1 Fakultät Biologie, Mikrobiologisches Institut, Mikrobiologie/Biotechnologie, Eberhard-Karls-Universität Tübingen, Auf der Morgenstelle 28, 72076 Tübingen, Germany
2 Leibniz-Institut für Naturstoff-Forschung und Infektionsbiologie-Hans-Knöll-Institut, Beutenbergstrasse 11, 07745 Jena, Germany
Correspondence
Dirk Schwartz
schwartz{at}pmail.hki-jena.de
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession number for the nucleotide sequence of the friulimicin biosynthetic genes reported in this paper is AJ488769.
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INTRODUCTION |
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Lipopeptide antibiotics are one class of antibiotics effective against Gram-positive bacteria, and with Cubicin (daptomycin), a first member of this class was approved by the US Food and Drug Administration in 2003.
Eight lipopeptides recently identified and isolated from the actinomycete Actinoplanes friuliensis (Aretz et al., 2000) are composed of an identical cyclic peptide structure of ten amino acids. The peptide core is N-terminally linked via diaminobutyric acid to an acylated, exocyclic amino acid, either asparagine or aspartate (Fig. 1a
) (Vértesy et al., 2000
). The acyl residue includes branched-chain (iso and anteiso) fatty acids with a chain length of C13 to C15, all of which have an unusual double bond at position
cis3 (Fig. 1b
). Four of the eight lipopeptides (A1437 A, A1437 B, A1437 E, A1437 G) have aspartate as the exocyclic amino acid and are identical to known peptide antibiotics of the amphomycin group; the other four lipopeptide structures (friulimicin AD) have asparagine as the exocyclic amino acid and represent a new class of antibiotics (Vértesy et al., 2000
) (Fig. 1b
).
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The clinical application of these antibiotics is often limited by their toxicity to eukaryotic cells. For example, friulimicin displays haemolytic activity against erythroplasts (H. Decker, Frankfurt, personal communication). The action and also the toxicity of lipopeptide antibiotics strongly depend on the moiety of the acyl residue. Therefore, the synthesis of lipopeptide antibiotics consisting of known peptide structures and a different acyl residue could lead to new antibiotics active against a wide range of bacteria, but with fewer side effects.
The biosynthesis of the peptide moiety of lipopeptide antibiotics via non-ribosomal peptide synthases has been well characterized; however, less is known about the synthesis of the acyl residue and its linkage to the peptide moiety. Several lipopeptide antibiotic biosynthetic gene clusters have been isolated and partially characterized, such as fengycin (Tosato et al., 1997), mycosubtilin (Duitman et al., 1999
), lichenysin (Konz et al., 1999
), syringomycin (Guenzi et al., 1998
), daptomycin (McHenney et al., 1998
) and CDA (Chong et al., 1998
; Hojati et al., 2002
; Ryding et al., 2002
). In addition to peptide synthase genes, genes probably involved in the synthesis of the lipid moiety of the antibiotics have been found in the gene clusters.
The synthesis of the 2,3-epoxyhexanoyl acyl residue of the CDA from Streptomyces coelicolor A3(2) has been postulated by Hojati et al. (2002) to involve primary and secondary metabolic enzymes. In their model, the precursor hexanoyl-acyl carrier protein (ACP) is synthesized by fatty acid synthases, enzymes of primary metabolism. Hexanoyl-ACP is then hydrolysed by a thioesterase, and free hexanoyl is activated by acyl-CoA synthase to form hexanoyl-CoA. These two enzymes might also be recruited from primary metabolism, since neither a gene encoding a thioesterase nor a gene encoding an acyl-CoA synthase has been found within the complete CDA biosynthetic gene cluster. In contrast, the subsequent two reactions responsible for the formation of the 2,3-epoxyhexanoyl group are carried out by two proteins of secondary metabolism, an oxidase and a monooxygenase, encoded by the genes hxcO and hcmO, both located within the CDA gene cluster. The peptide synthase CdaPS1, which activates the first amino acid, is postulated to transfer and attach 2,3-epoxyhexanoyl to the first amino acid (Hojati et al., 2002
).
Another example of an acylation mechanism in the synthesis of peptide structures has been described for mycosubtilin biosynthesis in Bacillus subtilis ATCC 6633 (Duitman et al., 1999). Here, a multifunctional hybrid of a peptide synthase, an amino transferase and a fatty acid synthase are responsible for the synthesis of mycosubtilin. In the hypothetical model of synthesis of the lipid moiety, a long-chain fatty acid is activated by an acyl-CoA synthase domain. In the next step, the activated acyl residue binds the 4'-phosphopantetheine cofactor of an ACP domain. Malonyl-CoA is loaded onto a second ACP domain, and the formation of a
-ketoacylthioester is catalysed by a
-ketoacylsynthase domain. After amination of the keto group, the lipid moiety is attached to the first amino acid of the peptide by the condensation domain of the peptide synthase (Duitman et al., 1999
).
