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Keywords:

  • enzyme activation;
  • polyketide;
  • phosphopantetheine;
  • antibiotics;
  • kirromycin

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusions
  7. Acknowledgements
  8. Authors' contribution
  9. References
  10. Supporting Information

The main steps in the biosynthesis of complex secondary metabolites such as the antibiotic kirromycin are catalyzed by modular polyketide synthases (PKS) and/or nonribosomal peptide synthetases (NRPS). During antibiotic assembly, the biosynthetic intermediates are attached to carrier protein domains of these megaenzymes via a phosphopantetheinyl arm. This functional group of the carrier proteins is attached post-translationally by a phosphopantetheinyl transferase (PPTase). No experimental evidence exists about how such an activation of the carrier proteins of the kirromycin PKS/NRPS is accomplished. Here we report on the characterization of the PPTase KirP, which is encoded by a gene located in the kirromycin biosynthetic gene cluster. An inactivation of the kirP gene resulted in a 90% decrease in kirromycin production, indicating a substantial role for KirP in the biosynthesis of the antibiotic. In enzymatic assays, KirP was able to activate both acyl carrier protein and petidyl carrier domains of the kirromycin PKS/NRPS. In addition to coenzyme A (CoA), which is the natural substrate of KirP, the enzyme was able to transfer acyl-phosphopantetheinyl groups to the apo forms of the carrier proteins. Thus, KirP is very flexible in terms of both CoA substrate and carrier protein specificity. Our results indicate that KirP is the main PPTases that activates the carrier proteins in kirromycin biosynthesis.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusions
  7. Acknowledgements
  8. Authors' contribution
  9. References
  10. Supporting Information

Kirromycin, which is produced by the actinomycete Streptomyces collinus Tü 365, is a potent protein biosynthesis inhibitor that blocks translation by interfering with the bacterial elongation factor EF-Tu (Wolf & Zähner, 1972; Wolf et al., 1974). In previous studies, the kirromycin biosynthetic gene cluster was identified using a genetic screening approach (Weber et al., 2003). The antibiotic is synthesized via a combined cis-/trans-AT type I polyketide synthase (PKS)/nonribosomal peptide synthetase (NRPS) mechanism (Weber et al., 2008; Laiple et al., 2009). Both PKS and NRPS megaenzymes have a modular architecture where multiple partial reactions involved in the biosynthesis take place at specific enzymatic domains.

PKS acyl carrier protein (ACP) and NRPS petidyl carrier (PCP) domains within these modules require a post-translational activation by the attachment of a phosphopantetheinyl group to a conserved serine residue within the active site. This reaction is catalyzed by phosphopantetheinyl transferases (PPTases) that use coenzyme A (CoA) as a substrate. PPTases can be divided into the three classes described below (Mootz et al., 2001). The members of the first class of PPTases are usually found in primary metabolism where they are responsible for the activation of fatty acid ACPs, which also require phosphopantetheinylation for catalytic activity. Due to their homology to the Escherichia coli holo-(ACP) synthase ACPS, this class is denoted as ACPS-type PPTases. ACPS-type PPTases have a relatively high specificity towards their cognate carrier protein. PPTases of the second class are required for the activation of carrier protein domains of modular NRPS and PKS enzymes involved in secondary metabolism (Finking et al., 2002; Finking & Marahiel, 2004). Their prototype, Sfp, which is found in Bacillus subtilis, activates the surfactin synthetase PCP domains (Quadri et al., 1998). Sfp has little target specificity. Therefore, this enzyme is widely used for the in vivo and in vitro phosphopantetheinylation of a variety of different heterologously expressed PCP and ACP domains of many biosynthetic gene clusters (for a review, see Sunbul et al., 2009). In addition, Sfp can not only use the native CoA as a substrate but also acyl- or peptidyl-CoA derivatives. This property of Sfp can be used to generate acyl- or peptidyl-holo ACPs or PCPs in vitro, which then can be applied in synthetic biology applications (e.g. Vitali et al., 2003). Based on phylogenetic reconstruction and the occurrence of short sequence motifs, Sfp-type PPTases were further subdivided into the following two families: the F/KES family, which includes mainly PPTases involved in NRPS and siderophore synthesis, and the W/KEA family, the majority of which are involved in PKS, glycolipid or lysine biosynthesis (Copp & Neilan, 2006). The third class of PPTases is found in integral domains of yeast type I fatty acid synthases. These domains are required to activate the ACP encoded in the same polypeptide (Fichtlscherer et al., 2000).

