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• W2103195220 abstract "Polyketide biosynthesis involves the addition of subunits commonly derived from malonate or methylmalonate to a starter unit such as acetate. Type I polyketide synthases are multifunctional polypeptides that contain one or more modules, each of which normally contains all the enzymatic domains for a single round of extension and modification of the polyketide backbone. Acyl carrier proteins (ACP(s)) hold the extender unit to which the starter or growing chain is added. Normally there is one ACP for each ketosynthase module. However, there are an increasing number of known examples of tandemly repeated ACP domains, whose function is as yet unknown. For the doublet and triplet ACP domains in the biosynthetic pathway for the antibiotic mupirocin from Pseudomonas fluorescens NCIMB10586 we have inactivated ACP domains by inframe deletion and amino acid substitution of the active site serine. By deletion analysis each individual ACP from a cluster can provide a basic but reduced activity for the pathway. In the doublet cluster, substitution analysis indicates that the pathway may follow two parallel routes, one via each of the ACPs, thus increasing overall pathway flow. In the triplet cluster, substitution in ACP5 blocked the pathway. Thus ACP5 appears to be arranged “in series” to ACP6 and ACP7. Thus although both the doublet and triplet clusters increase antibiotic production, the mechanisms by which they do this appear to be different and depend specifically on the biosynthetic stage involved. The function of some ACPs may be determined by their location in the protein rather than absolute enzymic activity. Polyketide biosynthesis involves the addition of subunits commonly derived from malonate or methylmalonate to a starter unit such as acetate. Type I polyketide synthases are multifunctional polypeptides that contain one or more modules, each of which normally contains all the enzymatic domains for a single round of extension and modification of the polyketide backbone. Acyl carrier proteins (ACP(s)) hold the extender unit to which the starter or growing chain is added. Normally there is one ACP for each ketosynthase module. However, there are an increasing number of known examples of tandemly repeated ACP domains, whose function is as yet unknown. For the doublet and triplet ACP domains in the biosynthetic pathway for the antibiotic mupirocin from Pseudomonas fluorescens NCIMB10586 we have inactivated ACP domains by inframe deletion and amino acid substitution of the active site serine. By deletion analysis each individual ACP from a cluster can provide a basic but reduced activity for the pathway. In the doublet cluster, substitution analysis indicates that the pathway may follow two parallel routes, one via each of the ACPs, thus increasing overall pathway flow. In the triplet cluster, substitution in ACP5 blocked the pathway. Thus ACP5 appears to be arranged “in series” to ACP6 and ACP7. Thus although both the doublet and triplet clusters increase antibiotic production, the mechanisms by which they do this appear to be different and depend specifically on the biosynthetic stage involved. The function of some ACPs may be determined by their location in the protein rather than absolute enzymic activity. Acyl carrier proteins (ACP(s)) 1The abbreviations used are: ACP, acyl carrier protein; TE, thioesterase; KS, ketosynthase; PKS, polyketide synthase; Mmp, mupirocin multifunctional polypeptide; HPLC, high performance liquid chromatography. 1The abbreviations used are: ACP, acyl carrier protein; TE, thioesterase; KS, ketosynthase; PKS, polyketide synthase; Mmp, mupirocin multifunctional polypeptide; HPLC, high performance liquid chromatography. are small (80–110 amino acids) acidic proteins or domains that are essential to a variety of metabolic pathways. The active site serine of the ACPs is joined to the 4′-phosphopantetheine prosthetic group by a phosphodiester linkage to form the holoenzyme ACP. Reaction intermediates are attached to the terminal thiol of the 4′-phosphopantetheine arm that swings between enzymes to perform its function (1Magnuson K. Jackowski S. Rock C.O. Cronan J.E.J. Micrbiol. Rev. 1993; 57: 522-542Crossref PubMed Google Scholar), allowing interaction with almost every other enzyme in the pathway. ACPs are involved in fatty acid synthesis in all living organisms. They can also act as acyl donors in the biosynthesis of lipids (2Rock C. Jackowski S. J. Biol. Chem. 1982; 257: 10759-10765Abstract Full Text PDF PubMed Google Scholar), can carry membrane-derived oligosaccharides (3Tang L. Weissborn A. Kennedy E. J. Bacteriol. 