To elucidate the acylation mechanism of friulimicin, we isolated a 6·2 kb DNA fragment flanking the previously isolated 9·2 kb portion of the friulimicin biosynthetic gene cluster (Heinzelmann et al., 2003) and identified genes belonging to this cluster that are involved in the synthesis of the acyl residue. We demonstrated the involvement of a potential FAD-dependent acyl-CoA dehydrogenase, encoded by one of these genes (lipB), in the introduction of the unusual
cis3 double bond within the acyl residue.
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METHODS |
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Intergeneric conjugation between Escherichia coli and A. friuliensis.
Intergeneric conjugation followed the procedure described by Heinzelmann et al. (2003).
Gene disruption, mutagenesis and transformation.
A gene disruption mutant was generated by intergeneric conjugation between A. friuliensis and E. coli ET12567/pUB307 (Heinzelmann et al., 2003) with plasmid pEHLBA2 (Table 1
). Streptomyces lividans T7 (Fischer, 1996
) protoplasts were transformed using PEG, as described by Hopwood et al. (1985)
. E. coli was transformed using the CaCl2 method described by Sambrook et al. (1989)
. For standard cloning experiments, E. coli XL-1 Blue was used.
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DNA sequencing and analysis.
DNA fragments of the friulimicin biosynthetic gene cluster were subcloned in the sequencing vectors pUC18 and pK18 or pK19. The nucleotide sequences were determined by standard techniques (Sanger et al., 1977). The codon usage programme of Staden & McLachlan (1982)
was used to identify ORFs. Homology searches and multiple sequence alignments were performed using the programs BLAST (Altschul et al., 1990
), CLUSTAL W (Thompson et al., 1994
) and Genedoc (Nicholas et al., 1997
).
Isolation of lipB.
For the construction of the lipB mutant MEHB and the overexpression strain S. lividans T7/pEHEX3, an internal lipB fragment (lipB*) and the complete lipB gene, respectively, each carrying a BglII and HindIII restriction site at the 5' and 3' termini, were isolated using PCR. Plasmid pOP2, which contains a 5·6 kb PstI fragment of the cluster carrying the 1·5 kb lipB gene, was used as template. The following reaction mixtures were used: 0·5 µg pOP2; primer M1 (5'-AATAGATCTGCGAAGCTCGACGAGGCCGAG-3') and primer M2 (5'-AATAAGCTTCTCCAGCAACGCCACGAGACG-3') for the isolation of lipB* or primer E1 (5'-AATAGATCTATGACGGACCTGTCCACCCTG-3') and primer E2 (5'-AATAAGCTTTCATCGGGCACCGACCCCG-3') for the isolation of lipB (sequences of restriction sites used for cloning are underlined; 100 pmol each); 10 µl of 10x reaction buffer (containing 20 mM MgCl2); 5 % DMSO; 0·2 mM deoxynucleoside triphosphates; and 1 U of Pwo polymerase (Roche). After a denaturation step (5 min, 98 °C), 25 cycles of amplification (1 min 94 °C, 1 min 69 °C, 1·5 min 72 °C) were performed. The 1·4 kb lipB* and the 1·5 kb lipB PCR products were electrophoretically separated in a 1 % agarose gel, eluted from the gel, and cloned into the vector pJOE890, resulting in plasmids pEHLB1 and pEHEX1, respectively (Table 1).
Construction of plasmids pEHLBA2 and pEHEX3.
For the construction of the lipB mutant, lipB* was isolated from pEHLB1 as a BglIIHindIII fragment and cloned into BamHIHindIII-digested vector pK19, resulting in pEHLB2 (Table 1). pEHLB2 was digested with NotI, whose recognition sequence is located approximately in the middle of lipB*, and the resulting overhanging ends were filled in with Klenow polymerase. The apramycin/PermE resistance cassette aprP was isolated by a StuIEcoRV restriction of plasmid pEH13 (Heinzelmann et al., 2001
). To lessen possible polar effects, aprP was inserted in the transcriptional direction of lipB* (lipBaprP) in plasmid pEHLB2, resulting in plasmid pEHLBA1 (Table 1
). A 3 kb KpnIHindIII fragment of pEHLBA1 carrying lipBaprP was cloned in the KpnIHindIII-digested vector pK18mob (Schäfer et al., 1990
). The resulting plasmid pEHLBA2 (Table 1
) was transferred to A. friuliensis by intergeneric conjugation between E. coli 12567/pUB307 and A. friuliensis, as described by Heinzelmann et al. (2003)
.
