Programa Multidisciplinario de Biología Experimental (PROMUBIE-CONICET) and Departamento de Microbiología, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Suipacha 531, 2000-Rosario, Argentina1
Author for correspondence: H. Gramajo. Tel: +54 341 4350661. Fax: +54 341 4390465. e-mail: gramajo{at}infovia.com.ar
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ABSTRACT |
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Keywords: acyl-CoA carboxylase, primary metabolism, propionyl-CoA carboxylase, biotinylated protein
Abbreviations: ACCase, acetyl-CoA carboxylase; Am, apramycin; Ap, ampicillin; BC, biotin carboxylase; BCCP, biotin-carboxyl carrier protein; Cm, chloramphenicol; Km, kanamycin; mmCoA, methylmalonyl-CoA; PCCase, propionyl-CoA carboxylase; Th, thiostrepton
The GenBank accession numbers for the accA1, aacA2 and pccB sequences determined in this work are AF113603, AF113604 and AF113605, respectively.
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INTRODUCTION |
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In Streptomyces species, malonyl-CoA and methylmalonyl-CoA (mmCoA) are the most common chain extender units for the biosynthesis of many polyketide antibiotics (Hopwood & Sherman, 1990 ). Since they can both be synthesized via different primary metabolic pathways, they occupy a key position in the linkage between primary and secondary metabolism. Substrate availability for synthesis of the carbon skeletons of some polyketide antibiotics is one of the points of control for polyketide metabolism (Katz & Donadio, 1993
). Therefore, knowledge of the enzymes involved in the supply of these precursors would enable the design of more rational approaches for the increased production of many useful secondary metabolites.
Several pathways leading to mmCoA have been described in bacteria (Gottschalk, 1986 ). In the genus Streptomyces, for example, mmCoA can be synthesized either from succinyl-CoA by mmCoA mutase (Birch et al., 1993
) or by carboxylation of propionyl-CoA by propionyl-CoA carboxylase (PCCase; EC 6 . 4 . 1 . 3) (Huanaiti & Kolattukudy, 1982; Donadio et al., 1996
; Bramwell et al., 1996
). It can also be derived from the catabolism of valine, methionine or threonine (Studier & Moffatt, 1986
) or from n-butyryl-CoA through isobutyryl-CoA mutase (Zerbe-Burkhardt et al., 1998
). Malonyl-CoA is synthesized in most species through the carboxylation of acetyl-CoA by an acetyl-CoA carboxylase (ACCase; EC 6 . 4 . 1 . 2) (Bloch & Vance, 1977
; Harwood, 1988
). However, in some Streptomyces species, a second pathway for the biosynthesis of malonyl-CoA has been described (Behal et al., 1977
; Laakel et al., 1994
). This route involves the anaplerotic enzymes phosphoenolpyruvate carboxylase and oxaloacetate dehydrogenase.
In Streptomyces coelicolor A3(2), four secondary metabolites have been characterized so far (Hopwood et al., 1994 ). Two of them are wholly or partly polyketide-derived (Wasserman et al., 1976
; Gorst-Allman et al., 1981
) and malonyl-CoA is the predicted substrate for all of the condensation reactions involved in their biosynthesis. No evidence for the presence of oxaloacetate dehydrogenase has been found in S. coelicolor; therefore biosynthesis of malonyl-CoA in this organism seems to occur exclusively via ACCase (Bramwell et al., 1993
). Since in bacteria malonyl-CoA is required for all the elongation steps of fatty acid biosynthesis, the ACCase of S. coelicolor should be a key enzyme linking primary and secondary metabolism. Interestingly, several complexes purified from actinomycetes with the ability to carboxylate acetyl-CoA also showed carboxylase activity with other substrates such as propionyl- and butyryl-CoA (Erfle, 1973
; Henrikson & Allen, 1979
; Huanaiti & Kolattukudy, 1982). This property has led to these enzyme complexes being called acyl-CoA carboxylases and all of them have been shown to consist of two subunits, a larger one (
chain) with the ability to carboxylate its covalently bound biotin group, and a smaller subunit (ß chain) bearing the carboxyl transferase activity.