In some cases, such as those of myxothiazol (Silakowski et al., 1999), surfactin (Nakano et al., 1992) or enterobactin (Coderre & Earhart, 1989), the biosynthetic gene clusters code for PPTases. Surprisingly, most gene clusters encoding PKS or NRPS biosynthetic pathways do not contain PPTase genes. Thus, the activation of their carrier protein domains must be carried out by enzymes that are encoded elsewhere in the genome. Such a spatial distribution is found, for example, in the cases of erythromycin (Weissman et al., 2004) and bleomycin biosynthesis (Sanchez et al., 2001). In the latter case, a PPTase, Svp, was identified, which has little substrate specificity for PKS and NRPS carrier proteins but a high specificity for CoA (Sanchez et al., 2001).

Although PPTase activity is essential for polyketide and nonribosomal peptide synthesis and the prototype PPTase Sfp is used routinely to convert apo CP to holo CP in vitro, only little is known about PPTases in other biosynthetic pathways. In the kirromycin biosynthetic gene cluster, a putative Sfp-type PPTase gene, kirP, was identified directly upstream of the kirromycin PKS/NRPS genes. In this work, the involvement and functional significance of kirP in the activation of the kirromycin PKS ACPs and NRPS PCPs was demonstrated using genetic and biochemical approaches.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusions
  7. Acknowledgements
  8. Authors' contribution
  9. References
  10. Supporting Information

Generation of the kirP gene replacement mutant EP-P1

The flanking regions of kirP and the thiostrepton resistance cassette were amplified by PCR using the primers denoted in Supporting Information,Table S1, and cloned into plasmid pA18 resulting in the gene inactivation vector pEP10. For detailed cloning procedure, see Supporting Information.

Transfer of pEP10 to wild-type S. collinus and selection of mutants were performed as described previously (Weber et al., 2008). One mutant, named EP-P1, in which the functional kirP was replaced by a thiostrepton resistance cassette, was obtained and checked by PCR and Southern hybridization. As a probe, the 671-bp internal fragment kirPint was amplified by PCR with the primers kirPint-5′ and kirPint-3′ and nonradioactively labeled using the Roche DIG PCR labeling kit.

Complementation of the mutant EP-P1 with native kirP

The complementation plasmid pEP11 and the empty vector pRM4 (Menges et al., 2007) (negative control) were transferred into wild-type S. collinus and mutant EP-P1 by intergeneric conjugation. The obtained complementation and control strains were tested for kirromycin production as described previously (Weber et al., 2008).

Expression and purification of KirP and the carrier proteins

To express KirP with N- and C-terminal His6-tags, the kirP gene was cloned into the vectors pET30 Ek/LIC (pMP02) and pET52 3C/LIC (pMP01), respectively. For the detailed cloning procedure, see Supporting Information. KirP expression was carried out in E. coli Rosetta2(DE3)pLysS (Novagen). For starting culture, the cells were grown in Luria–Bertani at 37 °C to an OD578 of 0.6. The main culture was inoculated with 10 vol% of a starting culture, induced immediately with 0.3 mM isopropyl-β-d-thiogalactopyranoside (IPTG) and grown for 16 h at 30 °C. The cells were harvested, treated with DNase I, protease inhibitors (Roche) and MgCl2, and passed twice through a French pressure cell at 1000 psi. The cell lysate was centrifuged for 1 h at 4 °C and 30 000 g. The proteins were purified using a Ni-NTA gravity flow column (IBA) in 50 mM Tris/HCl buffer (pH 7.6) containing 1 M NaCl and eluted with 300 mM imidazole in the same buffer. The purified proteins were concentrated using Amicon Ultra Centrifugal Filter Devices (Millipore) and dissolved in 50 mM Tris/HCl (pH 8) containing 25 mM NaCl, 10% glycerol and 20 mM dithiothreitol. Protein concentrations were determined using a BCA Protein Assay Kit (Thermo Scientific).

