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1 Pharmaceutical Biology, Pharmaceutical Institute, Eberhard-Karls-Universität Tübingen, Auf der Morgenstelle 8, 72076 Tübingen, Germany
2 Institute of Pharmacology and Toxicology, Department of Clinical Pharmacology, University Hospital Tübingen, 72076 Tübingen, Germany
Correspondence
Lutz Heide
heide{at}uni-tuebingen.de
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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A table of PCR primers used in this study is available as supplementary data with the online version of this paper.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) are potent inhibitors of DNA gyrase, thereby presenting interesting starting compounds for the development of new antibiotics by combinatorial biosynthesis and metabolic engineering (Flatman et al., 2006; Li & Heide, 2004, 2005). Their characteristic structural moiety is a 3-amino-4,7-dihydroxycoumarin unit. The same unit is also found in simocyclinone D8, an antibiotic which otherwise has a completely different structure than the aforementioned compounds (Fig. 1). Interestingly, simocyclinone D8 has also been identified as a potent inhibitor of gyrase (Flatman et al., 2005).
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; Pojer et al., 2002; Steffensky et al., 2000; Wang et al., 2000). Inactivation experiments as well as biochemical investigations allowed us to assign all ORFs contained in the novobiocin and the clorobiocin clusters to their functions in catalysis, regulation or self-resistance (Freitag et al., 2005; Li & Heide, 2004). The only gene of the clorobiocin cluster for which no functional assignment has been possible yet is the small ORF cloY.
As shown in Fig. 1
, cloY is situated downstream of the regulatory gene cloG (Eustáquio et al., 2005b) and upstream of the genes cloHIJK which encode enzymes involved in 3-amino-4,7-dihydroxycoumarin biosynthesis (Chen & Walsh, 2001; Pacholec et al., 2005). A similar arrangement of genes is found in the coumermycin A1 and the novobiocin gene clusters. However, while the coumermycin A1 cluster contains an orthologue of cloY (i.e. couY), the novobiocin cluster does not contain such a gene. In the simocyclinone D8 cluster, no orthologue of the regulatory gene cloG is found, but orthologues of all other mentioned genes of aminocoumarin biosynthesis are found, including the small ORF simY with high sequence similarity to cloY (Fig. 1).
CloY, CouY and SimY are predicted proteins of 71, 71 and 70 aa, respectively. All three show high similarity to the mbtH gene in the gene cluster for mycobactin, a non-ribosomally formed peptide siderophore from Mycobacterium tuberculosis (Quadri et al., 1998). Genes with high similarity to mbtH have been identified in many biosynthetic gene clusters for antibiotics and siderophores from bacteria. However, the function of these genes is unknown, and a recent publication on the gene cluster for the siderophore ornibactin aptly concluded that ‘the role of the mbtH-like proteins remains a mystery’ (Agnoli et al., 2006).
In a study using transposon mutagenesis in the biosynthetic gene cluster of the siderophore vicibactin in Rhizobium leguminosarum, insertion mutants of the mbtH-like gene vbsG were obtained. vbsG is the first gene of an operon which also comprises the NRPS gene vbsS and the mono-oxygenase gene vbsO. The latter enzyme has been suggested to hydroxylate ornithine to N5-hydroxyornithine (Carter et al., 2002). Vicibactin formation was abolished in the vbsG insertion mutant, but since the transposon insertion may also have affected the expression of vbsS and vbsO, this does not represent unequivocal proof for an essential function of vbsG in vicibactin biosynthesis.
Only a single experimental study has specifically addressed the function of an mbtH-like gene: Stegmann et al. (2006) recently reported the inactivation of the mbtH-like gene ORF1 from the biosynthetic gene cluster of the glycopeptide antibiotic balhimycin. The inactivation had no effect on balhimycin biosynthesis, and the authors concluded that ‘it can be assumed that MbtH-like proteins do not have an essential role in the specific secondary metabolite biosynthesis’. However, they also mentioned the possibility that mbtH-like genes situated elsewhere in the genome may take over the role of ORF1 in balhimycin biosynthesis.
We speculated that the strict conservation of mbtH-like genes throughout many secondary metabolic gene clusters of different bacterial taxa may suggest the existence of an evolutionary pressure favouring the maintenance of this ORF, and we therefore decided to investigate the importance of cloY for clorobiocin biosynthesis experimentally.