For the expression of lipB in S. lividans T7, the lipB gene was isolated as a BglIIHindIII fragment from pEHEX1 and cloned in BglIIHindIII-restricted pRSETB, resulting in plasmid pEHEX2 (Table 1). pEHEX2 was cloned as a HindIII fragment into vector pGM9, resulting in the StreptomycesE. coli shuttle plasmid pEHEX3 (Table 1
).
Bioassay for antibiotic production.
The antibiotic production of the mutant MEHB and of the wild-type was tested in a bioassay using B. subtilis as the test organism. The gene insertion mutant MEHB and the wild-type were each cultivated in M65 medium (Heinzelmann et al., 2003) for 7 days. The mutant and the wild-type grew at the same rate and to the same extent. Culture broth containing mycelium pellets was homogenized and the OD600 was determined. Both MEHB and the wild-type reached an OD600 of approx. 2·7. Ten millilitres of each culture was centrifuged and each cell pellet was spread uniformly on the surface of a defined M65 agar plate (35 ml per plate, plate diameter 9 cm). After 7 days of incubation at 30 °C, agar plugs of equal size were cut out and applied to the B. subtilis test medium according to the method of Alijah et al. (1991)
. The plates were incubated overnight at 37 °C and antibiotic production was detected by the formation of a zone of growth inhibition around the agar blocks.
Purification of friulimicin and the friulimicin derivative FR242 from culture supernatants.
For purification of friulimicin and the friulimicin derivative FR242 from culture supernatants, the wild-type and mutant MEHB were each cultivated in 100 ml seed medium (Aretz et al., 2000) in a 500 ml Erlenmeyer flask for 5 days at 30 °C on a rotary shaker at 180 r.p.m. Antibiotic production medium (Aretz et al., 2000
; 2x 500 ml in 1 l Erlenmeyer flasks) was inoculated with 5 ml of culture grown on seed medium and cultivated for 7 days at 30 °C at 180 r.p.m. Cells were removed by centrifugation at 5000 g and the supernatant was loaded onto a column packed with 100 ml degassed XAD-16 resin (Rohm and Haas) and equilibrated with 10 mM potassium phosphate buffer (pH 7·2). The column was washed and eluted as described by Vértesy et al. (2000)
. Fractions of 15 ml were collected, and the friulimicin content was determined by HPLC. The fractions containing native friulimicin or FR242 were pooled and concentrated to dryness using a rotary evaporator. The pellets were solubilized in methanol and analysed by GC and GC-MS.
HPLC analysis of friulimicin and FR242.
Friulimicin and FR242 were analysed by reverse-phase HPLC using a steel column (125 mmx4·6 mm) and a precolumn (20 mmx4·6 mm) packed with Nucleosil 100C-18·5 µm (Maisch) as the stationary phase. The mobile phase was 0·1 % phosphoric acid (A) and acetonitrile (B) [linear gradient: 015 min, (A) 1000 % and (B) 0100 %; 1 min, (A) 0 %, (B) 100 %]. The antibiotics were detected using a diode array detection system at various wavelengths (210, 230, 260, 280, 310, 360 and 435 nm) at a flow rate of 2 ml min1 and an injection volume of 10 µl.
Isolation of the fatty acid moiety of friulimicin and FR242.
To separate the acyl residue from the peptide core, purified friulimicin and FR242 were saponified by alkaline hydrolysis. Reagent I (1 ml; 22·5 g NaOH, 75 ml methanol, 75 ml H2O) was mixed with the samples and the reaction mixture was incubated at 100 °C for 35 min. After cooling, the free fatty acids were esterified by adding 10 ml of reagent II (162·5 ml 6 M HCl, 137·5 ml methanol) and incubated for 12 min at 80 °C. Fatty acid methyl esters were extracted with 1·25 ml hexane with shaking for 10 min. To the organic phase, 3 ml 3 M NaOH was added, and the upper phase was used for GC and GC-MS.
GC and GC-MS analyses of the acyl residue of friulimicin and FR242.