1997; 179: 3697-3705Crossref PubMed Google Scholar), and can be involved in the activation of RTX toxins in Gram-negative bacteria (4Issartel J.P. Koronakis V. Hughes C. Nature. 1991; 351: 759-761Crossref PubMed Scopus (253) Google Scholar). Specialized ACPs may also act as signal factors in rhizobial nodulation (5Geiger O. Spaink H.P. Kennedy E.P. J. Bacteriol. 1991; 173: 2872-2878Crossref PubMed Google Scholar) as well as substrates and carriers in the synthesis of lipoteichoic acid (6Heaton M.P. Neuhaus F.C. J. Bacteriol. 1994; 176: 681-690Crossref PubMed Google Scholar) and polyketide antibiotics (7Shen B. Summers R.G. Gramajo H. Bibb M.J. Hutchinson C.R. J. Bacteriol. 1992; 174: 3818-3821Crossref PubMed Google Scholar). Polyketides are assembled by successive condensations of small carboxylic acid derivatives catalyzed by enzymes called polyketide synthases (PKS), which are homologous to fatty acid synthases (8Staunton J. Weissman K.J. Nat. Prod. Rep. 2001; 18: 380-416Crossref PubMed Scopus (1278) Google Scholar). ACPs thioesterified at the phosphopantetheine cofactor with the chain extender unit are used to generate nucleophiles to be acted upon by PKSs during condensation reactions. There are basically three types of polyketide synthases, the multifunctional Type I modular systems, the discrete Type II iterative synthases (9Hopwood D.A. Chem. Rev. 1997; 97: 2465-2497Crossref PubMed Scopus (621) Google Scholar), and the Type III chalcone synthases, which consist of a single homodimeric enzyme working iteratively independent of ACPs (10Tropf S. Ka ̈rcher B. Schro ̈der G. Schro ̈der J. J. Biol. Chem. 1995; 270: 7922-7928Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). Programming exhibited by PKS can be modular, where each enzyme works just once in the biosynthetic process, and iterative, where the enzymes work many times. The iterative Type I system of fungi has a single large polypeptide with a set of active site domains as in Type I modular systems but working iteratively to produce the polyketide. Modular systems like erythromycin have a single ACP/module, one for each condensation cycle the growing polyketide chain undergoes before the completed chain is released, so that each ACP domain is used only once in the course of each biosynthetic cycle (11Donadio S. Staver M.J. McAlpine J.B. Swanson S.J. Katz L. Science. 1991; 252: 675-679Crossref PubMed Scopus (732) Google Scholar). Most of the Type II systems in bacteria contain a single ACP that works iteratively with other PKS enzymes performing the dual function of receiving the chain extender unit before condensation and of holding the growing polyketide chain after each condensation to produce the polyketide (12Revill W.P. Leadlay P.F. J. Bacteriol. 1991; 173: 4379-4385Crossref PubMed Google Scholar, 13Revill W. Bibb M. Hopwood D. J. Bacteriol. 1996; 178: 5660-5667Crossref PubMed Google Scholar). Despite the above generalizations about Type I PKS systems there are an increasing number of examples with tandem clusters of ACPs, for example in the naphthopyrone and sterigmatocystin PKS of Aspergillus nidulans (14Fujii I. Watanabe A. Sankawa U. Ebizuka Y. Chem. Biol. 2001; 8: 189-197Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar, 15Yu J. Leonard T. J. Bacteriol. 1995; 177: 4792-4800Crossref PubMed Google Scholar) and the albicidin biosynthetic cluster of Xanthomonas albilineans (16Huang G. Zhang L. Birch R.G. Microbiology. 2001; 147: 631-642Crossref PubMed Scopus (60) Google Scholar), although the role of such duplication is not known. Gene duplication is commonly proposed as a way that new functions arise, the second stage being either silencing of one of the gene copies by deleterious/degenerative mutations yielding pseudogenes or development of a novel function by one of the gene copies. Recently, however, the idea of subfunctionalization has evolved, which proposes the idea of partitioning the tasks of the ancestral gene between the pair (17Lynch M. O'Hely M. Walsh B. Force A. Genetics. 