobility-shift assays.
These assays were performed using the DIG Gel Shift Kit, 2nd Generation (Roche Molecular Biochemicals) according to the manufacturer's instructions. The DNA-binding reaction conditions were adapted from Retzlaff & Distler (1995). The reaction was carried out at 25 °C in 20 µl 12·5 mM Tris/HCl (pH 7·5), 5 % (w/v) glycerol, 62·5 mM KCl, 1 mM DTT, 5 mM MgCl2, 50 ng poly [d(I-C)] µl–1, 5 ng poly L-lysine µl–1. About 4 ng DIG-labelled DNA fragment and approximately 0·5 µg of purified His6-tagged NovG were used for each assay. For testing the specificity of binding, competitor plasmid DNA or the respective empty vector were added in approximately 125-fold molar excess in comparison to the labelled fragment. After 15 min incubation, the reaction mixture was applied to a pre-run (20 min at 35 V) 6 % (w/v) native polyacrylamide gel (85x75x0·75 mm) with 0·5x TBE (Sambrook & Russell, 2001) as running buffer. The gel was run at 35 V for about 3 h and transferred to a positively charged Hybond-N+ nylon membrane (Amersham Biosciences) by contact blotting. Cross-linking and detection were carried out following the manufacturer's instructions.

Computer-assisted sequence analysis.
The DNASIS software package (version 2.1; Hitachi Software Engineering, San Bruno, California) and the BLAST program were used for sequence analysis and for homology searches in the GeneBank database, respectively. The secondary structure of NovG was predicted using the PHD (Profile network prediction Heidelberg) method (Rost, 1996) (available on the web at http://www.embl-heidelberg.de/predictprotein).


   RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Sequence analysis of novG
novG encodes a protein which comprises 318 aa and shows 41 % identity at the amino acid level to StrR, the pathway-specific transcriptional activator of streptomycin biosynthesis in Streptomyces glaucescens and S. griseus (Retzlaff & Distler, 1995; Thamm & Distler, 1997). The predicted amino acid sequence of NovG shows a putative HTH motif in the central region of the protein, which is typical for a family of bacterial and phage DNA-binding proteins (Pabo & Sauer, 1992). This motif is also found in StrR (Retzlaff & Distler, 1995; Thamm & Distler, 1997). Prediction of the secondary structure of NovG using the PHD method confirmed the presence of an HTH motif (Fig. 1).



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Fig. 1. Putative helix–turn–helix (HTH) motif of NovG. The most highly conserved residues in the HTH motif are underlined and include a glycine (G) in the turn and several hydrophobic residues (I, A, L, V) (Pabo & Sauer, 1992). The secondary structure was predicted using the PHD method (Rost, 1996; http://www.embl-heidelberg.de/predictprotein).

 
novG contains the rare TTA leucine codon (codon 182), which indicates its dependence on bldA, the structural gene for tRNAUUA. This may represent a further indication for a regulatory role of NovG in novobiocin biosynthesis, since most of the known TTA-containing genes specify regulatory or resistance proteins associated with biosynthetic gene clusters for antibiotics (Leskiw et al., 1991). Orthologues of novG are found in the biosynthetic gene clusters of the aminocoumarin antibiotics clorobiocin and coumermycin (Pojer et al., 2002). They are termed cloG and couG and both code for proteins of 319 aa which share a mean 78 % sequence identity with novG. Just as for novG, both cloG and couG show the putative HTH motif in their central region. cloG contains three TTA codons (codons 28, 83 and 149), whereas couG contains none.

Inactivation of novG
In order to investigate the function of novG, we first examined the effects of its inactivation and its overexpression. Since the natural novobiocin producer Streptomyces spheroides is difficult to manipulate genetically (Hussain & Ritchie, 1991), we expressed the biosynthetic gene cluster of novobiocin in S. coelicolor M512 by previously described methods (Eustáquio et al., 2004, 2005). As shown in Fig. 2(a), cosmid nov-BG1, containing the entire novobiocin cluster as well as int and attP of phage {pi}C31, was used for this purpose.