We have developed methods for the heterologous expression of the clorobiocin biosynthetic gene cluster in Streptomyces coelicolor M512 (Eustáquio et al., 2005a). These methods not only allow the convenient investigation of gene replacements or in-frame deletions within the biosynthetic gene cluster, but also the expression of the modified clusters in a completely sequenced host and the rapid modification of any gene of interest in the host genome. Using these methods, we could now show that cloY is indeed required for efficient clorobiocin biosynthesis, specifically for the formation of the aminocoumarin moiety, and that it can be functionally replaced by various other mbtH-like genes. This is the first study proving the functional importance of members of the mbtH-like gene family.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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redD actII-ORF4 SCP1– SCP2–) was kindly provided by E. Takano (Gröningen, The Netherlands) and J. White (Norwich, UK), and routinely cultured in 50 ml Trypticase Soy Broth (30 g l–1) at 30 °C and 200 r.p.m. for 2 days. For the production of clorobiocin and novobiocin, a 3 ml sample from TSB cultures was precultured in 300 ml baffled flasks containing 50 ml corn starch medium (Kieser et al., 2000). After growth for 2 days at 30 °C and 180 r.p.m., 5 ml of the preculture was inoculated into 300 ml baffled flasks containing 50 ml production medium (Eustáquio et al., 2005a) and cultivated at 30 °C and 180 r.p.m. for 7 days. For feeding experiments, 2 mg 3-amino-4,7-dihydroxy-8-methylcoumarin dissolved in 100 % ethanol was added to 50 ml cultures 2 days and again 4 days after inoculation. After cultivation for a further 3 days, cultures were harvested and analysed for secondary metabolite formation.
Escherichia coli strains, as listed in Table 1, were cultured in liquid or on solid Luria–Bertani medium at 37 °C (Sambrook & Russell, 2001). E. coli BW25113 carrying the temperature-sensitive recombination plasmid pIJ790 was cultivated in SOB medium at 30 °C (Gust et al., 2004).
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DNA isolation, manipulation and cloning.
Standard procedures for DNA isolation and manipulation were performed as described previously (Sambrook & Russell, 2001). Isolation of DNA fragments from agarose gels and purification of PCR products were carried out with the NucleoSpin 2 in 1 Extract Kit (Macherey–Nagel). Genomic DNA isolation from Streptomyces strains, protoplast transformation and regeneration were carried out according to the procedures described by Kieser et al. (2000). REDIRECT technology for PCR targeting was obtained from Plant Bioscience Limited and used for inactivation experiments according to the PCR targeting protocol (Gust et al., 2003).
Generation of mbtH mutants
Construction of cloMW15 and cloMW16.
cloBG01 is a SuperCos1-based cosmid containing the intact clorobiocin biosynthetic gene cluster and the attP/int cassette from the bacteriophage 
C31 for stable integration into the S. coelicolor M512 host genome (Eustáquio et al., 2005a) (Fig. 2a). An apramycin resistance cassette [aac(3)IV] was amplified from plasmid pUG019 (Eustáquio et al., 2005a) using primers cloYF_Xba and cloYR_Spe (primer sequences are given in Table S1, available as supplementary data with the online version of this paper). The primers contained restriction sites for XbaI and SpeI for removal of the cassette. The resulting 1077 bp PCR product was used to replace the cloY gene from cosmid cloBG01 by using 
RED-mediated recombination as described previously, resulting in cosmid cloMW15 (Gust et al., 2003) (Fig. 2b).
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).
Deletion of the mbtH-like homologues in S. coelicolor.
For deletion of the mbtH orthologues from S. coelicolor, a 1576 bp gene disruption cassette, containing the viomycin resistance marker vph and oriT for conjugal transfer into Streptomyces strains, was amplified from plasmid pIJ780 using primers CDA_Spe_F and CDA_Xba_R (Table S1). This fragment was used to replace the gene SCO3218 (CDA-ORFX) in cosmid StE8 (kanamycin-resistant) (Redenbach et al., 1996) using the RED-mediated recombination system for PCR targeting (Gust et al., 2003), resulting in cosmid StE8CDA-ORFX (Fig. 3a).
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) as described above, resulting in StF76cchK (Fig. 3b).