2001; 159: 1789-1804PubMed Google Scholar). Elucidation of the reason for ACP domain duplication may not only shed light on the process of polyketide biosynthesis but may also provide more general insights. Two examples of tandemly repeated ACP clusters are found in the mupirocin biosynthetic gene cluster from Pseudomonas fluorescens, a Gram-negative soil-born bacterium (18El-Sayed A.K. Hothersall J. Cooper S.M. Stephens E. Simpson T.J. Thomas C.M. Chem. Biol. 2003; 10: 419-430Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar). Mupirocin or pseudomonic acid (PA) is an antibiotic that competitively inhibits isoleucyl-tRNA synthetase and prevents the incorporation of isoleucine into growing polypeptide chains (19Hughes J. Mellows G. Biochem. J. 1980; 191: 209-219Crossref PubMed Scopus (166) Google Scholar). It is used topically in the treatment of skin and burn wound infections or applied intranasally to control hospital outbreaks of methicillin-resistant Staphylococcus aureus (20Lamb Y.J. J. Hosp. Infect. 1991; 19: 27-30Abstract Full Text PDF PubMed Scopus (24) Google Scholar, 21Rode H. de Wet P.M. Millar A.J. Cywes S. Lancet. 1991; 338: 578Abstract PubMed Scopus (15) Google Scholar). Mupirocin is formed by the esterification of 9-hydroxynonanoic acid and monic acid (22Chain E.B. Mellows G. J. Chem. Soc. Perkin Trans. I. 1977; 1: 318-322Crossref Scopus (48) Google Scholar) (see Fig. 1). Oxygen labeling experiments confirmed that this ester linkage is formed between separate C17 and C9 moieties (23Martin F.M. Simpson T.J. J. Chem. Soc. Perkin Trans. 1989; 1: 207-209Crossref Scopus (25) Google Scholar). The ∼74-kb mupirocin biosynthetic cluster involved in mupirocin production was identified by transposon mutagenesis and reverse genetics (24Whatling C.A. Hodgson J.E. Martin K.R.B. Clarke N.J. Franklin C.H. Thomas C.M. Microbiology. 1995; 141: 973-982Crossref Scopus (25) Google Scholar). DNA sequencing (18El-Sayed A.K. Hothersall J. Cooper S.M. Stephens E. Simpson T.J. Thomas C.M. Chem. Biol. 2003; 10: 419-430Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar) revealing six long open reading frames typical of Type I PKS proteins, which we named mupirocin multifunctional polypeptides (MmpA–MmpF) as well as 27 genes (mupA-mupZ and macpA-macpE) encoding discrete proteins, some of which are similar to Type II PKSs. Quorum sensing regulates the expression of genes in the mupirocin biosynthetic cluster, which was repressed during the exponential phase and maximal on entry into stationary phase (25El-Sayed A.K. Hothersall J. Thomas C.M. Microbiology. 2001; 147: 2127-2139Crossref PubMed Scopus (99) Google Scholar). Monic acid synthesis is predicted to involve six condensation reactions catalyzed by six modules the last of which contains a pair of tandem ACPs, whereas the region currently hypothesized to be linked to 9-hydroxynonanoic acid production has a tandem cluster of three ACPs (18El-Sayed A.K. Hothersall J. Cooper S.M. Stephens E. Simpson T.J. Thomas C.M. Chem. Biol. 2003; 10: 419-430Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar). Other characteristics unique to the mupirocin cluster are the absence of a loading domain, the apparent absence of AT domains apart from AT1 and AT2 of MmpIII, the unusual position of the thioesterase domain, and the presence of 16 acyl carrier domains (18El-Sayed A.K. Hothersall J. Cooper S.M. Stephens E. Simpson T.J. Thomas C.M. Chem. Biol. 2003; 10: 419-430Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar). In this paper we establish for the first time a phenotype for the tandem repetition of Type I ACPs. Although the results show that in both cases the pathway can continue if just one of the ACPs is present, the results are not consistent with all ACPs being equal. Bacterial Strains and Growth Media Used—P. fluorescens NCIMB10586 (24Whatling C.A. Hodgson J.E. Martin K.R.B. Clarke N.J. Franklin C.H. Thomas C.M. Microbiology. 1995; 141: 973-982Crossref Scopus (25) Google Scholar) the producer of pseudomonic acid was used as the wild type strain for creating mutants. Escherichia coli DH5α (26Hanahan D. J. Mol. Biol. 1983; 166: 557-580Crossref PubMed Scopus (8146) Google Scholar) was used for plasmid transformation and propagation, and strain S17-1 (27Simon R. Priefer U. Puhler A. Bio/Technology. 