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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 -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 helixturnhelix (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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Kanamycin (15 µg ml1 in liquid medium and 50 µg ml1 in solid medium for Streptomyces species; 50 µg ml1 for E. coli), chloramphenicol (2550 µg ml1), apramycin (50 µg ml1), carbenicillin (50100 µg ml1) and thiostrepton (15 µg ml1 in liquid medium and 50 µg ml1 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 BamHIHindIII 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 (MachereyNagel). Isolation of cosmids and plasmids from E. coli was carried out with ion-exchange columns (Nucleobond AX kits, MachereyNagel) 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 -Red-mediated recombination, with a cassette from pIJ787 containing the integrase gene, int, and attachment site, attP, of phage
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
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 novG cosmid.
In cosmid nov-BG1, novG was replaced, via -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
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 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 novG mutants gave the following signals: m/z (relative intensity in %): 611 (4, [MH]), 568 (2, [MCONH]), 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 ml1 and 35 µg chloramphenicol ml1 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 ml1) 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 44634787 in AF170880, i.e. upstream of novE); (2) the 341 bp PvuISalI fragment obtained from pMS32 (position 42314571 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 52385509 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 64516715 in AF170880, i.e. upstream of novG); (5) the 180 bp Van91IHindIII fragment obtained from pMS63 (position 77257904 in AF170880, i.e. upstream of novH); (6) 209 bp SalIEcoRI fragment obtained from pMS33 (position 76137821 in AF170880, i.e. upstream of novH); (7) the 244 bp BamHI fragment obtained from pMS61 (position 17 26417 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 10117 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 168398 in AF205854, i.e. upstream of gyrBR); (10) the 277 bp SmaI fragment obtained from pMS62 (position 25 60825 617 in AF170880 and 4270 in AF205854, i.e. upstream of gyrBR); (11) the 235 bp NotIMluI fragment obtained from pAE9 (Table 1) (position 12 65412 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 94613 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 12213 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)] µl1, 5 ng poly L-lysine µl1. 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
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
C31, was used for this purpose.
|
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 [MH] 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 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
-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 24).
Analysis of secondary metabolites by HPLC showed that the resulting novG strains still produced novobiocin, which was identified by negative-ion FAB MS analysis in comparison to an authentic standard (molecular ion [MH] at m/z 611; novobiocin, C31H36N2O11, has a molecular mass of 612). However, the amount of novobiocin produced by the
novG mutants was reduced by 98 % in comparison to S. coelicolor M512 strains carrying the intact novobiocin cluster (Table 2
).
|
Transformants of the 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
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.
|
|
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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
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 9199·5 % 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 |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Arias, P., Fernández-Moreno, M. A. & Malpartida, F. (1999). Characterization of the pathway-specific positive transcriptional regulator for actinorhodin biosynthesis in Streptomyces coelicolor A3(2) as a DNA-binding protein. J Bacteriol 181, 69586968.
Bentley, S. D., Chater, K. F., Cerdeño-Tárraga, A. M. & 40 other authors (2002). Complete genome sequence of the model actinomycete Streptomyces coelicolor A3(2). Nature 417, 141147.[CrossRef][Medline]
Chater, K. F. & Bibb, M. J. (1997). Regulation of bacterial antibiotic production. In Biotechnology, vol. 7. Products of Secondary Metabolism, pp. 57105. Edited by H. Kleinkauf & H. von Döhren. Weinheim: VCH.
Chater, K. F. & Horinouchi, S. (2003). Signalling early developmental events in two highly diverged Streptomyces species. Mol Microbiol 48, 915.[CrossRef][Medline]
Chen, H. & Walsh, C. T. (2001). Coumarin formation in novobiocin biosynthesis: -hydroxylation of the aminoacyl enzyme tyrosyl-S-NovH by a cytochrome P450 NovI. Chem Biol 8, 301312.[CrossRef][Medline]
Chiu, H.-T., Hubbard, B. K., Shah, A. N., Eide, J., Fredenburg, R. A., Walsh, C. T. & Khosla, C. (2001). Molecular cloning and sequence analysis of the complestatin biosynthetic gene cluster. Proc Natl Acad Sci U S A 98, 85488553.
Datsenko, K. A. & Wanner, B. L. (2000). One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci U S A 97, 66406645.
Eustáquio, A. S., Gust, B., Luft, T., Li, S.-M., Chater, K. F. & Heide, L. (2003a). Clorobiocin biosynthesis in Streptomyces. Identification of the halogenase and generation of structural analogs. Chem Biol 10, 279288.[CrossRef][Medline]
Eustáquio, A. S., Luft, T., Wang, Z.-X., Gust, B., Chater, K. F., Li, S.-M. & Heide, L. (2003b). Novobiocin biosynthesis: inactivation of the putative regulatory gene novE and heterologous expression of genes involved in aminocoumarin ring formation. Arch Microbiol 180, 2532.[CrossRef][Medline]
Eustáquio, A. S., Gust, B., Li, S.-M., Pelzer, S., Wohlleben, W., Chater, K. F. & Heide, L. (2004). Production of 8'-halogenated and 8'-unsubstituted novobiocin derivatives in genetically engineered Streptomyces coelicolor strains. Chem Biol 11, 15611572.[CrossRef][Medline]
Eustáquio, A. S., Gust, B., Galm, U., Li, S.-M., Chater, K. F. & Heide, L. (2005). Heterologous expression of novobiocin and clorobiocin biosynthetic gene clusters. Appl Environ Microbiol 71 (in press).