After electroporation of cosmid StE8CDA-ORFX into E. coli ET12567/pUZ8002 for conjugal transfer into Streptomyces, recombinant E. coli strains carrying StE8CDA-ORFX were selected using 30 mg viomycin l–1. Conjugal transfer of the cosmid into S. coelicolor M512 and screening for double cross-over mutants (viomycin-resistant, kanamycin-sensitive) was carried out as described previously (Freitag et al., 2004), resulting in the CDA-ORFX mutant S. coelicolor MW01. Subsequently, cosmid StF76cchK was introduced into S. coelicolor MW01 by conjugation, using apramycin (50 µg ml–1) and viomycin (30 µg ml–1) for selection. Screening for double cross-over mutants (apramycin- and viomycin-resistant; kanamycin-sensitive) resulted in the CDA-ORFXcchK double mutant S. coelicolor MW03.
Construction of plasmids for complementation of S. coelicolor MW01 and S. coelicolor MW03.
For complementation of the mbtH S. coelicolor mutants, the genes cloY, SCO0489 (cchK), SCO3218 (CDA-ORFX) and couY were amplified by PCR using the primer pairs cloY_Hind_F and cloY_Spe_R, cchK_Hind_F and cchK_Spe_R, CDA_Hind_F and CDA_Spe_R, couY_Hind_F and couY_Spe_R, respectively (primer sequences are given in Table S1, available as supplementary data with the online version of this paper).
The PCR products were purified using the Nucleospin Kit (Macherey–Nagel), incubated with HindIII and SpeI, and subsequently ligated into the HindIII/SpeI sites of expression vector pUWL201 (Doumith et al., 2000). This resulted in plasmids pMW01, pMW02, pMW03 and pMW04, respectively. DNA sequencing of these plasmids using the sequencing primer ermE_seq confirmed the correct sequence of all constructs. For protoplast transformation, the four plasmids were transferred into the non-methylating E. coli strain ET12567 and DNA was isolated by standard procedures after growth at 37 °C overnight. Transformation of S. coelicolor strains was carried out by polyethylene glycol-mediated protoplast transformation (Kieser et al., 2000). Selection of recombinant strains was performed on DNA solid medium containing 8 µg thiostrepton ml–1.
Analysis of secondary metabolites.
Standard procedures for isolation of clorobiocin and novobiocin were used as described previously (Eustáquio et al., 2003a, 2005a). A 1 ml sample of bacterial culture from production medium was acidified to pH 4 with HCl and extracted twice with an equal volume of ethyl acetate. After centrifugation and separation of the organic phase, the solvent was evaporated and the residue was dissolved in 200 µl methanol. Metabolites were analysed by HPLC using a Multosphere RP 18-5 column (250x4 mm, 5 µm; C+S Chromatographie Service, Düren, Germany) at a flow rate of 1 ml min–1 using a linear gradient from 40 to 100 % of solvent B in solvent A over 37 min (solvent A, MeOH/H2O/HCOOH, 50 : 49 : 1; solvent B, MeOH/HCOOH, 99 : 1) with detection for clorobiocin at 340 nm.
For HPLC analysis of novclobiocin 102, a linear gradient from 60 to 100 % of solvent B in solvent A (solvent A, H2O/HCOOH, 99 : 1; solvent B, MeOH/HCOOH, 99 : 1) and detection at 305 nm were used (Eustáquio et al., 2003b). Authentic clorobiocin or novobiocin (Aventis) was used as the standard.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). For the inactivation of cloY, an apramycin resistance cassette was amplified by PCR and used to replace the entire ORF of cloY in cosmid cloBG01, resulting in cosmid cloMW15 (Fig. 2b).
To avoid a possible polar effect, the cassette was removed by XbaI/SpeI digestion and religation as described previously (Eustáquio et al., 2005a). As shown in Fig. 2(b), the resulting cosmid, cloMW16, carries only a minimal 18 bp ‘scar’ sequence, situated in between the original start and stop codon of cloY.
The cloY replacements within cloMW15 and cloMW16 were verified both by restriction analysis and PCR. Transfer of cloMW16 into S. coelicolor M512 was carried out by conjugation. Three individual Streptomyces exconjugants were selected, and the integration of cloMW16 was verified by PCR with chromosomal DNA.
Subsequently, both S. coelicolor (cloBG01) and the cloY strain S. coelicolor (cloMW16) were cultured and analysed for clorobiocin formation by HPLC (Fig. 4a). The strain carrying the intact cluster produced 54 mg clorobiocin l–1, while the cloY deletion mutants produced on average 12 mg l–1.
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; Li & Heide, 2005). The present result, however, was remarkably inconclusive. It neither proved that cloY was required nor that it was unnecessary for clorobiocin production. Therefore, we considered the hypothesis that, after deletion of cloY, this gene may have been functionally replaced by other genes contained in the genome of the producer strain. For an investigation of this hypothesis, our heterologous expression system offers a distinct advantage: the complete genome sequence of S. coelicolor has been determined (Bentley et al., 2002) and very efficient methods for the genetic manipulation of this strain are available.