1983; 1: 784-791Crossref Scopus (5611) Google Scholar) was used to mobilize suicide and expression plasmids into the wild type P. fluorescens NCIMB10586. Bacillus subtilis 1064 was used as the sensitive organism in the bioassays to detect the antibacterial activity of mupirocin produced by wild type as well as mutants if any were produced (24Whatling C.A. Hodgson J.E. Martin K.R.B. Clarke N.J. Franklin C.H. Thomas C.M. Microbiology. 1995; 141: 973-982Crossref Scopus (25) Google Scholar). P. fluorescens NCIMB10586 was grown at 30 °C in L-broth/agar containing 50 μg/ml ampicillin or 50 μg/ml kanamycin. Mupirocin production medium containing 2.3 g/liter yeast extract, glucose 1.1 g/liter, Na2HPO4 2.6 g/liter, KH2PO4 2.4 g/liter, and (NH)2SO4 5.0/liter was used in preparing cultures for HPLC. DNA Isolation and Manipulation—Plasmid DNA was extracted using the alkaline SDS method (28Birnboim H.C. Doly J. Nucleic Acids Res. 1979; 7: 1513-1523Crossref PubMed Scopus (9886) Google Scholar) or by Wizard Plus SV Minipreps DNA Purification Systems (Promega). The digestion of DNA was carried out with appropriate restriction enzymes (MBI Fermentas). The PCR products were cloned into vectors using T4 DNA ligase (29Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Construction of Deletion Mutants Using Suicide Vector Strategy— The suicide vector pAKE604 containing the AmpR, KanR, lacZα, sacB as selectable markers with an oriT was used to construct the deletion mutants (18El-Sayed A.K. Hothersall J. Cooper S.M. Stephens E. Simpson T.J. Thomas C.M. Chem. Biol. 2003; 10: 419-430Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar). The general procedure involves designing two sets of primers with 5′-restriction sites to amplify 500 bp-flanking regions on either side of the gene to be deleted by PCR and ligating them. The ∼1-kb insert was introduced into pAKE604 and E. coli S17-1 was transformed to mobilize the plasmid into P. fluorescens. Point mutations in mACP3, mACP4, mACP5, and mACP7 were constructed by replacing the active site codon TCG encoding serine in the wild type by GCC encoding alanine in one of the primers, and a restriction site was introduced by altering the third codon without changing the amino acid it encodes. The suicide derivatives of pAKE604 constructed in this way were transferred from E. coli S17-1 into wild type P. fluorescens NCIMB10586. Both of the strains were mixed and filtered through 0.45 μm sterile Millipore filters, which were then placed on L-agar plates to allow conjugation. P. fluorescens with the suicide plasmid integrated into the chromosome was then selected on M9 minimal medium with kanamycin. Medium containing 5% (w/v) sucrose was subsequently used to select strains in which the suicide plasmid had been excised from the chromosome. Screening for sensitivity to kanamycin identified bacteria where excision of the suicide plasmid had occurred. DNA Sequencing—Automated sequencing of the plasmid DNA was carried out by ABI Prism BigDye V3 Terminator ready reaction kit (PE-ABI), which has ampliTaq DNA polymerase, dye terminators, deoxynucleoside triphosphates, magnesium chloride, and buffer premixed into a single tube of Ready Reaction Mix and is based on the chain termination method of Sanger et al. (30Sanger F. Nicklen S. Coulson A.R. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 4563-4567Crossref Scopus (52505) Google Scholar). The sequencing reaction was performed on ABI 3700 DNA analyzer (Genomics facility, University of Birmingham). Bioassay—Single colonies of P. fluorescens NCIMB10586 wild type/mutants were inoculated into L-broth containing ampicillin and incubated overnight at 30 °C. After normalizing the optical density measured at 600 nm, cultures were spotted onto L-agar plates and incubated at room temperature for 18–24 h. These plates were overlaid with a mixture of L-agar, B. subtilis culture (strain 1064 grown at 37 °C to late log phase), and 2,3,5-triphenyl tetrazolium chloride (0.025%) as an indicator and incubated at 37 °C overnight. The zone of clearance around the spot culture was taken as an estimate of the amount of mupirocin produced. Culture Conditions for Isolation of Compounds—To obtain the seed culture a loop full of wild type and mutant cultures of P. fluorescens NCIMB10586 were inoculated