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1 Department of Chemistry, University of Warwick, Coventry CV4 7AL, UK
2 Institut de Génétique et Microbiologie, UMR CNRS 8621, Université Paris-Sud 11, 91405 Orsay Cedex, France
Correspondence
Gregory L. Challis
G.L.Challis{at}warwick.ac.uk
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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A table of oligonucleotide primers used in this study is available as supplementary data with the online version of this paper.
Present address: Institut de Génétique et Microbiologie, UMR CNRS 8621, Université Paris-Sud 11, 91405 Orsay Cedex, France.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Quadri et al., 1998). However, by no means all gene clusters encoding non-ribosomal peptide synthetase (NRPS) biosynthetic systems contain a gene encoding an MbtH-like protein and some MbtH-like proteins do not appear to be associated with NRPS gene clusters (e.g. NFA5500 from Nocardia farcinica). The MbtH-like family is rapidly expanding and searches of protein sequence databases indicate that about 130 members have been identified thus far, all in bacteria. Interestingly, no MbtH-like proteins have been identified in fungal NRPS gene clusters.
In a few cases, it has been shown that genes encoding MbtH homologues are cotranscribed with other genes encoding biosynthetic proteins. Thus, in the vicibactin gene cluster, vbsG (an mbtH-like gene) is cotranscribed with vbsS (an NRPS gene) (Carter et al., 2002). The ybdZ (mbtH-like) gene in the Escherichia coli enterochelin biosynthetic gene cluster is cotranscribed with the fes and entF genes encoding enterochelin esterase and an NRPS, respectively (Pettis & McIntosh, 1987), and mbtH from the peptidoglycolipid biosynthetic gene cluster of Mycobacterium smegmatis is cotranscribed with two NRPS-encoding genes (Sondén et al., 2005).
Two studies have examined whether a gene encoding an MbtH-like protein that is clustered with an NRPS-encoding gene is required for metabolite biosynthesis in vivo. In the first study, interruption of the vbsG gene within the vicibactin biosynthetic gene cluster by transposon insertion abrogated vicibactin production (Carter et al., 2002). vbsG is the first gene of an operon containing the vbsS gene encoding the vicibactin NRPS. Therefore, this observation could stem from a polar effect on vbsS expression. However, insertions of Tnlac transposons are not normally polar on downstream genes. In a more recent study, Wohlleben and coworkers showed that an in-frame deletion of orf1 of the balhimycin biosynthetic gene cluster (encoding a homologue of MbtH) does not abolish balhimycin production in Amycolatopsis balhimycina (Stegmann et al., 2006). This seems to indicate that Orf1 is not required for balhimycin biosynthesis, although the authors do not exclude the possibility of complementation by the other mbtH homologues (encoding AmyBal2 and AmyBal3) present in the genome of A. balhimycina.
Streptomyces coelicolor possesses two homologues of MbtH. The first one, CchK, is encoded by a gene within the cch cluster that directs coelichelin biosynthesis (Fig. 1a). Coelichelin is a peptide siderophore assembled by an unusual NRPS system encoded by cchH and cchJ (Lautru et al., 2005). The second S. coelicolor MbtH homologue is CdaX, encoded by a gene within the calcium-dependent antibiotic (CDA) biosynthetic gene cluster (Fig. 1b) that directs NRPS-mediated assembly of an anionic lipopeptide (Hojati et al., 2002).
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. Escherichia coli and Micrococcus luteus strains were grown in LB medium supplemented as necessary with appropriate antibiotics. Genetic manipulations of S. coelicolor and spore stock preparations were carried out on SFM medium (Kieser et al., 2000). Oxoid nutrient agar was used for CDA bioassays and HPLC analyses of coelichelin production were carried out on culture supernatants of S. coelicolor strains grown for 4 days at 30 °C in the dark in an iron-deficient liquid medium (Müller & Raymond, 1984).
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; Kieser et al., 2000). The Expand high-fidelity DNA polymerase kit (Roche) was used to amplify cchK and cdaX from cosmids SCF34 and SCE8, respectively, and the oriT-aac(3)IV cassette from pIJ773. PCR reactions for verification of the replacement or deletion of genes in S. coelicolor were carried out using Taq polymerase from Fermentas. DNA fragments were purified from solution or agarose gels using the Qiagen Qiaquick gel extraction kit. S. coelicolor chromosomal DNA for mutant analyses was obtained using the FastDNA Spin kit for soil from Q-biogene.