Deletion of mbtH homologues in S. coelicolor
An analysis of the Streptomyces coelicolor genome sequence revealed two ORFs that show high similarity to cloY, i.e. SCO3218 (CDA-ORFX) from the calcium-dependent antibiotic (CDA) gene cluster (Hopwood & Wright, 1983) and SCO0489 (cchK) from the coelichelin gene cluster (Challis & Ravel, 2000). These two genes show 68 and 62 % identity to cloY at the amino acid level, respectively. Both CDA-ORFX and cchK are annotated in the database as mbtH-like genes, but the function of these genes is unknown. CDA-ORFX was inactivated in S. coelicolor M512 (see Methods), resulting in the desired CDA-ORFX mutant S. coelicolor MW01. Three individual recombinants were isolated and the correct genotype was verified by PCR in all of them. Subsequently, cchK was inactivated in S. coelicolor MW01, resulting in the CDA-ORFXcchK double mutant MW03. The absence of the two mbtH-like genes in S. coelicolor MW03 was confirmed by PCR.
Cosmid cloMW16 was introduced into S. coelicolor MW03, resulting in the triple mutant S. coelicolor MW03(cloMW16), an integration mutant containing the cloY clorobiocin cluster in a host lacking all mbtH homologues. Three individual mutants of this strain were cultivated in clorobiocin production medium and secondary metabolite formation was analysed by HPLC. All mbtH triple mutants showed essentially negligible amounts of clorobiocin (Fig. 4a). While S. coelicolor M512(cloBG01) produced approximately 50 mg clorobiocin l–1, the mbtH triple mutants produced a mean of only 0.4 mg l–1. This demonstrated that deletion of all three mbtH-like genes from this heterologous clorobiocin producer reduced clorobiocin production by 99 % in comparison to the reference strain.
Complementation of the mbtH triple mutant with cloY, CDA-ORFX and cchK
To prove that the observed reduction of clorobiocin biosynthesis was indeed due to the inactivation of the three mbtH-like genes cloY, cchK and CDA-ORFX, we complemented the mbtH triple mutant by expression of intact copies of these three genes, each cloned into the expression vector pUWL201 (Doumith et al., 2000) (see Methods). The three plasmids were introduced into S. coelicolor MW03(cloMW16) and three independent transformants were obtained for each construct. Cultivation of the transformants in clorobiocin production medium and analysis of secondary metabolites by HPLC analysis showed that clorobiocin production was restored in all strains, reaching 8 mg l–1 with the cloY expression plasmid, 4.1 mg l–1 with the cchK plasmid and 5.1 mg l–1 with the CDA-ORFX plasmid (Fig. 4b). In contrast, a control strain transformed with the empty vector pUWL201 produced <0.5 mg l–1.
Therefore, complementation of the mbtH triple mutant with each of the three mbtH homologues resulted in an at least 10-fold increase of clorobiocin production in the mbtH triple mutant, showing for the first time that mbtH-like genes from different clusters and organisms can functionally replace each other. However, complementation did not fully restore clorobiocin production to the original level. This may be attributed to an inappropriate regulation of the transcription and translation of the mbtH-like genes from the multicopy vectors with constitutive promoters.
Complementation with couY from the coumermycin gene cluster
The cloY gene from the clorobiocin gene cluster shows 81 % identity at the amino acid level to couY from the gene cluster for the aminocoumarin coumermycin A1 (Fig. 1). To prove whether couY can also complement the mbtH triple mutant, we cloned couY into the expression vector pUWL201. After introduction of the resulting plasmid into S. coelicolor MW03(cloMW16) three independent transformants were selected and examined for clorobiocin production. Again, clorobiocin production was restored, reaching a level of 5.3 mg l–1 (Fig. 4b). Therefore, also couY can functionally replace cloY in clorobiocin biosynthesis.
Proof of the involvement of cloY in the formation of the 3-amino-4,7-dihydroxy-coumarin moiety
The experiments described above have shown that cloY is required for clorobiocin biosynthesis and that it can be functionally replaced by other mbtH orthologues. This raises the question whether it acts as a global regulator for the biosynthesis of clorobiocin, or whether it has a catalytic, regulatory or other function for the formation of a specific moiety of this antibiotic.