In S. coelicolor, a complex exhibiting only PCCase activity has recently been purified (Bramwell et al., 1996 ) and shown to contain a biotinylated protein of 88 kDa as well as a non-biotinylated component, the carboxyl transferase, of 66 kDa. However, there is good evidence to suggest the existence of other acyl-CoA carboxylase(s) in this micro-organism. This evidence is the readily detectable levels of ACCase activity in crude extracts of S. coelicolor (Bramwell et al., 1996
) and the presence of a biotinylated protein with a molecular mass (65 kDa) similar to that of the
component of several acyl-CoA carboxylase complexes isolated from actinomycetes (Erfle, 1973
; Henrikson & Allen, 1979
; Huanaiti & Kolattukudy, 1982). In order to bring new insights to the characterization of the acyl-CoA carboxylases present in S. coelicolor, we set out to clone the gene(s) encoding the 65 kDa biotinylated protein and a gene encoding a carboxyl transferase (highly homologous to other known carboxyl transferases; Donadio et al., 1996
; Cole et al., 1998
). These studies led us to the genetic and biochemical characterization of a new PCCase complex from S. coelicolor.
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METHODS |
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Escherichia coli strain DH5 was used for routine subcloning and was transformed according to Sambrook et al. (1989)
. Transformants were selected on media supplemented with the appropiate antibiotics: ampicillin (Ap) 100 µg ml-1; chloramphenicol (Cm) 25 µg ml-1 or kanamycin (Km) 30 µg ml-1. Strain BL21(DE3) is an E. coli B strain [F- ompT (
) (DE3)] lysogenized with
DE3, a prophage that expresses the T7 RNA polymerase downstream of the IPTG-inducible lacUV5 promoter (Studier & Moffat, 1986
). Unmodified plasmids purified from the dam dcm E. coli strain ET12567 (MacNeil et al., 1992
) were used to transform S. coelicolor. ET12567/pUZ8002 (a gift from M. Paget, John Innes Centre, Norwich, UK) was used for E. coliS. coelicolor conjugation experiments (Bierman, 1992). For selection of Streptomyces transformants and exconjugants, media were overlaid with thiostrepton (Th) (300 µg per plate) or apramycin (Am) (1 mg per plate). Strains and recombinant plasmids are listed in Table 1
.
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For the expression of heterologous proteins, E. coli strains harbouring the appropriate plasmids were grown at 37 °C in shake flasks in LB medium in the presence of 25 µg Cm ml-1 or 100 µg Ap ml-1 for plasmid maintenance. For the expression of biotinylated proteins, 10 µM D-biotin was added to the medium. Overnight cultures were diluted 1:10 in fresh medium and grown to A600 0·40·5 before the addition of IPTG to a final concentration of 0·1 mM. Induction was allowed to proceed for 24 h. The cells were then harvested, washed and resuspended in 1 ml buffer A. Cell-free extracts were prepared as described above.
Protein methods.
Cell-free extracts were analysed by denaturing (SDS)-PAGE (Laemmli, 1970 ) using the Bio Rad mini-gel apparatus. The final acrylamide monomer concentration was 12% (w/v) for the separating gel and 5% for the stacking gel. Coomassie brilliant blue was used to stain protein bands. The biotinylated proteins were detected by a modification of the Western blotting procedure described by Nikolau et al. (1985)
. After electrophoretic separation, proteins were electroblotted onto nitrocellulose membranes (Bio-Rad) and probed with alkaline phosphatasestreptavidin conjugate (Bio-Rad) diluted 1:10000. Immunoblotting was performed according to Burnette (1981)
using anti-PccB in a 1:500 dilution. Antigenic polypeptides were visualized using an alkaline-phosphatase-tagged secondary antibody. Antisera against PccB were elicited in rabbits following conventional procedures (Burnette, 1981
). Protein contents were determined by the method of Bradford (1976)
with BSA as standard.
ACCase/PCCase assay.
ACCase and PCCase activities in cell-free extracts were measured following the incorporation of H14 into acid non-volatile material (Huanaiti & Kolattukudy, 1982; Bramwell et al., 1996
). The reaction mixture contained 100 mM potassium phosphate pH 8·0, 300 µg BSA, 3 mM ATP, 5 mM MgCl2, 50 mM NaH14CO3 [specific activity 200 µCi mmol-1 (740 kBq mmol-1)], 1 mM substrate (acetyl-CoA or propionyl-CoA) and 100 µg cell-free protein extract in a total reaction volume of 100 µl. The reaction was initiated by the addition of NaH14CO3, allowed to proceed at 30 °C for 15 min and stopped with 200 µl 6 M HCl. The contents of the tubes were then evaporated to dryness at 95 °C. The residue was resuspended in 100 µl water, 1 ml of Optiphase liquid scintillation (Wallac Oy) was added and 14C radioactivity determined in a Beckman scintillation liquid counter. Non-specific CO2 fixation by crude extracts was assayed in the absence of substrate. One unit of enzyme activity catalysed the incorporation of 1 µmol 14C into acid-stable products per min.