Disruptions of cchK and cdaX.
PCR-based REDIRECT technology was used to construct the cchK and cdaX mutants (Gust et al., 2004). In the SCF34 and SCE8 cosmids from the S. coelicolor ordered genomic library, the cchK and cdaX genes, respectively, were replaced with the oriT-aac(3)IV cassette from pIJ773. The oligonucleotides used for the replacements were as follows (bases identical to regions flanking cchK and cdaX, respectively, are underlined): for cchK, 5'-TTAGGCGAGCCTAACCTAATCCACTGGGAGGTACCGGGTATTCCGGGGATCCGTCGACC-3' (forward primer) and 5'-AGTTGGGAGTTCACGGGCGACGCTTGACGGGGCTCGGCCTGTAGGCTGGAGCTGCTTC-3' (reverse primer); and for cdaX, 5'-GAGTCTCCAGCCCGACGCTCCCGGAAGGAATGCGACGTGATTCCGGGGATCCGTCGACC-3' (forward primer) and 5'-CCCCCTGCCGGGGACGTACGGGCCGTCGTGGTCCGGTCATGTAGGCTGGAGCTGCTTC-3' (reverse primer).
The mutagenized cosmids (SCF34-cchK : : aac and SCE8-cdaX : : aac) were introduced into S. coelicolor M145 by conjugation from E. coli ET12567 containing pUZ8002, selecting for apramycin resistance. The primary transconjugants were screened for kanamycin sensitivity resulting in the double cross-over mutants W7 and W9. The desired gene replacements were confirmed in these mutants by PCR and sequencing using the following oligonucleotides: for cchK, 5'-CACGGCAGTTGGGAGTTCAC-3' (forward primer) and 5'-CCGGAAGACTAAGCTCATCG-3' (reverse primer); and for cdaX, 5'-GGCGGGATGCGCTTTAAGTG-3' (forward primer) and 5'-GGAAGGAAAGACGGTCTCAG-3' (reverse primer).
To construct the cchK and cdaX double mutant, an in-frame cchK deletion mutant was first created. The SCF34-cchK : : aac cosmid was introduced into E. coli BT340 to excise the disruption cassette, resulting in SCF34-
cchK. To introduce this cosmid into S. coelicolor M145 by conjugation, it was re-engineered to replace the neo gene in the superCos1 vector backbone with the oriT-aac(3)IV cassette, as described by Barona-Gómez et al. (2004), yielding SCF34-cchK-aac. After conjugal transfer of this plasmid from E. coli ET12567/pUZ8002 to S. coelicolor M145, single cross-over recombination events were first selected for with apramycin. After a round of non-selective growth, potential double cross-over recombinants were identified by their sensitivity to apramycin. PCR using the same oligonucleotides as for W7 was used to identify a double cross-over mutant W8 from this pool of mutants.
cdaX in S. coelicolor W8 was disrupted using the same procedure for construction of S. coelicolor W9, resulting in the double cchK– and cdaX– mutant W10.
Complementation of W10.
The two genes cchK and cdaX were cloned into the E. coli/Streptomyces shuttle vector pUWL201 under the control of the ermE* promoter. To ensure good translation initiation, a 17 bp long sequence corresponding to the one immediately upstream of the initiation codon in the expression vector pIJ6021 (Takano et al., 1995) was introduced into the forward primers. The genes were amplified by PCR using the following oligonucleotides: for cchK, 5'-GGGGGAAGCTTGAGAAGGGAGCGGACATATGAGCACCAACCCCTTC-3' (forward primer, HindIII site underlined) and 5'-TTTTTACTAGTTCAGGCGTCCGCGGTCCG-3' (reverse primer, SpeI site underlined); and for cdaX, 5'-GGGGGAAGCTTGAGAAGGGAGCGGACATATGACCAATCCGTTCGAAGA-3' (forward primer, HindIII site underlined) and 5'-GGGTTACTAGTTCAGTTGCCGGTGCTCAT-3' (reverse primer, SpeI site underlined).