Since in coumermycin A1 and simocyclinone D8 gene clusters cloY and its orthologues couY and simY are localized in close proximity to the genes of 3-amino-4,7-dihydroxy-coumarin formation (Fig. 1), we speculated that the function of cloY may be related to the formation of this moiety. To test this hypothesis, we decided to feed the mbtH triple mutant, which carries the cloY-deficient clorobiocin cluster in a completely mbtH-deficient host strain, with the aminocoumarin moiety of novobiocin. This moiety carries a methyl group instead of a chlorine atom at position 8 of the 3-amino-4,7-dihydroxycoumarin molecule (Fig. 1). Previous experiments had shown that this compound was readily accepted by the enzymes of clorobiocin biosynthesis, resulting in a structural analogue of clorobiocin, i.e. novclobiocin 102, which differs from clorobiocin only by the methyl group instead of the chlorine (Eustáquio et al., 2003a, 2004).
A total of 2 mg 8-methyl-3-amino-4,7-dihydroxycoumarin was fed to a 50 ml culture of the mbtH triple mutant S. coelicolor MW03(cloMW16) (see Methods). After cultivation for 7 days, secondary metabolite formation was analysed by HPLC and HPLC-MS. Without feeding, very low formation of clorobiocin was observed again (0.4 mg l–1). After feeding, however, an accumulation of novclobiocin 102 was clearly observed, reaching 5.2 mg l–1. The identity of this compound was confirmed by LC-MS analysis in comparison with an authentic reference sample. Therefore, our experiment showed that deletion of mbtH specifically affected the formation of the aminocoumarin moiety of clorobiocin, while the biosynthesis of the other structural moieties and their linkage reactions were still possible in the mbtH triple mutant.
Heterologous expression of the novobiocin biosynthetic gene cluster in the mbtH double mutant
The biosynthetic gene clusters of clorobiocin, coumermycin A1 and simocyclinone D8 contain an mbtH-like gene (i.e. cloY, couY and simY, respectively), but no mbtH-like gene is found in the novobiocin gene cluster (Fig. 1). Novobiocin can be produced by heterologous expression of the novobiocin gene cluster in Streptomyces coelicolor M512, using the integrative cosmid novBG01 (Eustáquio et al., 2005a). However, given our observation that cloY was required for clorobiocin biosynthesis, this raised the question whether novobiocin biosynthesis really proceeds without involvement of an mbtH homologue, or whether mbtH-like genes encoded in the host genome cooperate with this process. To investigate this question we introduced the integrative cosmid novBG01 (Eustáquio et al., 2005a) by site-specific integration into the mbtH-double mutant strain S. coelicolor MW03. Four independent integration mutants were selected for further investigation, and the presence of the gene cluster for novobiocin biosynthesis was confirmed by PCR.
The mbtH+ strain S. coelicolor M512(novBG01) and the four mbtH double mutant strains of S. coelicolor MW03(novBG01) were cultured as described above and analysed by HPLC, using novobiocin as standard. The mbtH double mutants containing the novobiocin gene cluster still produced novobiocin (5.7 mg l–1), i.e. approximately 50 % of the amounts found in the mbtH+ strain S. coelicolor M512(novBG01) (12.5 mg l–1). These results show that novobiocin biosynthesis can take place even in the absence of mbtH-like genes, although it appears to be enhanced by the presence of an intact copy of cchK or CDA-ORFX.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). They are found in the biosynthetic gene clusters of peptide and aminocoumarin antibiotics and of siderophores from different bacterial taxa. Fig. 5 shows an alignment of the predicted amino acid sequences of 24 MbtH-like proteins from the gene clusters of well characterized compounds, including peptide and glycopeptide antibiotics, phosphinotricin, enterochelin, mycobactin and aminocoumarins. While mbtH genes mostly represent separate ORFs, there are three notable exceptions: the N terminus of the NikP1 protein of nikkomycin biosynthesis has obvious sequence similarity to MbtH-like proteins (Fig. 5). The enzyme NikP1 activates histidine by thioester formation, which is followed by 
-hydroxylation of the amino acid under catalysis of the cytochrome P450 enzyme NikQ (Chen et al., 2002). The reaction sequence catalysed by NikP1 and NikQ, i.e. activation and -hydroxylation of histidine, is very similar to the reactions catalysed by CloH and CloI in clorobiocin biosynthesis, i.e. activation and -hydroxylation of tyrosine (Chen & Walsh, 2001). The second example where MbtH forms the N-terminal part of a larger protein is LtxB from lyngbyatoxin biosynthesis (Edwards & Gerwick, 2004). The predicted amino acid sequence of LtxB shows similarity to cytochrome P450 enzymes. This protein has been suggested to catalyse an epoxidation of tryptophan during the non-ribosomal biosynthesis of the dipeptide lyngbyatoxin. The third example is the mbtH orthologue from the bleomycin cluster, but no functional data are available on this gene.