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Fig. 2. Inactivation of novG. (a) Schematic representation of cosmids nov-BG1, containing the intact novobiocin biosynthetic gene cluster, and nov-AE10, in which novG was deleted. The novobiocin cluster is presented in grey, the flanking regions in white and novG in black. P, PstI restriction site; T3, T7, promoters of the SuperCos1 vector; tet, tetracycline resistance gene; neo, neomycin/kanamycin resistance gene; int, attP, integrase and attachment sites of phage {pi}C31. Cosmid backbone not to scale. (b) Schematic representation of site-specific integration. The integrase, int, derived from Streptomyces phage {pi}C31, catalyses integration via recombination between attP (from phage or vector) and attB (from Streptomyces genome) sites, generating the hybrid sites attL and attR (Thorpe et al., 2000). The sizes of junction fragments which allow proof of site-specific integration into the {pi}C31 attachment site of the S. coelicolor genome are indicated (4·8 and 14·0 kb). Not to scale. (c) Schematic representation of novG inactivation. novG (957 bp) was replaced by an apramycin resistance (aac(3)IV) cassette. The cassette was then excised by digestion with XbaI and SpeI and religated, leaving an in-frame ‘scar’ of 18 nt between the start and stop codons of novG. Not to scale. (d) Southern blot analysis of S. coelicolor M512 (lane 1) and M512-derived integration mutants bearing cosmid nov-BG1 (lane 2) or the novG cosmid nov-AE10 (lanes 3 and 4). M, DIG-labelled DNA molecular mass Marker VII (Roche). Genomic DNA was digested with PstI. The DIG-labelled cosmid nov-BG1 was used as a probe. The novG inactivation is confirmed by the 7·2 kb band (lane 3 and 4) instead of an 8·1 kb band (lane 2); otherwise the band pattern is expected to be identical.

 
Cosmid nov-BG1 was introduced into S. coelicolor M512 (Floriano & Bibb, 1996) by PEG-mediated protoplast transformation and integrated into the attB site of the chromosome. Southern blot analysis confirmed the site-specific integration of the entire cosmid into the genome of the heterologous host (Fig. 2b, d).

The analysis of secondary metabolites by HPLC showed that the integration mutants, in contrast to the parental strain S. coelicolor M512, accumulated novobiocin. The identity of the produced novobiocin was confirmed by 1H-NMR and negative-ion FAB MS analysis (molecular ion [M–H] at m/z 611; novobiocin, C31H36N2O11, has a molecular mass of 612). The obtained spectroscopic data were identical to those from authentic novobiocin.

Functional investigations of novG could now be carried out by modification of the cosmid nov-BG1 prior to its introduction into S. coelicolor M512. Thus, we generated a {Delta}novG strain by deletion of novG in cosmid nov-BG1 and introduction of this modified cosmid into the genome of S. coelicolor M512. For this purpose, novG was replaced by an apramycin resistance cassette flanked by XbaI and SpeI recognition sites via {lambda}-Red-mediated recombination (Datsenko & Wanner, 2000; Gust et al., 2003). The cassette was then removed by digestion with XbaI and SpeI, enzymes which create compatible ends, allowing re-ligation of the outer ends and consequent excision of the cassette (Fig. 2c). The modified cosmid (named nov-AE10) was introduced into S. coelicolor M512 by protoplast transformation. Southern blot analysis confirmed the site-specific integration into the genome, and the deletion of novG was clearly shown by the size of the relevant PstI restriction fragment in comparison to nov-BG1 strains (Fig. 2d, lanes 2–4).

Analysis of secondary metabolites by HPLC showed that the resulting {Delta}novG strains still produced novobiocin, which was identified by negative-ion FAB MS analysis in comparison to an authentic standard (molecular ion [M–H] at m/z 611; novobiocin, C31H36N2O11, has a molecular mass of 612). However, the amount of novobiocin produced by the {Delta}novG mutants was reduced by 98 % in comparison to S. coelicolor M512 strains carrying the intact novobiocin cluster (Table 2).