Expression plasmids for the carrier proteins KirAIIACP4, KirAIIACP5, KirAIIIPCP and KirBPCP were constructed as described in the Supporting Information. The carrier proteins were expressed in E. coli Rosetta2(DE3)pLysS at 23 °C by induction with 0.3 mM IPTG for 16 h. The purification of the proteins was performed as described above.

In vitro phosphopantetheinylation assays

To analyze the function of KirP, in vitro phosphopantetheinylation assays were performed. The reactions analyzed by MS contained 20 μM KirP, 100–150 μM acyl or peptidyl carrier protein (KirAIIACP4, KirAIIACP5, KirAIIIPCP or KirBPCP), 300 μM CoA (or malonyl-/methylmalonyl-CoA), 50 mM MgCl2 and 50 mM Tris/HCl (pH 7.5) in a total reaction volume of 50 μL. The assays were incubated for 1.5 h at 30 °C and then analyzed via HPLC-ESI-MS on a Reprosil Gold 300 C18 column 200 × 3 mm ID, 5 μm) in an Agilent HPLC-MS system. The analytes were separated by gradient elution as follows: (t0=40% B, t20=t35=100% B, Post-time 15 min 40% B; flow rate 500 μL min−1; injection volume 5 μL) using buffer A (0.1% formic acid) and buffer B (0.06% formic acid in methanol) as mobile phase. Mass spectra deconvolution was performed using the Zscore algorithm (Zhang & Marshall, 1998) implemented in magtran 1.03 (kindly provided by Dr Z. Zhang).

For autoradiographic analysis, the reaction mixtures contained 5 μM KirP, 30 μM acyl or peptidyl carrier protein, 12.5 mM MgCl2, 50 mM Tris/HCl (pH 7.5) and 7 μM [1,3-14C](methyl)malonyl-CoA (50 mCi mmol−1/0.1 mM Ci mL−1). As a control, assays were performed without KirP. The reactions were incubated for 30 min at 32 °C and then quenched with 800 μL of cold acetone. The proteins were centrifuged and redissolved in sample buffer. The samples were loaded onto and separated on a 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis gel. The proteins were then blotted onto a nitrocellulose membrane. The membrane was air-dried, and the signals were visualized by phosphorimaging with a GE phosphor screen.

Results and discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusions
  7. Acknowledgements
  8. Authors' contribution
  9. References
  10. Supporting Information

Bioinformatic analysis of KirP

The kirP gene is encoded directly upstream of the kirromycin PKS gene kirAI. Its product is similar to actinomycetal Sfp-type PPTases, having 56% identity and 65% similarity to the characterized PPTase Svp of Streptomyces verticillus (Sanchez et al., 2001). KirP contains all three conserved sequence motifs described by Lambalot et al. (1996) and Sanchez et al. (2001). Based on the presence of a conserved FSxKESLxK in motif P3 and its phylogenetic relationship to other PPTases, KirP can be assigned to the F/KES subfamily (Copp & Neilan, 2006) of Sfp-type PPTases.

Effects of the inactivation of kirP on kirromycin production

To analyze the role of KirP in vivo, kirP was inactivated by gene replacement. The gene replacement plasmid pEP10 was introduced into the wild-type strain S. collinus Tü 365. Homologous recombination resulted in the replacement of kirP with the thiostrepton resistance cassette of pEP10. The genotype of the resulting mutant strain, EP-P1, was confirmed by Southern analysis with a kirP probe (Fig. 1a and b). Extracts from wild-type and EP-P1 cultures were analyzed for kirromycin production by HPLC. The mutant strain showed a substantial reduction in kirromycin yield of approximately 90%. The identity of kirromycin was confirmed by comparison with an HPLC-UV/Vis spectra library (Fiedler, 1993) and by MS (m/z of kirromycin=795 [M-H]).

image

Figure 1.  (a) Genetic organization of the kirP region in wild-type Streptomyces collinus and the mutant strain EP-P1. The probe used for Southern blots is indicated by a box. (b) Southern blot of total genomic DNA from wild-type S. collinus (lanes 1 and 3) and EP-P1 (lanes 2 and 4) cut with BamHI (lanes 1 and 2) and MluI (lanes 3 and 4).