The acyl residues were analysed by GC (injector and flame-ionization detector 250 °C, split 1 : 10, injection volume 10 µl) using a polar capillary column [Carbowax 20-M (PEG), length 30 m, i.d. 0·32 mm, layer thickness 0·25 µm; JW Scientific] and the following temperature programme: 160200 °C at 4 °C min1, 200240 °C at 8 °C min1 and 240 °C for 5 min. Fatty acids were analysed by GC-MS on a non-polar capillary column (Optima 5, methylsilicone phase, length 15 m, i.d. 0·25 mm, layer thickness 0·25 µm; Macherey-Nagel) under the following conditions: temperature gradient (3 s at 90 °C, 90200 °C at 26 °C min1, 200300 °C at 8 °C min1 and 10 min at 300 °C); injector temperature, 230 °C; interface temperature, 220 °C; column pressure, 4 kPa; split, 1 : 4; injection volume, 1 µl; flow rate, 0·8 ml min1; total flow rate, 4 ml min1. The data were analysed using the GC-17A program (Shimadzu) and BenchTop-PBM (Palisade Corporation). Fatty acids were identified by comparison to the fatty acid standard NHI-D-Mix (Supelco).
Hydrogenation of the acyl residue of friulimicin.
Friulimicin (5 mg) was dissolved in 0·5 ml methanol. After the addition of a spatula tip of palladium-activated carbon, molecular hydrogen was slowly added for 5 min. The reaction mixture was then centrifuged for 5 min at 15 000 g and the supernatant was placed in a 10 ml glass cup and dried under a stream of nitrogen gas. The hydrogenated friulimicin was dissolved in 500 µl hexane and used for GC and GC-MS analyses and bioassay.
Heterologous expression of lipB in S. lividans and purification of His-tagged LipB.
The recombinant lipB gene (hislipB) cloned on plasmid pEHEX2 was expressed in S. lividans T7, which carries a thiostrepton-inducible T7 RNA polymerase gene (Fischer, 1996) and the His-tagged LipB protein was purified following the procedures described by Heinzelmann et al. (2001)
. For the GC-MS analysis of the recombinant S. lividans T7/pEHEX3 and S. lividans T7/pEHK (control) (Heinzelmann et al., 2003
), cells were grown in TSB medium (Bacto tryptic soy broth; Becton-Dickinson) instead of YEME medium.
Determination of acyl-CoA dehydrogenase activity.
Acyl-CoA dehydrogenase activity was assayed spectrophotometrically using a standard acyl-CoA dehydrogenase assay described by Kieweg et al. (1997) and Lea et al. (2000)
, which is based on the reduction of FAD to FADH2 during the dehydrogenase reaction. In this reaction, the blue dye 2,3-dichlorophenol indophenol (Cl2PIP), as final electron acceptor, is reduced and becomes colourless, which can be followed spectroscopically at 600 nm. Commercially available CoA-activated straight-chain fatty acids (nC14nC16; Sigma), were used as substrates.
Nucleotide sequences and accession numbers.
The nucleotide sequence of the friulimicin biosynthetic genes reported in this paper has been assigned accession no. AJ488769 at EMBL.
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RESULTS |
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Gene disruption mutagenesis of the acyl-CoA dehydrogenase gene (lipB)
To determine whether the lipB gene product is involved in the biosynthesis of the acyl residue, and specifically in the introduction of the unusual cis3 double bond, the lipB gene was subjected to insertion mutagenesis using plasmid pEHLBA2 following a protocol described by Heinzelmann et al. (2003)
. pEHLBA2 carries an apramycin/PermE resistance cassette aprP in the middle of the lipB fragment. This disrupted fragment is designated lipB*. Apramycin-resistant, kanamycin-sensitive transformants of A. friuliensis were analysed by Southern hybridization, and clones showing a double-crossover event between the chromosomal copy of lipB and the mutated lipB* located on pEHLBA2 were identified (Fig. 2b
). The generated mutant, MEHB, was analysed for antibiotic production using a B. subtilis bioassay. The zone of growth inhibition around mutant MEHB was reproducibly smaller than that around the wild-type (approx. 62 % of the wild-type inhibition zone) (Fig. 3a
), which suggested either a reduced friulimicin production or that the mutant produced a modified, less biologically active friulimicin.
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To analyse whether the difference in the retention times can really be attributed to the presence or absence of an unsaturated bond, the masses were determined by GC-MS. An overlay of the iC14 : 0 (from FR242) and iC14 : 1 (from friulimicin) spectra demonstrated the different retention times (Fig. 4a). Owing to the non-polar character of the column used, iC14 : 1 elutes before iC14 : 0. The GC spectra and the corresponding characteristic GC-MS spectra of iC14 : 1 and iC14 : 0 are shown in Fig. 4(b)
, (c) (marked by arrows). The mass difference of 2 indicated that the iC14 : 1 fatty acid of friulimicin possesses two hydrogen atoms fewer than the iC14 : 0 acyl residue of FR242, in other words, that the iC14 : 0 fatty acid is saturated. Therefore, the GC and GC-MS spectra showed that the acyl residue of FR242 from mutant MEHB lacks the characteristic
cis3 double bond, in other words, that LipB actually is responsible for or involved in the introduction of the double bond.