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Table 2. Effect of novG inactivation and expression on novobiocin production by recombinant S. coelicolor strains carrying the novobiocin cluster

 
Complementation of the novG mutation
To prove that the inactivation of novG was indeed responsible for the low productivity of the {Delta}novG strains, we attempted to complement one of these strains by expressing novG under the control of its own promoter. For this purpose, we used a derivative of the promoter-less, replicative shuttle vector pWHM3 (Vara et al., 1989), into which the coding region of novG and 336 bp of the DNA region upstream of the putative translational start point was cloned, resulting in pAE8.

Transformants of the {Delta}novG strain carrying pAE8 produced on average 80 % of the novobiocin amount accumulated by S. coelicolor M512 strains bearing the intact novobiocin cluster (Table 2). Therefore, complementation was successful, establishing that the low productivity of {Delta}novG strains was due to lack of novG and not to possible polar effects of the deletion on downstream genes.

Notably, a pWHM3 construct containing novG with only 100 bp of the upstream DNA region (termed pAE-G2_2) was much less effective in restoration of novobiocin productivity (Table 2). If the orientation of its insert was reversed (i.e. placed against the lacZ orientation), the resulting plasmid pAE-G2_1 was completely unable to enhance novobiocin production. Apparently, these constructs did not contain a functional promoter upstream of novG, and the low increase of productivity observed in pAE-G2_2 transformants may be due to low activity of the lacZ promoter in the S. coelicolor host.

Overexpression of novG in S. coelicolor (nov-BG1) leads to overproduction of novobiocin
The results presented above indicate that NovG could act as a positive regulator in novobiocin biosynthesis. It has been reported that overexpression of pathway-specific activators can lead to overproduction of the respective antibiotic (Gramajo et al., 1993; Stutzman-Engwall et al., 1992). Therefore, we introduced the multicopy plasmids pAE-G2_2 and pAE8 into S. coelicolor(nov-BG1) by protoplast transformation. As presented in Table 2, pAE-G2_2 led to a 1·9-fold and pAE8 a 2·7-fold increase in novobiocin biosynthesis in comparison to strains carrying only the empty vector pWHM3.

Overexpression and purification of NovG as a His6 fusion protein
Retzlaff & Distler (1995) successfully expressed the regulator protein StrR both in E. coli and in Streptomyces lividans, and found no difference in the DNA binding property between the proteins from either expression system. For further investigation of the function of novG, its gene product was therefore expressed as an N-terminal His6 fusion protein in E. coli (see Methods). Upon cultivation at 30 °C and induction with 1 mM IPTG for 5 h, most of the resulting protein was insoluble. The amount of soluble protein did not improve significantly when the growth temperature was reduced to 15 °C and the IPTG concentration to 0·25 mM. However, useful amounts of soluble protein could be obtained reproducibly when the induction period was shortened to only 1·5 h, using 0·5 mM IPTG at 30 °C (see Methods). The His6-tagged NovG protein was purified from the soluble fraction by nickel affinity chromatography. SDS-PAGE analysis showed a band of 40 kDa (calculated mass 38·1 kDa) in the eluate (Fig. 3). The protein yield of purified NovG was about 15 µg per g cells (fresh weight), determined by SDS-PAGE.



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Fig. 3. Purification by SDS/12 % PAGE of NovG after overexpression as a fusion protein with an N-terminal His6 tag in E. coli BL21(DE3)pLysS. Lane 1, soluble protein before induction; lane 2, soluble protein after induction with 0·5 mM IPTG for 1·5 h; lane 3, eluate from nickel affinity chromatography; M, molecular mass marker. The molecular masses (kDa) of the marker proteins are indicated.