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To prove that the significant reduction in kirromycin yield is due to the inactivation of kirP, plasmid pEP11 expressing the intact wild-type kirP gene under control of the consitutive ermE* promoter was used to complement the inactivated kirP gene. The pEP11 construct was introduced into the mutant strain EP-P1. In the complemented strain, kirromycin production was partially restored, increasing by a factor of 3 compared with the mutant and reaching approximately 30% of the wild-type production level. Observations that gene replacement mutations in streptomycetes can be only partially complemented have been made in many pathways, for example daptomycin biosynthesis (Coeffet-Le Gal et al., 2006) when genes are deleted and subsequently reintroduced in a different context (for a review, also see Baltz, 1998).

The partial complementation of the kirP deletion in mutant EP-P1 indicated that the loss of kirP activity was responsible for the large decrease in kirromycin production and thus that kirP plays an important role in the biosynthesis of kirromycin.

However, the kirP gene replacement mutant was viable and produced low amounts of kirromycin. This finding implies that the genome of the producer strain S. collinus Tü 365 includes additional PPTase genes. Indeed, analysis of preliminary data of an ongoing whole genome sequencing project of S. collinus enabled the identification of at least six additional Sfp-type PPTase genes and one ACPS-type PPTase gene in the genome of the kirromycin producer strain. Thus, one or more of these enzymes might provide some phosphopantetheinylation of the kirromycin PKS/NRPS enzyme, albeit with a much lower efficiency than KirP, as indicated by the 90% drop in kirromycin yield in the kirP deletion mutant EP-P1.

In vitro analysis of KirP

The effects of kirP inactivation indicate that KirP plays a functional role in kirromycin biosynthesis. To characterize the enzyme biochemically, KirP was overexpressed in and purified from the E. coli strain Rosetta2(DE3)pLysS for use in in vitro studies. The kirP gene was amplified from cosmid 6O07 using PCR and cloned into the expression vector pET52, yielding the plasmid pMP01, which allows expression of KirP as a fusion protein with a His6-tagged C-terminus. For the expression of KirP as N-terminal His6-tag fusion protein, kirP was introduced into pET30, yielding pMP02.

The analysis of all cellular proteins in IPTG-induced cells showed that KirP was almost completely insoluble under all conditions tested when it was expressed with a C-terminal His6-tag. The expression of KirP in pET-30 with an N-terminal His6 tag (pMP02) led to an increase of soluble protein, which could be purified via affinity chromatography on Ni-NTA agarose.

Because the kirP gene is localized in the kirromycin biosynthetic gene cluster, the cognate substrates of KirP are likely the carrier proteins of the kirromycin PKS/NRPS. For this reason, we chose the following four carrier proteins as substrates to test the PPTase activity of KirP: the ACPs of the PKS modules 4 and 5 (KirAIIACP4 and KirAIIACP5) and two PCPs, KirAIIIPCP and KirBPCP, which are located in NRPS modules 6 and 16, respectively. KirAIIACP4 (pEM4ACP4) and KirAIIACP5 (pEM5ACP5) were expressed with C-terminal His6-tags in pET52, and KirAIIIPCP (pMP03) and KirBPCP (pMP04) were expressed in pET30 as fusion proteins with N-terminal His6-tags. All carrier proteins were obtained in soluble forms and purified on Ni-NTA agarose.

KirP and the four carrier proteins were then used in in vitro phosphopantetheinylation assays. To test the PPTase activity, each carrier protein was incubated with KirP and CoA, and the reaction was then analyzed by HPLC-ESI-MS.