into 25 ml of L-broth in 250-ml conical flasks and were shaken at 25 °C for 24 h. Five ml of the seed culture was inoculated into 25 ml of mupirocin production media and shaken at 22 °C for 20–60 h. The cultures were collected at different times, and bacteria were removed by centrifugation at 25 °C. The supernatant was used for HPLC analysis. Before injection the samples were filtered through 0.2 μm Acrodisc Nylon filters (Gelman Laboratory-Fischer). Analysis of Compounds by Reverse Phase High Performance Liquid Chromatography—HPLC analysis was carried out to compare product profiles of the wild type P. fluorescens NCIMB10586 and mutant derivatives. HPLC (Gilson) with UV detector was used at a sensitivity of 0.002 absorbance units full scale and a wavelength of 233 nm. The DiscoveryR C-18 silica column (Supelco) used had a pore size of 5 μm. The solvents used were HPLC-grade water (Fischer) and HPLC-grade 100% acetonitrile (Fischer). Trifluoroacetic acid was added to adjust the pH of the mobile phase. A 70% acetonitrile gradient was used to elute mupirocin and the intermediates at a flow rate of 1 ml/min. Amino Acid Sequence Similarities between Doublet and Triplet ACPs—For all the doublet and triplet ACPs, the active site serine residue lies in the highly conserved DSV motif characteristic of acyl carrier proteins (Fig. 2). The modular mACPs contain almost equal numbers (∼90) of amino acids, but show a high degree of sequence divergence (19–42% sequence identity), ruling out very recent gene duplication. Pairwise alignments (Fig. 2B) showed that mACP3 and mACP4 are more related to each other (40% identical) than to mACP5/6/7. Within the triplet, mACP5 and mACP7 (42% identical) are more closely related, but mACP5, mACP6, and mACP7 as a group are more closely related to each other than to mACP3/4. The similarity/identity is consistent with the mean similarity observed between tandemly duplicated genes of prokaryotes and eukaryotes (25–30%) (31Coissac E. Maillier E. Netter P. Mol. Biol. Evol. 1997; 14: 1062-1074Crossref PubMed Scopus (48) Google Scholar). The tandem ACPs can, therefore, be described as paralogous genes and might have diverged in sequence after ancient tandem duplication events. We were not able to identify any heterologous ACPs more closely related, which might have supported the idea of recent acquisition from an alternative source. Thus the closest identified relative of mACP3 is mACP4 and therefore they might have originated by gene duplication. A similar situation is found with mACP5 and mACP7, whereas mACP6, which seems to have originated from a different ancestor, might have been inserted between the two other mACPs of the triplet by a random recombination process. Alternatively it could be that mACP6 and mACP5/7 are the result of an initial duplication with mACP5/7 resulting from a later duplication. Construction of Mutants of the Putative Acyl Carrier Proteins—The mACPs in this study are part of multifunctional megaproteins and so it was important to achieve inactivation without affecting the other functions in the protein. Initial analysis was therefore by in-frame deletions. These deletions were constructed in the suicide plasmid pAKE604 individually and introduced into P. fluorescens NCIMB10586 as described under “Experimental Procedures.” Because the mmpA and mmpB genes appear to be part of a larger polycistronic transcriptional unit, all deletion mutations were constructed in-frame to remove only specific runs of amino acids and avoid polar effects on the transcription of the downstream genes. Within the Mmp proteins the spacer regions between domains are usually 100 residues long, but mACP3 and mACP4 are separated by 12 amino acids, whereas mACP5 and mACP6 and mACP6 and mACP7 are separated by only 3 amino acids in both cases. The double and triple mutants were constructed by deleting completely the mACP doublet and mACP triplet including the spacer regions between these domains, respectively, from the mupirocin cluster. The single/individual deletions were made in such a manner that the spacer regions between the individual domains of an Mmp were retained to allow correct three-dimensional folding and orientation of domains of the polypeptide. The most complicated construction was that removing mACP7 from the mutant lacking mACP5, so