 
DNA-binding activity
As described above, we speculated that NovG may act as a positive regulator of novobiocin biosynthesis by binding at or near the promoter region of biosynthetic genes and thereby activating their transcription. In order to verify whether NovG does indeed have DNA-binding activity, gel mobility-shift assays were carried out using the purified His6-tagged NovG and selected DNA fragments from the novobiocin biosynthetic gene cluster. In the novobiocin cluster, the intergenic regions between most genes are very short, and often the coding regions even overlap, indicating a possible translational coupling (Steffensky et al., 2000). Only six of the intergenic regions are larger than 70 bp (Fig. 4a) and those were chosen for gel shift experiments. As shown in Fig. 4(b), DNA fragments extending into the coding sequences upstream of these intergenic regions were also investigated. The NovG fusion protein showed very clear binding activity for one of the two fragments obtained from the novGnovH intergenic region (Fig. 4), but not for any other of the ten DNA fragments investigated.



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Fig. 4. Gel mobility-shift assays with DNA fragments from the novobiocin biosynthetic gene cluster. (a) Schematic representation of the novobiocin biosynthetic gene cluster. All intergenic regions larger than 70 bp are shown and their exact size is given. (b) Autoradiogram of gel mobility-shift assays using purified His6-tagged NovG. The DNA-binding activity was analysed as described in Methods, using about 4 ng of the indicated DIG-end-labelled fragment and no protein (–) or 0·5 µg of purified His6-tagged NovG protein (+). A map showing the location of the DNA fragments used is given above each autoradiogram (not to scale; the intergenic regions are oversized in comparison to the ORFs). The fragments were obtained by digestion of appropriate plasmids or by PCR amplification. For fragments obtained by digestion, restriction sites are indicated: P, PvuI; S, SalI; A, AvaI; V, Van91I; E, EcoRI; H, HindIII; B, BamHI; Sm, SmaI.

 
The novobiocin and the clorobiocin clusters show very high similarity between each other. For most of the nov genes, orthologues exist in the clorobiocin cluster, which show 75–80 % sequence identity to the genes of the novobiocin cluster at the amino acid level (Pojer et al., 2002). However, in the novobiocin cluster, the gene novH, which encodes a biosynthetic enzyme (Chen & Walsh, 2001), is situated directly downstream of the regulatory gene novG, whereas in the clorobiocin cluster their orthologues cloG and cloH are separated by a small ORF of unknown function, i.e. cloY (Fig. 5). As shown in Fig. 5, we tested whether NovG could also bind to the DNA regions upstream of cloY and cloH. Very clearly, NovG did bind to the DNA region upstream of cloY, but not to those upstream of cloH.



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Fig. 5. Gel mobility-shift assays with DNA fragments from the clorobiocin biosynthetic gene cluster. The fragments were obtained by digestion of pAE9 (fragment 1) or by PCR amplification from cosmid K1F2 (fragments 2 and 3). N, NotI; M MluI. See legend of Fig. 4 for further explanations.

 
NovG binds specifically to the novGnovH and cloGcloY intergenic regions
To provide proof that binding of NovG to the novGnovH and cloGcloY intergenic regions is specific, a gel mobility-shift assay was carried out in which a competitor plasmid containing the respective binding site was added in approximately 125-fold molar excess. As a negative control, the same amount of empty vector was used. As shown in Fig. 6, the presence of the specific competitor plasmid strongly reduced the intensity of the band representing the NovG–DNA complex, while the empty vector had no significant effect.



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Fig. 6. Specifity of His6-tagged NovG binding to DNA. Approximately 4 ng DIG-end-labelled fragment (novH, fragment 6 of Fig. 4; cloY, fragment 1 of Fig. 5) and no protein (–) or 0·5 µg purified His6-tagged NovG protein (+) were used in each lane for a standard DNA-binding assay. When indicated (+), an additional 125-fold molar excess of specific competitor plasmid DNA (pAE7 or pAE9, respectively) or non-specific empty vector DNA (pBluescript SK(–) or pSPORT1, respectively) was added.