In a control reaction (KirAIIACP5 without KirP), only the mass of the KirAIIACP5 apo form (16 248.7 Da) was detected by MS (Table 1). Addition of KirP to the reaction mixture led to the formation of the KirAIIACP5 holo form (16 589.6 Da). This form corresponds to a mass shift of 340 Da, which is expected upon attachment of a phosphopantetheinyl group to the active site serine of the apo-ACP. Thus, KirP is responsible for the conversion of apo-KirAIIACP5 to holo-KirAIIACP5. The conversion from apo-KirAIIACP5 to holo-KirAIIACP5 by KirP was also visible in the UV chromatogram of HPLC analyses because of a shift in retention time of the KirAIIACP5 peak (Fig. 2a and b). Apo-KirAIIACP5 was eluted from the HPLC column at 15.8 min, while holo-KirAIIACP5 was eluted at 16.1 min.

Table 1.   Calculated and observed masses of proteins used in the in vitro phosphopantetheinylation assays
Carrier proteinCalculated massObserved mass
  • *

    Due to noise in the mass spectral data, methylmalonyl-holo-KirAIIACP4 could not be unambiguously detected by the MS approach. Using autoradiography, the loading of KirAIIACP4 with [1,3-14C]methylmalonyl-CoA was clearly detectable (see Fig. 3).

Apo-KirAIIACP516 248.316 248.7
Holo-KirAIIACP516 588.616 589.6
Malonyl-holo-KirAIIACP516 674.616 676.1
Methylmalonyl-holo-KirAIIACP516 688.716 690.3
Apo-KirAIIACP417 992.117 994.0
Holo-KirAIIACP418 332.518 333.8
Malonyl-holo-KirAIIACP418 418.518 420.7
Methylmalonyl-holo-KirAIIACP418 432.5*
Apo-KirAIIIPCP16 347.916 350.0
Holo-KirAIIIPCP16 688.216 686.3
Malonyl-holo-KirAIIIPCP16 774.216 777.3
Methylmalonyl-holo-KirAIIIPCP16 788.216 789.1
Apo-KirBPCP12 619.812 621.8
Holo-KirBPCP12 960.112 960.7
Malonyl-holo-KirBPCP13 046.213 048.0
Methylmalonyl-holo-KirBPCP13 060.213 061.9
image

Figure 2.  HPLC UV-Vis chromatograms and MS spectra of the KirAIIACP5 in vitro phosphopantetheinylation assays. (a) Control reaction: KirAIIACP5+CoA. (b) Activation reaction: KirP+KirAIIACP5+CoA. (c) Loading of malonyl-CoA: KirP+KirAIIACP5+malonyl-CoA. (d) Loading of methylmalonyl-CoA: KirP+KirAIIACP5+methylmalonyl-CoA.

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The ability of KirP to activate ACPs in the kirromycin PKS/NRPS was also confirmed using KirAIIACP4 from PKS module 4 of the kirromycin megasynthase as a substrate for KirP. KirP was able to convert the apo form of ACP4 (17 994.0 Da) to its holo form (18 333.8 Da), as monitored by MS analyses (Table 1). Due to the physicochemical properties of KirAIIACP4, no shift in retention time was detected in HPLC-UV-Vis traces when the phosphopantetheine group was attached. But still the mass shift occurring on conversion of apo to holo KirAIIACP4 was clearly detectable in the MS data.

PPTases of hybrid PKS/NRPS normally exhibit broad substrate specificity because they must activate both ACPs and PCPs. To test whether KirP also transfers phosphopantetheine to the PCP domains within the kirromycin PKS/NRPS, KirAIIIPCP and KirBPCP were expressed in E. coli and used in in vitro activation assays. HPLC-ESI-MS analyses of the reaction mixtures revealed that KirP was able to activate these two apo-PCPs by addition of the 340 Da phosphopantetheine moieties. Control reactions without KirP confirmed that the conversion to their holo forms are the result of KirP phosphopantetheinylation activity, because in the control reaction lacking KirP, only apo-PCPs were detected by MS analyses.