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Heterologous expression of lipB in S. lividans
To demonstrate the assumed acyl-CoA dehydrogenase activity of LipB, we expressed the lipB gene heterologously in S. lividans T7, which carries the gene encoding T7 RNA polymerase under the control of the thiostrepton-inducible tipA promoter (Fischer, 1996). S. lividans T7 was transformed with the lipB expression plasmid pEHEX3 (Table 1
). The expression of the lipB gene was under the control of the T7 promoter and resulted in the production of N-terminally His-tagged LipB protein (HisLipB) after induction with thiostrepton. HisLipB was purified by metal-chelate-affinity chromatography using Ni-NTA resin under native conditions, as described by Heinzelmann et al. (2001)
. The eluted fractions were analysed by SDS-PAGE (Fig. 5a
). Using the spectroscopic acyl-CoA dehydrogenase assay according to the method of Kieweg et al. (1997)
and Lea et al. (2000)
, no specific enzyme activity was detected when the protein-containing fractions were incubated with straight-chain activated fatty acids (nC14, nC15, nC16) not even after modifying the reaction buffer conditions. Therefore, it seems likely that under in vitro conditions, the substrate specificity of the putative acyl-CoA dehydrogenase is directed toward activated branched-chain fatty acids instead of activated straight-chain fatty acids. This is in keeping with the fact that branched fatty acids are attached to the peptide core of friulimicin. However, this hypothetical activity could not be biochemically investigated, since activated branched-chain fatty acids of a chain length of C13C14 are not commercially available.
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DISCUSSION |
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It is also possible that friulimicin [as described for daptomycin (Silverman et al., 2003) or CDA (Lakey et al., 1983
)] is involved in Ca2+-dependent pore formation, which leads to a disruption of the functional integrity of the cytoplasmic membrane. It can be speculated that the
cis3 double bond of the acyl residue of friulimicin supports such a mechanism. Further experiments are needed to determine whether the haemolytic effect of FR242 on erythroblasts is also reduced.
The involvement of LipB in the introduction of double bonds in fatty acids was also confirmed by lipB overexpression in S. lividans T7. Under these conditions, higher amounts of unsaturated fatty acids were found in S. lividans T7/pEHEX3 than in the control S. lividans T7/pEHK (Fig. 5b). The major unsaturated fatty acids produced by S. lividans/pEHEX3 were iC17 : 1, aiC17 : 1 and iC16 : 1; nC16 : 1 was produced in lower amounts (Fig. 5b
).
The in vitro spectroscopic enzyme activity assay with activated nC16 : 0 as substrate produced negative results, and the detection of nC16 : 1 was unexpected. The low concentration of nC16 : 1 produced in vivo suggests that oxidation of nC16 : 0 is only a side activity of the enzyme, which is probably not detectable in the in vitro enzyme assay using purified LipB.
Acyl-CoA dehydrogenases (ACADs) differ in their substrate specificity and are separated into short-chain (SCAD, <C6), medium-chain (MCAD, C6C11), long-chain (LCAD, C12C18) and very-long-chain (VLCAD, >C18) acyl-CoA dehydrogenases, which reflects the acyl-chain length of their preferred substrates (Lea et al., 2000). According to this classification, LipB might be an LCAD that oxidizes fatty acids with a chain length of C12C18. Structural analyses of different ACADs SCADs, MCADs, and branched-short-chain acyl-CoA dehydrogenases, such as iso(3)valeryl-CoA dehydrogenase (i3VCD) indicate that all members of this enzyme class show a characteristic polypeptide fold of 11
-helices (helices AK) and 7
-strands (17) (Kim & Miura, 2004
). According to the determined protein structure, a substrate prediction seems to be possible for these enzymes (Kim & Miura, 2004
).
The characteristic secondary structure of acyl-CoA dehydrogenases was also found in LipB [PROFsec (Rost & Sander, 1993; Rost, 1996
), data not shown]. However, since no crystal structure data for straight long-chain and branched-long-chain acyl-CoA dehydrogenases are available, it would be helpful to solve the protein structure of LipB to determine the regions conferring substrate specificity.