 
In silico analysis of the NovG binding DNA fragments
When the DNA sequences of the novGnovH and cloGcloY intergenic regions were compared, similar palindromic structures could be found in both fragments (Fig. 7). Notably, similar inverted repeats were identified as the binding site for StrR by DNase I footprinting assay (Retzlaff & Distler, 1995). The putative NovG-binding sites and the StrR-binding sites all possess a conserved palindromic structure with the consensus sequence GTTCRACTG(N)11CRGTYGAAC.



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Fig. 7. Sequences of putative NovG-binding sites of S. spheroides (upstream of novH) and Streptomyces roseochromogenes (upstream of cloY) in comparison to the consensus sequence of StrR-binding sites of S. griseus and S. glaucescens (see Retzlaff & Distler, 1995). Nucleotides conserved in at least two of the sequences are in bold. Nucleotides conserved in all three sequences are underlined. The inverted repeats are indicated with arrows. A putative consensus sequence for NovG- and StrR-binding sites is indicated.

 

   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The present work provides experimental proof for the function of NovG as a DNA-binding protein which acts as a positive regulator of novobiocin biosynthesis. Inactivation of novG led to a 98 % reduction of the novobiocin productivity of the heterologous producer strain. Since {Delta}novG strains still produced some novobiocin, an essential catalytic role for novG can be ruled out. By complementation with novG under control of its own promoter, novobiocin production was restored nearly to the same level as observed before novG inactivation, indicating that the observed phenotype of {Delta}novG strains was indeed caused by lack of novG.

The inactivation of positive regulators in the biosynthetic gene clusters of various antibiotics has been reported to result in a complete loss of antibiotic production (Antón et al., 2004; Lombó et al., 1999; Otten et al., 1995, 2000; Pérez-Llarena et al., 1997; Wilson et al., 2001). The {Delta}novG strains constructed in this study, however, still produced some novobiocin (see above). This indicates a low level transcription of the biosynthetic genes in the absence of NovG. Similarly, in vivo analysis of regulatory genes of the nystatin biosynthetic gene cluster showed that nystatin production in the deletion mutants was reduced by 91–99·5 % compared to that in the wild-type strain; i.e. some nystatin production could still be detected (Sekurova et al., 2004).

Expression of novG from a multicopy plasmid in a S. coelicolor M512 strain carrying the intact novobiocin cluster led to almost threefold overproduction of the antibiotic, suggesting that novobiocin biosynthesis in the heterologous expression host investigated is limited by availability of the activator protein, as has been shown for other pathway-specific activators in their respective natural antibiotic producers (Gramajo et al., 1993; Stutzman-Engwall et al., 1992).

Furthermore, it was demonstrated that His6-tagged NovG protein binds specifically to the novGnovH intergenic region and, in so doing, probably activates the transcription of novH, just as described for StrR, which activates transcription of streptomycin biosynthetic genes by binding to their promoter regions (Retzlaff & Distler, 1995). The putative binding site of NovG is located directly downstream of the translational stop codon of novG, i.e. between –165 and –194 bp upstream of the putative translational start codon of novH. Since nickel affinity purified protein, generated in E. coli, was used for the DNA-binding assays, it is very likely that NovG can bind to DNA with no further macromolecular factor involved. However, our results do not completely rule out the possibility that other proteins may be required for activating the transcription of novH.

The novobiocin and clorobiocin clusters show very high similarity between each other. Indeed, NovG could also bind to a DNA region of the clorobiocin cluster, i.e. the cloGcloY intergenic region; the putative binding site is located between positions –160 and –189 upstream of the putative translational start of cloY. The function of the small ORF cloY remains to be elucidated. Genes with obvious similarity to cloY have been found in many other clusters, such as those for coumermycin A1 (Wang et al., 2000), teicoplanin (Sosio et al., 2004), complestatin (Chiu et al., 2001), CDA (Hojati et al., 2002) and balhimycin (Pelzer et al., 1999), but not in the novobiocin cluster.