as to leave mACP6 intact. Point mutations were also constructed by replacing the active site serine of tandem mACPs with an alanine, so as not to alter the natural folding of the polypeptide. The mutations were checked by PCR amplification and confirmed by sequencing. Qualitative Analysis of Mutants by Bioassay—To compare the antibacterial activity possessed by the wild type and mutant strains, a bioassay was performed using B. subtilis 1064 as the test organism. Indicator plates containing 2,3,5-triphenyl tetrazolium chloride, which acts as a terminal electron acceptor and turns red when bacteria are actively growing, were used to estimate the zone of inhibition produced around the spot culture of wild type P. fluorescens (Fig. 3A). By contrast, the Δmacp3/4 double domain deletion mutant phenotype was indistinguishable from that of the mupI mutant (lacking the quorum regulation autoinducer needed to switch on mup expression in stationary phase) that is used as a negative control to demonstrate the loss of antibiotic production. The zones of inhibition of the individual Δmacp3, Δmacp4, Δmacp5, Δmacp6, and Δmacp7 mutants were comparable with or only slightly smaller than that of wild type P. fluorescens, whereas the Δmacp5/6/7 once again was similar to the negative mupI mutant (Fig. 3, B and C). Pairwise deletions constructed within the mACP triplet cluster (Δmacp5/6, Δmacp6/7, Δmacp5/7) did not abolish mupirocin production, but there was a significant decrease in the area of the zone of inhibition (Fig. 3C). The conclusion from this is that any one of the doublet and the triplet ACPs is sufficient for the formation of the polyketide chain but that reduction of the triplet cluster to a single mACP does have a quantitative effect on production. Quantitative Analysis of Compounds Produced by Wild Type and Mutants—The compounds secreted into the supernatant of the wild type and mutant cultures were analyzed by reverse phase HPLC with an acetonitrile-water gradient elution on a silica column (Fig. 4). The system was calibrated with known amounts of mupirocin standard as described under “Experimental Procedures.” P. fluorescens produces a mixture of PAs (Fig. 1). Mupirocin, which is PA-A, elutes between 20 and 21 min and is seen as a single discrete peak on the chromatogram. The peak at ∼19.5 min is predicted to be PA-B, and there are additional minor peaks seen after PA-A, which might be PA-C and PA-D, the other minor constituents of the antibiotic pool. All the single and pairwise deletions that retained antibacterial activity showed the same peaks as the wild type, whereas the double and the triple mutants (Δmacp3/4, Δmacp5/6/7), no longer produced any of the same compounds (as shown in Fig. 4). The time course of mupirocin production by the wild type strain was established by analyzing the samples collected between 16 and 64 h. Mupirocin production reached a plateau after 18 h, and the level then remained more or less constant until 64 h. The variables were reduced to a minimum while performing the quantitative analysis of mutants by equalizing the optical density of the seed cultures and maintaining similar culture conditions. The average area under the PA-A peaks of two clones of wild type and each mutant was taken at each time point. Production from the mutant cultures between 18–64 h was also analyzed and found to follow the same kinetics as seen for the wild type, reaching a plateau at ∼18 h. The area under the PA-A peak was used as an estimate of the amount of antibiotic produced. As the peak areas were more or less constant once the plateau was reached, all of the peak areas were pooled to maximize the statistical significance of the results. With the doublet mACPs in MmpA the deletion of either mACP3 and mACP4 had a similar effect. The Δmacp3 and Δmacp4 mutant strains produced 0.68 ± 0.03 and 0.62 ± 0.07 of the PA-A level of the wild type, the reduction being highly significant (p ≪ 0.001) (Fig. 5A). With the triplet mACP cluster in MmpB the Δmacp5, Δmacp6, and Δmacp7 mutant strains showed 0.36 ± 0.05, 0.32 ± 0.03, and 0.25 ± 0.02, respectively, when compared with wild type, which is a highly significant decrease in mupirocin production. The pairwise mutants Δmacp5/6, Δmacp6/7, and Δmacp5/7 produced 0.11 ± 0.01, 0.16 ± 0.01, and 0.10 ± 0.02, respectively, compared