Since only branched-chain fatty acids of a chain length of C13C15 are attached to the peptide core of friulimicin (Vértesy et al., 2000), a substrate specificity of LipB for branched-chain C13C15 fatty acids was expected. However, why no iC14, iC15 and aiC15 : 1 fatty acids were found in S. lividans T7/pEHEX3 is unclear. It can be speculated that these fatty acids are formed and then degraded for unknown reasons. Such an instability and possible degradation of iC14, iC15 : 1 and aiC15 : 1 fatty acids has also been discussed by Cropp et al. (2000)
regarding the induction of unsaturated fatty acid metabolism by cold shock in S. avermitilis. Another possible explanation for the lack of these fatty acids could be that the acyl-CoA synthase LipA, which probably activates fatty acids to form acyl-CoA thioesters, the substrates of acyl-CoA dehydrogenases (LipB), and which is essential for the synthesis of friulimicin (data not shown), has a narrow substrate range for branched-chain C13C15 fatty acids. A possible LipA/LipB protein complex formation could prevent the oxidation and incorporation of other unsaturated fatty acids. A functional correlation of LipA and LipB is supported by the translational coupling of lipA and lipB in the friulimicin biosynthetic gene cluster. Similar situations have been observed in many antibiotic gene clusters, for example, within the biosynthetic gene cluster of the glycopeptide antibiotic balhimycin from Amycolatopsis mediterranei, in which the gene products of the four translationally coupled genes dpgABCD are involved in the synthesis of the unusual amino acid 3,5-dihydroxyphenylglycine (Pfeifer et al., 2001
).
The chemical structure of the acyl residue of FR242 in the lipB mutant indicates that the acyl-CoA dehydrogenase LipB is responsible for the introduction of the cis3 double bond, but the mechanism is unknown. Two main pathways can be postulated for the formation of the
cis3 double bond in the acyl residue of friulimicin (Fig. 6
): 1) LipB introduces the
cis3 double bond in one reaction by a new mechanism or LipB is a bifunctional enzyme with both dehydrogenase and isomerase activity (Fig. 6
, pathway 1), or 2) LipB introduces the double bond in the
trans2 position as acyl-CoA dehydrogenases of primary metabolism and the double bond is then isomerized to the
cis3 position by an unknown
trans2-
cis3-isomerase, for which an encoding gene has not been identified within the biosynthetic gene cluster (Fig. 6
, pathway 2). The activity of such an enzyme would be similar to that of FabM, a
trans2-
cis3-isomerase from Streptococcus pneumoniae (Marrakchi et al., 2002
).
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ACKNOWLEDGEMENTS |
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REFERENCES |
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---|
Altenbuchner, J., Viell, P. & Pelletier, I. (1992). Positive selection vectors based on palindromic DNA sequences. Methods Enzymol 216, 457466.[Medline]
Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. (1990). Basic local alignment search tool. J Mol Biol 215, 403410.[CrossRef][Medline]
Aretz, W., Meiwes, G., Seibert, G., Vobis, G. & Wink, J. (2000). Friulimicins: novel lipopeptide antibiotics with peptidoglycan synthesis inhibiting activity from Actinoplanes friuliensis sp. nov. I. Taxonomic studies of the producing microorganism and fermentation. J Antibiot (Tokyo) 53, 807815.[Medline]
Barie, P. S. (1998). Antibiotic-resistant Gram-positive cocci: implications for surgical practice. World J Surg 22, 118126.[CrossRef][Medline]
Bentley, S. D., Chater, K. F., Cerdeno-Tarraga, A. M. & 40 other authors (2002). Complete genome sequence of the model actinomycete Streptomyces coelicolor A3(2). Nature 417, 141147.[CrossRef][Medline]
Bullock, W. O., Fernandez, J. M. & Short, J. M. (1987). Xl1-Blue, a high efficiency plasmid transforming recA Escherichia coli strain with beta galactosidase selection. Focus 5, 376378.
Chong, P. P., Podmore, S., Kieser, H. M., Redenbach, M., Turgay, K., Marahiel, M., Hopwood, D. A. & Smith, C. (1998). Physical identification of a chromosomal locus encoding biosynthetic genes for the lipopeptide calcium-dependent antibiotic (CDA) from Streptomyces coelicolor A3(2). Microbiology 144, 193199.[Medline]
Cole, S. T., Brosch, R., Parkhill, J. & 39 other authors (1998). Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 393, 537544.[CrossRef][Medline]
Cole, S. T., Eiglmeier, K., Parkhill, J. & 41 other authors (2001). Massive gene decay in the leprosy Bacillus. Nature 409, 10071011.[CrossRef][Medline]
Cropp, T. A., Smogowicz, A. A., Hafner, E. W., Denoya, C. D., McArthur, H. A. & Reynolds, K. A. (2000). Fatty-acid biosynthesis in a branched-chain alpha-keto acid dehydrogenase mutant of Streptomyces avermitilis. Can J Microbiol 46, 506514.[CrossRef][Medline]
Dieckmann, R. & von Döhren, H. (1997). Structural model of acyl carrier domains in integrated biosynthetic system forming peptides, polyketides and fatty acids based on analogy to the E. coli acyl carrier protein. In Developments in Industrial MicrobiologyGMBIM 1996, pp. 7985. Edited by R. Baltz, G. Hegemann & P. Skatrud. Washington, DC: Society for Industrial Microbiology.