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, shows a remarkable sequence conservation, most obvious from the three conserved tryptophan residues. In many predicted proteins there is a conservation of the sequence X2–4NPF(D/E)(D/N)X2GX(F/Y)LVLVNXEXQHSLWPXFAXVPXGW(T/R)X7RX(D/E)CLX(Y/F)V(E/N)X2WTDXRPXSLX2–12, or of similar amino acids. This strongly indicates the existence of an evolutionary pressure which favours the maintenance of this sequence, suggesting a definite function of the mbtH-like genes. But at present, neither database comparisons nor experimental data can suggest what this function may be. As for aminocoumarin antibiotic biosynthesis, the different enzymic steps for enterochelin formation (Crosa & Walsh, 2002), phosphinotricin formation (Schwartz et al., 2004) and glycopeptide antibiotic formation (Sosio et al., 2003) have been investigated in considerable detail in vitro, without revealing an essential catalytic function which may be assigned to the MbtH proteins encoded in the respective clusters. Therefore, it has been speculated that MbtH-like proteins may be involved in regulatory or transport processes (Yeats et al., 2003) or in protein–protein interactions (Stegmann et al., 2006), but no experimental support has been provided for these hypotheses.
The biosynthetic pathways of all compounds mentioned in the alignment in Fig. 5 involve the activation of amino acids, or of aromatic acids, by thioester formation with the 4'-phosphopantetheinyl cofactor of an enzyme, often followed by chemical modification of the activated acid by other enzymes and its subsequent transfer to amino or hydroxyl groups, forming peptide or ester bonds. Clorobiocin biosynthesis comprises two such reaction sequences: the activation and -hydroxylation of tyrosine by CloH and CloI, which are the first steps of the biosynthesis of the aminocoumarin moiety (Chen et al., 2002; Pacholec et al., 2005), and the activation of proline and its subsequent oxidation to pyrrole-2-carboxyl-S-CloN5, catalysed by CloN3, CloN4 and CloN5 (Garneau et al., 2005). Our study showed that feeding of an intact aminocoumarin moiety to a heterologous clorobiocin producer strain, defective in all three mbtH-like genes, restored formation of the antibiotic to a similar level to that obtained by complementation with cloY or other mbtH orthologues. This indicates that, except for the formation of the aminocoumarin moiety, all other biosynthetic steps were intact in the mbtH-defective mutant, including biosynthesis and transfer of the pyrrole-2-carboxyl moiety. In contrast, earlier inactivation experiments of genes involved in the biosynthesis and transfer of the pyrrole-2-carboxyl moiety invariably led to strains accumulating novclobiocin 102, i.e. a clorobiocin derivative lacking that moiety (Freitag et al., 2005; Xu et al., 2003). Therefore, we can conclude that cloY is required for the efficient formation of the aminocoumarin moiety of clorobiocin rather than for the formation or transfer of the pyrrole-2-carboxyl moiety. However, we cannot define whether cloY is involved in catalysis, in protein–protein interaction or in the regulation of transcription and translation.
The inactivation of cloY and two other mbtH-like genes in a heterologous producer of clorobiocin dramatically reduced clorobiocin formation. In contrast, Stegmann et al. (2006) found that inactivation of the mbtH-like gene ORF1 did not impair balhimycin biosynthesis. However, the authors mentioned that the genome of the balhimycin producer did contain at least two further mbtH-like genes, and they could not exclude the possibility that these genes may functionally replace ORF1 in their mutant. Our present results on clorobiocin biosynthesis suggests that different mbtH-like genes can functionally replace each other. Therefore, it will be necessary to inactivate all mbtH-like genes in the balhimycin producer strain before a conclusion can be drawn whether or not these genes are required for balhimycin biosynthesis. Any further investigation on the role of mbtH-like genes in vivo must consider the contribution of all copies of this gene family in the genome and should therefore be carried out only in completely sequenced strains.
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ACKNOWLEDGEMENTS |
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Edited by: D. M. Gordon
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Received 4 October 2006;
accepted 17 January 2007.
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