with wild type as shown in Fig. 5B, a further significant decrease. This observation is more or less consistent with the expected additive effect of a double deletion except for Δmacp6/7 that showed higher production than for the other double knock-outs. However, it would not be surprising if there were variations in the activities of the different mACPs especially because as argued below mACP5 may work in series rather than in parallel with mACP6 and mACP7. The effect of removing mACP domains could be due not just to loss of 1 unit of ACP activity but could also be because of secondary effects on the remaining enzymic activities in MmpA or MmpB because of conformational changes in the protein. We therefore determined the effect of inactivating an ACP by a point mutation that would minimize structural changes. The point mutants macp3S>A (Mmp I S2664A), macp4S>A (MmpA S2769A), macp5S>A (Mmp II S1390A), macp6S>A (Mmp II S1478A) and macp7S>A (Mmp II S1574A) produced 0.61 ± 0.01, 0.72 ± 0.03, 0.03 ± 0.01, 0.66 ± 0.03, and 0.85 ± 0.13 when compared with the wild type as shown in Fig. 5. For mACP3 and mACP4 these results are generally in line with the deletion of whole mACP domains. The results were unexpected in the triplet cluster. Surprisingly macp5S>A showed no detectable mupirocin production, whereas the effect of the point mutations in macp6 and macp7 was much less than expected. This study has focused on why there are tandemly repeated mACP domains in the Type I Mmps of the mupirocin biosynthetic cluster when normally only one such domain would be expected. Our strategy was to inactivate the mACP domains and determine the effects by antibacterial bioassay and by HPLC. Although we were able to detect changes in clearing zone for the double deletions in the triplet cluster, the major conclusion from the plate bioassay was that each ACP in a cluster is functional and is capable of substituting for the other ACPs in that cluster. This seems much higher than the basal level of interchangeability expected between very different ACPs observed in previous work. For example, only weak complementation by the fatty acid synthase ACP was obtained for an ACP mutant in the act system of Streptomyces coelicolor where the level of identity was 31% identity between the ACPs (13Revill W. Bibb M. Hopwood D. J. Bacteriol. 1996; 178: 5660-5667Crossref PubMed Google Scholar). The finding that any of the ACP domains in the mup tandem clusters can suffice means that they can substitute for each other. It also indicates that these tandem clusters represent a single step in the biosynthetic pathway or that a single ACP can support more than one step. HPLC analysis gave more reproducible results than the plate assay and provided the basis for the detailed quantitative analysis that we have performed. The consistent result was that the wild type with intact doublet or triplet clusters gave higher yields of mupirocin than any of the mutants and in the case of the triplet cluster progressive deletions of pairs generally gave bigger effects than deletion of single mACPs. Before detailed consideration of the quantitative data it is important to consider that the effect of deleting a domain could either be because of the loss of the function of that domain or could arise from conformational changes on the rest of the protein arising from the deletion. To address this issue a point mutation was introduced into the active sites of ACP3, ACP4, ACP5, ACP6, and ACP7, resulting in the inability of the mutated ACPs to be converted into a holoenzyme by the addition of the phosphopantetheine arm. In the case of doublet ACPs this had a similar effect to that of deleting the whole domain. The similar loss of activity caused by deletion or point mutation for both mACP3 and mACP4 sugge" @default.
• W2103195220 created "2016-06-24" @default.
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• W2103195220 creator A5067269192 @default.
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• W2103195220 date "2005-02-01" @default.
• W2103195220 modified "2023-10-02" @default.
• W2103195220 title "Tandemly Duplicated Acyl Carrier Proteins, Which Increase Polyketide Antibiotic Production, Can Apparently Function Either in Parallel or in Series" @default.
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