Duitman, E. H., Hamoen, L. W., Rembold, M. & 10 other authors (1999). The mycosubtilin synthetase of Bacillus subtilis ATCC6633: a multifunctional hybrid between a peptide synthetase, an amino transferase, and a fatty acid synthase. Proc Natl Acad Sci U S A 96, 1329413299.
DuPlessis, E. R., Pellet, J., Stankovich, M. T. & Thorpe, C. (1998). Oxidase activity of the acyl-CoA dehydrogenases. Biochemistry 37, 1046910477.[CrossRef][Medline]
Fischer, J. (1996). Entwicklung eines regulierbaren Expressionssystems zur effizienten Synthese rekombinanter Proteine in Streptomyces lividans. PhD thesis, University of Stuttgart, Germany.
Flett, F., Mersinias, V. & Smith, C. P. (1997). High efficiency intergeneric conjugal transfer of plasmid DNA from Escherichia coli to methyl DNA-restricting streptomycetes. FEMS Microbiol Lett 155, 223229.[CrossRef][Medline]
Guenzi, E., Galli, G., Grgurina, I., Gross, D. C. & Grandi, G. (1998). Characterization of the syringomycin synthetase gene cluster. J Biol Chem 273, 3285732863.
Heinzelmann, E., Kienzlen, G., Kaspar, S., Recktenwald, J., Wohlleben, W. & Schwartz, D. (2001). The phosphinomethylmalate isomerase gene pmi, encoding an aconitase-like enzyme, is involved in the synthesis of phosphinothricin tripeptide in Streptomyces viridochromogenes. Appl Environ Microbiol 67, 36033609.
Heinzelmann, E., Berger, S., Puk, O., Reichenstein, B., Wohlleben, W. & Schwartz, D. (2003). A glutamate mutase is involved in the biosynthesis of the lipopeptide antibiotic friulimicin in Actinoplanes friuliensis. Antimicrob Agents Chemother 47, 447457.
Hiltunen, J. K. & Qin, Y.-M. (2000). -Oxidation-strategies for the metabolism of a wide variety of acyl-CoA esters. Biochim Biophys Acta 148, 117128.
Hojati, Z., Milne, C., Harvey, B. & 9 other authors (2002). Structure, biosynthetic origin, and engineered biosynthesis of calcium-dependent antibiotics from Streptomyces coelicolor. Chem Biol 9, 11751187.[CrossRef][Medline]
Hopwood, D. A., Bibb, M. J., Chater, K. F. & 7 other authors (1985). Genetic Manipulation of Streptomyces: a Laboratory Manual. Norwich: John Innes Foundation.
Hu, Y., Helm, J. S., Chen, L., Ye, X. Y. & Walker, S. (2003). Ramoplanin inhibits bacterial transglycosylases by binding as a dimer to lipid II. J Am Chem Soc 125, 87368737.[CrossRef][Medline]
Kieweg, V., Kräutle, F. G., Nandy, A. & 8 other authors (1997). Biochemical characterization of purified, human recombinant Lys304Glu medium-chain acyl-CoA dehydrogenase containing the common disease-causing mutation and comparison with the normal enzyme. Eur J Biochem 246, 548556.[Abstract]
Kim, J.-J. & Miura, R. (2004). Acyl-CoA dehydrogenases and acyl-CoA oxidases. Structural basis for mechanistic similarities and differences. Eur J Biochem 271, 483493.
Konz, D., Doekel, S. & Marahiel, M. A. (1999). Molecular and biochemical characterization of the protein template controlling biosynthesis of the lipopeptide lichenysin. J Bacteriol 181, 133140.
Lakey, J. H., Lea, E. J., Rudd, B. A., Wright, H. M. & Hopwood, D. A. (1983). A new channel-forming antibiotic from Streptomyces coelicolor A3(2) which requires calcium for its activity. J Gen Microbiol 129, 35653573.[Medline]
Lea, W., Abbas, A. S., Sprecher, H., Vockley, J. & Schulz, H. (2000). Long-chain acyl-CoA-dehydrogenase is a key enzyme in the mitochondrial -oxidation of unsaturated fatty acids. Biochim Biophys Acta 1485, 121128.[Medline]
Marrakchi, H., Choi, K. H. & Rock, C. O. (2002). A new mechanism for anaerobic unsaturated fatty acid formation in Streptococcus pneumoniae. J Biol Chem 277, 4480944816.