PCR products were first cloned into the pGEM-T Easy vector, yielding pSL73 and pSL74. After sequencing of the inserts, the HindIII–SpeI fragments from these plasmids were subcloned into HindIII/SpeI digested pUWL201 vector (resulting in plasmids pSL75 and pSL76). The plasmids were introduced separately into S. coelicolor W10 by protoplast transformation.
HPLC analyses.
A 1 M FeCl3 solution (20 µl) was added to 50 ml culture supernatants of S. coelicolor strains M145, W7, W8, W9, W10, W10/pSL75 and W10/pSL76 grown in iron-deficient medium, to form the ferricoelichelin complex. Supernatants were concentrated using a rotary evaporator and the residues were redissolved in the minimum amount of water. Samples were filtered through a Vivaspin 0.5 ml concentrator (10 000 Da molecular mass cut-off) and analysed on a Supelco Discovery HSF5 column (150x4.6 mm, 5 µm, column temperature 20 °C) using an Agilent 1100 HPLC instrument equipped with a binary pump. Isocratic elution was carried out at 1 ml min–1 with 1 : 9 10 mM ammonium carbonate (pH 7.0)/MeOH. The ferricoelichelin complex was detected by monitoring A435 (Lautru et al., 2005). Coelichelin production was evaluated in wild-type S. coelicolor and the W7 mutant by quantification of the peak area and expressed as mAU (g wet cells)–1.
CDA bioassay.
Production of CDA was detected using the bioassay adapted from Kieser et al. (2000). Patches of strains to be tested were grown on Oxoid nutrient agar (ONA) containing 200 µM of the iron chelator 2,2'-dipyridyl. In addition, S. coelicolor W9 was grown on ONA with 100 µM FeCl3 (without 2,2'-dipyridyl). After 2 days incubation at 30 °C, each plate (20 ml) was overlaid with 2.5 ml soft nutrient agar containing the indicator strain Micrococcus luteus, FeCl3 to a final concentration of 300 µM and Ca(NO3)2 to a final concentration of 15 mM. In the overlay of the control plate, Ca(NO3)2 was omitted. Inhibition of growth of M. luteus was observed after overnight incubation at 37 °C.
Transcriptional analysis.
The S. coelicolor M145 and W10 strains were grown on cellophane sheets placed on plates of ONA medium containing 200 µM 2,2'-dipyridyl. After 48 h growth at 30 °C, the mycelium was collected and RNA was extracted using the method described by Oh & So (2003). The purified RNA samples were treated with DNase (Ambion) followed by extraction twice with phenol and once with chloroform. Oligonucleotides were designed to amplify gene fragments of about 400 bp (see Table S1 available as supplementary data with the online version of this paper) from the genes sco3210, sco3216, sco3219, cdaR, absA2, cdaPS1, cdaPS2 and fabH4 for the cda gene cluster, and cchA, cchB, cchG, cchH and cchJ for the cch gene cluster. They were first tested by carrying out PCR using wild-type S. coelicolor M145 genomic DNA and then used in PCRs with DNase-treated RNA samples from S. coelicolor M145 and W10 to ensure that no DNA was left in the preparations. The RT-PCR reactions were carried out using the One step RT-PCR kit (Qiagen) on 1 µg RNA using the following conditions: 50 °C for 30 min, 95 °C for 15 min, then 25 cycles of 95 °C for 45 s, 54 °C for 45 s and 72 °C for 40 s, and finally 10 min at 72 °C. RT-PCR products were analysed on a 1 % agarose gel by electrophoresis.
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RESULTS |
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), a cytochrome P450-encoding gene in the lyngbyatoxin biosynthetic gene cluster (LtxB, AAT12284) (Edwards & Gerwick, 2004) and a biotin carboxylase-encoding gene (Plu1218, CAE13512) in Photorhabdus luminescens. In the bleomycin gene cluster, orf13, encoding an MbtH homologue, has a C-terminal extension of about 100 aa showing no similarities to known proteins (Du et al., 2000).