Sfp was reported to use different CoA derivatives (acetyl-CoA, desulfo-CoA, benzoyl-CoA and phenylacetyl-CoA) as substrates (Quadri et al., 1998). A similar flexibility has also been described for AcpS from E. coli, which uses acetyl-CoA, propionyl-CoA, butyryl-CoA, malonyl-CoA, benzoyl-CoA and phenylacetyl-CoA as substrates for phosphopantetheinylation of type II ACPs (Carreras et al., 1997).

Therefore, the specificity of KirP with respect to its CoA substrate was investigated. The purified apo-carrier proteins (KirAIIACP4, KirAIIACP5, KirAIIIPCP and KirBPCP) were incubated with [1,3-14C]methylmalonyl-CoA and KirP. Autoradiographic analyses were performed to examine the incorporation of [1,3-14C]methylmalonyl-pantetheine moieties into the carrier proteins. Strong signals were detected in all tested carrier proteins, indicating efficient incorporation of the radioactively labeled substrate. In the absence of KirP, no incorporation of [1,3-14C]methylmalonyl-CoA was observed (Fig. 3).

image

Figure 3. In vitro phosphopantetheinylation assay using [1,3-14C]methylmalonyl-CoA as substrate. Tested carrier proteins: KirAIIACP5, KirAIIACP4, KirAIIIPCP and KirBPCP. Left: sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel. Right: autoradiogram.

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The utilization of modified CoAs by KirP was also detected in HPLC-ESI-MS analyses. Both malonyl- and methylmalonyl-CoA were found to be substrates for KirP (Fig. 2c and d). The enzyme transferred the acyl-phosphopantetheinyl group of each substrate to the carrier proteins KirAIIACP4, KirAIIACP5, KirAIIIPCP and KirBPCP. The observed mass shifts in the HPLC-MS data corresponded exactly to the expected values for attachment of a malonylated or methylmalonylated phosphopantetheinyl group (Table 1).

Conclusions

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusions
  7. Acknowledgements
  8. Authors' contribution
  9. References
  10. Supporting Information

The significant drop in kirromycin yield in S. collinus EP-P1, a kirP gene replacement mutant, shows that KirP plays an important role in kirromycin biosynthesis and can only be weakly complemented by other PPTases encoded elsewhere in the genome.

In vitro phosphopantetheinylation assays demonstrated that KirP can activate both ACPs and PCPs within the kirromycin PKS/NRPS, thus exhibiting a broad specificity towards cognate ACP and PCP domains. A relaxed specificity was also observed with respect to CoA substrates; KirP was able to attach the phosphopantetheine group of not only CoA but also acyl-CoA substrates to the active site serine of the CP. In the latter cases, KirP directly catalyzed the loading of each tested CP with acyl-phosphopantetheine.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusions
  7. Acknowledgements
  8. Authors' contribution
  9. References
  10. Supporting Information

We would like to thank Thomas Härtner and David Worbs for excellent technical assistance. This work was funded by the BMBF grants GenoMikPlus/GenBioCom (FKZ0313805J/FKZ0315585A) to W.W. and T.W., and a PhD scholarship to E.K.P. by the DFG graduate school ‘Infection Biology’ GK675.

Authors' contribution

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusions
  7. Acknowledgements
  8. Authors' contribution
  9. References
  10. Supporting Information

M.P. carried out the CP and KirP expressions, performed the mutant complementation and the loading experiments and wrote parts of the manuscript. E.M.M. developed the ACP expression protocols and the HPLC-MS-based assays and performed the autoradiography analyses. E.K.P. generated the kirP replacement mutant EP-P1, constructed the complementation plasmid and wrote parts of the manuscript. A.K. performed HPLC-MS analyses. W.W. and T.W. planned and supervised the experiments and wrote parts of the manuscript. M.P., E.K.P. and E.M.M. contributed equally to this work.

References

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  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusions
  7. Acknowledgements
  8. Authors' contribution
  9. References
  10. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusions
  7. Acknowledgements
  8. Authors' contribution
  9. References
  10. Supporting Information

Table S1. Oligonucleotide primers used in this study.

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FilenameFormatSizeDescription
FML_2263_sm_supmat.doc79KSupporting info item

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.