The in silico analysis of the DNA fragments from the novobiocin and clorobiocin clusters which bind NovG showed the presence of a perfectly conserved 9 bp inverted repeat, separated by a somewhat less-conserved (two mismatches) 11 bp spacer sequence (Fig. 7). Notably, the previously identified StrR binding sites in S. griseus and S. glaucescens contain a similar palindromic structure, i.e. conserved inverted repeats of 9 bp each, separated by a non-conserved 11 bp spacer (Retzlaff & Distler, 1995). The same putative NovG binding site, with exactly the same inverted repeat and spacer sequences as found upstream of cloY, is also present in the coumermycin A1 cluster, between genes couG and couY. The close similarity of the putative NovG/CloG/CouG binding sites in the novobiocin, clorobiocin and coumermycin A1 clusters further indicates a common evolutionary origin for these clusters (Eustáquio et al., 2003b).

The consensus sequence GTTCRACTG(N)11CRGTYGAAC or similar motifs were not found anywhere else in the gene clusters of novobiocin or clorobiocin, except in the mentioned regions upstream of novH and cloY, respectively. This is in accordance with the results of the gel shift assays with the novobiocin cluster depicted in Fig. 4. In contrast, four StrR binding sites have been identified in the streptomycin biosynthetic gene cluster in S. griseus and three in the 5'-OH-streptomycin cluster in S. glaucescens (Retzlaff & Distler, 1995). It cannot be completely excluded that NovG may also bind to additional sequences which are different from the motif shown above. However, it is quite possible that only a single transcription unit is expressed under control of novG. In the novobiocin and clorobiocin clusters, all genes are orientated in the same direction and may, in principle, be transcribed as a single mRNA from novH to novW or from cloY to cloZ, respectively. In contrast, previous results suggest that the resistance gene gyrBR, at the right border of the cluster depicted in Fig. 4, is under control of its own promoter and may be regulated by changes in DNA superhelical density (Thiara & Cundliffe, 1989). NovG was not required for expression of gyrBR in S. lividans (Thiara & Cundliffe, 1988) and, correspondingly, we did not observe binding of NovG to the promoter sequence of gyrBR in our gel-shift assays.

Analysis of different gene clusters encoding biosynthetic pathways for antibiotics has revealed the existence of distinct families of pathway-specific regulatory proteins. One of these families comprises the strR-like genes and includes, besides novG, cloG and couG, also dbv4 of the glycopeptide antibiotic A40926 (Sosio et al., 2003), bbr of the balhimycin (Pelzer et al., 1999) and tcp28 of the teicoplanin (Sosio et al., 2004) clusters. Another family comprises the SARPs, i.e. ActII-ORF4 and DnrI (see Introduction) and SnoA of the nogalamycin, RedD of the undecylprodigiosin and CcaR of the cephamycin clusters, respectively (Wietzorrek & Bibb, 1997). However, no genes with sequence similarity to this family have been found in the gene clusters of the aminocoumarin antibiotics.

An understanding of the complex regulatory mechanisms that determine the onset of antibiotic biosynthesis in actinomycetes is only just beginning to emerge (Wietzorrek & Bibb, 1997). Activation of any particular pathway in any particular organism might be expected to require its own combination of signals (Chater & Bibb, 1997). Therefore, analysis of regulatory genes from different species is crucial to build a comprehensive picture of these processes.

The aminocoumarin antibiotics may provide useful model systems for such studies, due to their very stringent genetic organization (Pojer et al., 2002) and the availability of detailed data on the function of most genes contained therein (Li & Heide, 2004). The fact that they can be successfully expressed in S. coelicolor M512, and the genome sequence of its wild-type strain S. coelicolor A3(2) is available (Bentley et al., 2002), makes them even more attractive for investigating the regulation cascades for improved antibiotic production in streptomycetes.


   ACKNOWLEDGEMENTS
 
We thank E. Takano, R. Shawky and W. Wohlleben for valuable discussions. We are also grateful to E. Takano and Janet White for kindly providing S. coelicolor M512. This work was supported by a grant from the European Community (No. 503466 to L. H. and S.-M. L.).


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Received 30 September 2004; revised 24 January 2005; accepted 24 February 2005.



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