A sequence alignment of selected MbtH-like proteins is shown in Fig. 2. Sequence alignment of all members of this protein family reveals an overall high degree of sequence conservation among the various MbtH homologues. In particular, CchK and CdaX share 74 % amino acid identity and 83 % similarity. The two tryptophan residues W36 and W56 (CchK numbering), separated by 19–27 amino acid residues, are completely conserved in all members of the family. Removing the sequences of the MbtH homologues that are fused to other proteins (especially Plu1218, but also NikP1) allows two new universally conserved residues, W26 and S24, to be identified. This suggests that MbtH homologues fused to other proteins may no longer be functional. Among other well conserved residues are G35 (conserved in all homologues except VbsG from Rhizobium etli CFN 42; conserved in VbsG from Rhizobium leguminosarum), N18 (conserved in all but three homologues) and P61 (conserved in all but four homologues). Finally, MbtH homologues seem to possess two regions rich in residues with acidic side chains (Fig. 2).
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). Thus any polar effects on the expression of other cch genes in this mutant are excluded. HPLC analysis of coelichelin production by the mutant W7 showed that coelichelin is still produced (Fig. 3b), although a clear reduction of coelichelin titre is observed (about 2.5-fold from a mean of five measurements) compared to coelichelin production by the wild-type strain M145.
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), suggesting the involvement of CdaX in CDA biosynthesis.
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).
cchK or cdaX is required for coelichelin and CDA biosynthesis
The W7 (cchK–) mutant still produced coelichelin and the W9 (cdaX–) mutant still produced CDA under appropriate conditions. The results reported above suggested that this was probably due to complementation of the cchK mutation by cdaX and vice versa. Consequently, we undertook the construction of a cchK/cdaX double knockout mutant. A cchK in-frame deletion mutant was constructed first, and cdaX was then replaced in this mutant with the oriT-aac(3)IV cassette used for construction of the W9 mutant to create S. coelicolor W10. HPLC analysis of this double mutant showed that coelichelin production was completely abolished (Fig. 3d). CDA bioassays carried out on ONA medium containing 200 µM 2,2'-dipyridyl indicated that W10 did not produce any CDA either (Table 2).
In trans complementation of S. coelicolor W10 with cchK and cdaX
To confirm that the abolition of coelichelin and CDA production in the W10 mutant was due to the disruption of cchK and cdaX, respectively, both genes were separately reintroduced into this strain. The cchK and cdaX genes were cloned into pUWL201, an E. coli/Streptomyces shuttle vector containing a Streptomyces origin of replication under the control of the strong constitutive ermE* promoter. To ensure good translation, an artificial ribosome-binding site was introduced into the forward PCR primer 7 bp upstream of the start codon of each gene. Production of coelichelin was restored in S. coelicolor W10/pSL75 (complementation with cchK, Fig. 3e) and production of CDA was restored in S. coelicolor W10/pSL76 (complementation with cdaX, Table 2) confirming that CchK and CdaX are involved in coelichelin and CDA biosynthesis, respectively. In addition, W10/pSL75 produced CDA and W10/pSL76 produced coelichelin, consistent with the hypothesis that CchK and CdaX can functionally complement each other.