Floriano, B. & Bibb, M. (1996). afsR is a pleiotropic but conditionally required regulatory gene for antibiotic production in Streptomyces coelicolor A3(2). Mol Microbiol 21, 385396.[CrossRef][Medline]
Galm, U., Dessoy, M. A., Schmidt, J., Wessjohann, L. A. & Heide, L. (2004). In vitro and in vivo production of new aminocoumarins by a combined biochemical, genetic and synthetic approach. Chem Biol 11, 173183.[CrossRef][Medline]
Gramajo, H. C., Takano, E. & Bibb, M. J. (1993). Stationary-phase production of the antibiotic actinorhodin in Streptomyces coelicolor A3(2) is transcriptionally regulated. Mol Microbiol 7, 837845.[Medline]
Gust, B., Challis, G. L., Fowler, K., Kieser, T. & Chater, K. F. (2003). PCR-targeted Streptomyces gene replacement identifies a protein domain needed for biosynthesis of the sesquiterpene soil odor geosmin. Proc Natl Acad Sci U S A 100, 15411546.
Hojati, Z., Milne, C., Harvey, B. & 9 other authors (2002). Structure, biosynthetic origin, and engineered biosynthesis of calcium-dependent antibiotics from Streptomyces coelicolor. Chem Biol 9, 11751187.[CrossRef][Medline]
Hussain, H. A. & Ritchie, D. A. (1991). High frequency transformation of Streptomyces niveus protoplasts by plasmid DNA. J Appl Bacteriol 71, 422427.[Medline]
Kieser, T., Bibb, M. J., Buttner, M. J., Chater, K. F. & Hopwood, D. A. (2000). Practical Streptomyces Genetics, 2nd edn. Norwich: John Innes Foundation.
Kominek, L. A. (1972). Biosynthesis of novobiocin by Streptomyces niveus. Antimicrob Agents Chemother 1, 123134.[Medline]
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680685.[Medline]
Leskiw, B. K., Bibb, M. J. & Chater, K. F. (1991). The use of a rare codon specifically during development? Mol Microbiol 5, 28612867.[Medline]
Li, S.-M. & Heide, L. (2004). Functional analysis of biosynthetic genes of aminocoumarins and production of hybrid antibiotics. Curr Med Chem Anti-Infective Agents 3, 279295.[CrossRef]
Lombó, F., Braña, A. F., Méndez, C. & Salas, J. A. (1999). The mithramycin gene cluster of Streptomyces argillaceus contains a positive regulatory gene and two repeated DNA sequences that are located at both ends of the cluster. J Bacteriol 181, 642647.
MacNeil, D. J., Gewain, K. M., Ruby, C. L., Dezeny, G., Gibbons, P. H. & MacNeil, T. (1992). Analysis of Streptomyces avermitilis genes required for avermectin biosynthesis utilizing a novel integration vector. Gene 111, 6168.[CrossRef][Medline]
Maxwell, A. (1993). The interaction between coumarin drugs and DNA gyrase. Mol Microbiol 9, 681686.[Medline]
Otten, S. L., Ferguson, J. & Hutchinson, C. R. (1995). Regulation of daunorubicin production in Streptomyces peucetius by the dnrR2 locus. J Bacteriol 177, 12161224.
Otten, S. L., Olano, C. & Hutchinson, C. R. (2000). The dnrO gene encodes a DNA-binding protein that regulates daunorubicin production in Streptomyces peucetius by controlling expression of the dnrN pseudo response regulator gene. Microbiology 146, 14571468.[Medline]
Pabo, C. O. & Sauer, R. T. (1992). Transcription factors: structural families and principles of DNA recognition. Annu Rev Biochem 61, 10531095.[CrossRef][Medline]
Pelzer, S., Süßmuth, R., Heckmann, D., Recktenwald, J., Huber, P., Jung, G. & Wohlleben, W. (1999). Identification and analysis of the balhimycin biosynthetic gene cluster and its use for manipulating glycopeptide biosynthesis in Amycolatopsis mediterranei DSM5908. Antimicrob Agents Chemother 43, 15651573.
Pérez-Llarena, F. J., Liras, P., Rodríguez-García, A. & Martín, J. F. (1997). A regulatory gene (ccaR) required for cephamycin and clavulanic acid production in Streptomyces clavuligerus: amplification results in overproduction of both -lactam compounds. J Bacteriol 179, 20532059.