McHenney, M. A., Hosted, T. J., Dehoff, B. S., Rosteck, P. R., Jr & Baltz, R. H. (1998). Molecular cloning and physical mapping of the daptomycin gene cluster from Streptomyces roseosporus. J Bacteriol 180, 143151.
Moellering, R. C., Jr (1998). The specter of glycopeptide resistance: current trends and future considerations. J Med 104, 3S6S.[CrossRef]
Muth, G., Nußbaumer, B., Wohlleben, W. & Pühler, A. (1989). A vector system with temperature-sensitive replication for gene disruption and mutational cloning in streptomycetes. Mol Gen Genet 219, 341348.[CrossRef]
Nicholas, K. B., Nicholas, H. B., Jr & Deerfield, D. W., II (1997). GeneDoc: analysis and visualization of genetic variation. EMBNEW NEWS 4, 14.
Omura, S., Ikeda, H., Ishikawa, J. & 11 other authors (2001). Genome sequence of an industrial microorganism Streptomyces avermitilis: deducing the ability of producing secondary metabolites. Proc Natl Acad Sci U S A 98, 1221512220.
Pfeifer, V., Nicholson, G. J., Ries, J., Recktenwald, J., Schefer, A. B., Shawky, R. M., Schröder, J., Wohlleben, W. & Pelzer, S. (2001). A polyketide synthase in glycopeptide biosynthesis: the biosynthesis of the non-proteinogenic amino acid (S)-3,5-dihydroxyphenylglycine. J Biol Chem 276, 3837038377.
Rost, B. (1996). PHD: predicting one-dimensional protein structure by profile-based neural networks. Methods Enzymol 266, 525539.[CrossRef][Medline]
Rost, B. & Sander, C. (1993). Prediction of protein secondary structure at better than 70% accuracy. J Mol Biol 20, 584599.[CrossRef]
Ryding, N. J., Anderson, T. B. & Champness, W. C. (2002). Regulation of the Streptomyces coelicolor calcium-dependent antibiotic by absA, encoding a cluster-linked two-component system. J Bacteriol 184, 794805.
Sambrook, J., Fritsch, T. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Sanger, F., Nicklen, S. & Coulson, A. R. (1977). DNA sequencing with chain terminating inhibitors. Proc Natl Acad Sci U S A 74, 54635467.
Schäfer, A., Kalinowski, J., Simon, R., Seep-Feldhaus, A.-H. & Pühler, A. (1990). High-frequency conjugal plasmid transfer from Gram-negative Escherichia coli to various Gram-positive coryneform bacteria. J Bacteriol 172, 16631666.[Medline]
Schauwecker, F., Pfennig, F., Schroeder, W. & Keller, U. (1998). Molecular cloning of the actinomycin synthetase gene cluster from Streptomyces chrysomallus and functional heterologous expression of the gene encoding actinomycin synthetase II. J Bacteriol 180, 24682474.
Silverman, J. A., Perlmutter, N. G. & Shapiro, H. M. (2003). Correlation of daptomycin bactericidal activity and membrane depolarization in Staphylococcus aureus. Antimicrob Agents Chemother 47, 25382544.
Staden, R. & McLachlan, A. D. (1982). Codon preference and its use in identifying protein coding regions in large DNA sequences. Nucleic Acids Res 10, 141156.[Medline]
Steller, S., Vollenbroich, D., Leenders, F., Stein, T., Conrad, B., Hofemeister, J., Jacques, P., Thonart, P. & Vater, J. (1999). Structural and functional organization of the fengycin synthetase multienzyme system from Bacillus subtilis b213 and A1/3. Chem Biol 6, 3141.[CrossRef][Medline]
Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994). CLUSTALW: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22, 46734680.[Abstract]
Tosato, V., Albertini, A. M., Zotti, M., Sonda, S. & Brushi, C. V. (1997). Sequence completion, identification and definition of the fengycin operon in Bacillus subtilis 168. Microbiology 143, 34433450.[Medline]
Vértesy, L., Ehlers, E., Kogler, H., Kurz, M., Meiwes, J., Seibert, G., Vogel, M. & Hammann, P. (2000). Friulimicins: novel lipopeptide antibiotics with peptidoglycan synthesis inhibiting activity from Actinoplanes friuliensis sp. nov. II. Isolation and structural characterization. J Antibiot (Tokyo) 53, 816827.[Medline]
Weber, M. H., Klein, W., Müller, L., Niess, U. M. & Marahiel, M. A. (2001). Role of the Bacillus subtilis fatty acid desaturase in membrane adaptation during cold shock. Mol Microbiol 39, 13211329.[CrossRef][Medline]
Received 20 December 2004;
revised 22 February 2005;
accepted 2 March 2005.
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