Inactivation of cchK and cdaX does not abolish transcription of the cda or cch gene clusters
To examine whether CdaX and CchK are involved in the transcriptional regulation of the CDA and coelichelin biosynthetic gene clusters, RNA from the wild-type M145 strain and the W10 double mutant was extracted after 48 h growth on ONA medium containing 200 µM 2,2'-dipyridyl (to relieve ferrous iron-promoted repression of the cch cluster). Purified RNA was used to examine transcription of a number of genes from the cda (sco3210, sco3216, sco3219, cdaR, absA2, cdaPS1, cdaPS2 and fabH4) and cch (cchA, cchB, cchG, cchH and cchJ) gene clusters using RT-PCR. These genes, which are indicated in Fig. 1 and mostly belong to different transcriptional units, were chosen to span the entire clusters and include the NRPS-encoding genes. Fig. 4 shows that all the genes examined in the two clusters are transcribed in both strains, indicating that inactivation of cckK and cdaX has no major effect on the transcription of the cda and cch genes.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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), an analogous gene in the bahlimycin biosynthetic gene cluster has been reported not to be required for bahlimycin production in Amycolatopsis bahlimycinia (Stegmann et al., 2006). A possible explanation for the latter result is that other MbtH homologues encoded by genes outside the bahlimycin gene cluster in A. bahlimycinia are able to functionally replace the protein encoded by the gene within the bahlimycin cluster (Stegmann et al., 2006). Our results provide strong support for this hypothesis by demonstrating that CchK can function in place of CdaX and vice versa in the biosynthesis of CDA and coelichelin in S. coelicolor. We suggest that such MbtH-like protein-mediated cross-talk between non-ribosomal peptide biosynthetic pathways is likely to be a general phenomenon in bacteria containing multiple NRPS gene clusters. It is not immediately obvious what selective pressure maintains two genes encoding functionally interchangeable proteins in the genome of S. coelicolor. The cda cluster directs biosynthesis of an antibiotic and its expression is activated during the transition from exponential to stationary phase in S. coelicolor. On the other hand, expression of the cch cluster is activated by intracellular ferrous iron deficiency and results in production of the siderophore coelichelin which sequesters extracellular ferric iron and transports it into the cell (Barona-Gómez et al., 2006). Once iron homeostasis has been re-established by this iron uptake mechanism, the expression of this cluster should be repressed. Thus, the preservation of two genes encoding MbtH-like proteins in S. coelicolor probably stems from the differential regulation of the cda and cch clusters. If the cchK gene was absent from the genome, coelichelin-mediated iron acquisition could only occur during stationary phase. Similarly, if the cdaX gene was absent from the genome, CDA production could only occur in the stationary phase under conditions of iron deficiency. Our data speak to the importance of coelichelin for ferric iron acquisition during vegetative growth of S. coelicolor (Barona-Gómez et al., 2006).
Although our study demonstrates the requirement for CchK and CdaX for the biosynthesis of coelichelin and CDA in vivo in S. coelicolor, the role of these proteins in biosynthesis remains unclear. Another family of small proteins in Streptomyces species (the Wbl family) is involved in regulation, probably acting as transcriptional activators (Soliveri et al., 2000). Moreover, a small domain, the Myb-like DNA-binding domain, which contains three conserved tryptophans, is found in the Myb family of eukaryotic proteins, a family of transcriptional regulators. Thus, a role for MbtH homologues in transcriptional regulation, either direct or as a ‘helper’ of another regulatory protein, as recently identified for tylU in tylosin biosynthesis regulation (Bate et al., 2006), could be envisaged. However, our results show that neither CchK nor CdaX is required for transcription of either the cda or the cch gene clusters, suggesting that these proteins do not play a significant role in transcriptional regulation. A role in post-transcriptional regulation, however, cannot be ruled out. The E. coli enterochelin biosynthetic gene cluster contains a gene (ybdZ) encoding an MbtH-like protein. However, enterochelin biosynthesis in vitro can be reconstituted from EntB, EntE and EntF, suggesting that YbdZ may not play a significant role in this catalytic process (Gehring et al., 1998). It would therefore be interesting to examine whether YbdZ is required for enterochelin production in E. coli. Other possible roles for MbtH-like proteins include participation in export of the metabolites and the mediation of protein–protein interactions that are important for metabolite assembly in vivo. Small domains with conserved tryptophan residues in other proteins have been shown to mediate such protein–protein interactions: for example, the W2 domain (two conserved tryptophans separated by 22–30 aa), found at the C-terminal extremity of some eukaryotic initiation factors and the WW domain (two conserved tryptophans separated by 20–23 aa) that binds to proline-rich regions of some proteins.
While the precise function of MbtH-like proteins remains unclear, it seems likely that they play an important role in the production of many non-ribosomal peptide metabolites in bacteria. Future studies aimed at examining the role of these proteins in post-transcriptional regulation, metabolite export and in mediating protein–protein interactions between components of NRPS biosynthetic systems should help to elucidate their function. Structural studies of these proteins may also provide valuable insight into their mode of action.
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ACKNOWLEDGEMENTS |
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Edited by: D. M. Gordon
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Received 4 October 2006;
revised 8 January 2007;
accepted 17 January 2007.
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