Peschke, U., Schmidt, H., Zhang, H. Z. & Piepersberg, W. (1995). Molecular characterization of the lincomycin-production gene cluster of Streptomyces lincolnensis 78-11. Mol Microbiol 16, 11371156.[Medline]
Pojer, F., Li, S.-M. & Heide, L. (2002). Molecular cloning and sequence analysis of the clorobiocin biosynthetic gene cluster: new insights into the biosynthesis of aminocoumarin antibiotics. Microbiology 148, 39013911.[Medline]
Retzlaff, L. & Distler, J. (1995). The regulator of streptomycin gene expression, StrR, of Streptomyces griseus is a DNA binding activator protein with multiple recognition sites. Mol Microbiol 18, 151162.[CrossRef][Medline]
Rost, B. (1996). PHD: predicting one-dimensional protein structure by profile-based neural networks. Methods Enzymol 266, 525539.[CrossRef][Medline]
Sambrook, J. & Russell, D. W. (2001). Molecular Cloning: a Laboratory Manual, 3rd edn. New York: Cold Spring Harbor Laboratory.
Sekurova, O. N., Brautaset, T., Sletta, H., Borgos, S. E., Jakobsen, M. O., Ellingsen, T. E., Strom, A. R., Valla, S. & Zotchev, S. B. (2004). In vivo analysis of the regulatory genes in the nystatin biosynthetic gene cluster of Streptomyces noursei ATCC 11455 reveals their differential control over antibiotic biosynthesis. J Bacteriol 186, 13451354.
Sheldon, P. J., Busarow, S. B. & Hutchinson, C. R. (2002). Mapping the DNA-binding domain and target sequences of the Streptomyces peucetius daunorubicin biosynthesis regulatory protein, DnrI. Mol Microbiol 44, 449460.[CrossRef][Medline]
Sosio, M., Stinchi, S., Beltrametti, F., Lazzarini, A. & Donadio, S. (2003). The gene cluster for the biosynthesis of the glycopeptide antibiotic A40926 by Nonomuraea species. Chem Biol 10, 541549.[CrossRef][Medline]
Sosio, M., Kloosterman, H., Bianchi, A., de Vreugd, P., Dijkhuizen, L. & Donadio, S. (2004). Organization of the teicoplanin gene cluster in Actinoplanes teichomyceticus. Microbiology 150, 95102.[CrossRef][Medline]
Steffensky, M., Mühlenweg, A., Wang, Z.-X., Li, S.-M. & Heide, L. (2000). Identification of the novobiocin biosynthetic gene cluster of Streptomyces spheroides NCIB 11891. Antimicrob Agents Chemother 44, 12141222.
Stutzman-Engwall, K. J., Otten, S. L. & Hutchinson, C. R. (1992). Regulation of secondary metabolism in Streptomyces spp. and overproduction of daunorubicin in Streptomyces peucetius. J Bacteriol 174, 144154.[Abstract]
Thamm, S. & Distler, J. (1997). Properties of C-terminal truncated derivatives of the activator, StrR, of the streptomycin biosynthesis in Streptomyces griseus. FEMS Microbiol Lett 149, 265272.[CrossRef][Medline]
Thiara, A. S. & Cundliffe, E. (1988). Cloning and characterization of a DNA gyrase B gene from Streptomyces sphaeroides that confers resistance to novobiocin. EMBO J 7, 22552259.[Abstract]
Thiara, A. S. & Cundliffe, E. (1989). Interplay of novobiocin-resistant and -sensitive DNA gyrase activities in self-protection of the novobiocin producer, Streptomyces sphaeroides. Gene 81, 6572.[CrossRef][Medline]
Thorpe, H. M., Wilson, S. E. & Smith, M. C. (2000). Control of directionality in the site-specific recombination system of the Streptomyces phage C31. Mol Microbiol 38, 232241.[CrossRef][Medline]
Vara, J., Lewandowska-Skarbek, M., Wang, Y. G., Donadio, S. & Hutchinson, C. R. (1989). Cloning of genes governing the deoxysugar portion of the erythromycin biosynthesis pathway in Saccharopolyspora erythraea (Streptomyces erythreus). J Bacteriol 171, 58725881.[Medline]
Wang, Z.-X., Li, S.-M. & Heide, L. (2000). Identification of the coumermycin A1 biosynthetic gene cluster of Streptomyces rishiriensis DSM 40489. Antimicrob Agents Chemother 44, 30403048.
Wietzorrek, A. & Bibb, M. (1997). A novel family of proteins that regulates antibiotic production in streptomycetes appears to contain an OmpR-like DNA-binding fold. Mol Microbiol 25, 11811184.[CrossRef][Medline]
Wilson, D. J., Xue, Y., Reynolds, K. A. & Sherman, D. H. (2001). Characterization and analysis of the PikD regulatory factor in the pikromycin biosynthetic pathway of Streptomyces venezuelae. J Bacteriol 183, 34683475.
Xu, H., Heide, L. & Li, S. M. (2004). New aminocoumarin antibiotics formed by a combined mutational and chemoenzymatic approach utilizing the carbamoyltransferase NovN. Chem Biol 11, 655662.[CrossRef][Medline]
Received 30 September 2004;
revised 24 January 2005;
accepted 24 February 2005.
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
J MED MICROBIOL | ALL SGM JOURNALS |