DNA manipulations.
Isolation of chromosomal and plasmid DNA, restriction enzyme digestion and agarose gel electrophoresis were carried out by conventional methods (Sambrook et al., 1989 ; Hopwood et al., 1985
). Southern analyses were performed by using 32P-labelled probes made by random oligonucleotide priming (Prime-a-gene kit; Promega).
Gene cloning and plasmid construction.
The synthetic oligonucleotides HG5, 5'-CATGGATCCCTCCATCTTCAT(GC)GC(CT)TC, and HG2, 5'-CATGGATCCATCCACCC(GC)CG(GC)TA(CT)CG(GC)TTCCT, (bracketed nucleotides indicate positions of degeneracy) were used in PCR to amplify an internal fragment of a gene encoding the 65 kDa biotinylated protein. The reaction mixture contained 10 mM Tris/HCl pH 8·3, 50 mM KCl, 1 mM MgCl2, 6% glycerol, 25 µM of each of the four dNTPs, 2·5 U Taq DNA polymerase, 20 pmol of each primer and 50 ng of S. coelicolor chromosomal DNA in a final volume of 100 µl. After denaturation at 95 °C for 5 min, the samples were subjected to 30 cycles of denaturation (95 °C, 1 min), annealing (55 °C, 1 min) and extension (72 °C, 2 min). PCR products were analysed by agarose (0·9 %) gel electrophoresis. The PCR product was digested with BamHI and cloned in BamHI-cleaved pBluescript SK(+) (Stratagene) in E. coli DH5, yielding pTR10. The pTR10 insert was isolated as a BamHI fragment, labelled with 32P by random oligonucleotide priming and used as a probe to isolate the accA1 and accA2 genes from S. coelicolor chromosomal DNA. A 7·5 kb SstI fragment hybridizing to the 1·4 kb probe was isolated from a size-enriched library and cloned in SstI-cleaved pBluescript SK(+), yielding pTR9. An EcoRIKpnI fragment from pTR9 was isolated and cloned into EcoRIKpnI-cleaved pBluescript SK(+), yielding pCL1. A second hybridizing band was also isolated from a size-enriched library and cloned as a 4 kb PstI fragment, yielding pTR45.
Oligonucleotides TC1, 5'-CAGAATTCAAGCAGCACGCCAAGGGCAAG, and TC2, 5'-CAGAATTCGATGCCGTCGTGCTCCTGGTC, were used to amplify an internal fragment of the S. coelicolor pccB gene. The reaction mixture was the same as the one indicated above. Samples were subjected to 30 cycles of denaturation (95 °C, 30 s), annealing (65 °C, 30 s) and extension (72 °C, 1 min). A 1 kb PCR fragment was used as a 32P-labelled probe to screen a size-enriched library. A 4·5 kb PstI fragment containing the complete pccB gene was cloned in PstI-cleaved pBluescript SK(+), yielding pTR58.
The synthetic oligonucleotide HG14, 5'-CGAGGATCCATATGTGGGAAAGACCGC ACTGCC, used to introduce an NdeI site at the translational start codon of the S. coelicolor pccB gene, and HG15, 5'-CGAGGATCCTTGCGGCGGAAGATCTCG, were used to amplify an internal fragment of S. coelicolor pccB gene. The reaction mixture was the same as the one indicated above. Samples were subjected to 35 cycles of denaturation (95 °C, 30 s), annealing (65 °C, 30 s) and extension (72 °C, 1 min). The PCR product was digested with BamHI and cloned in BamHI-cleaved pBluescript SK(+) in E. coli DH5, yielding pTR66. This plasmid was digested with BglII and HindIII, ligated with a BglIIHindIII fragment cleaved from pTR58 and introduced by transformation into E. coli DH5
, yielding pTR67. An NdeIHindIII fragment from the plasmid pTR67 was cloned in NdeIHindIII-cleaved pET22b(+) (Novagen) (pTR68), thus placing the pccB gene under the control of the powerful T7 promoter and ribosome-binding sequences. To obtain the same gene under the control of the lac promoter, an XbaIHindIII fragment from pTR68 was cloned in XbaIHindIII-cleaved pIJ2926 (Janssen & Bibb, 1993
) and introduced by transformation into E. coli DH5
, yielding pTR71. To co-express accA1 and pccB genes, we cloned a BamHIHindIII fragment from pTR71 containing pccB into BamHIHindIII-cleaved pSU18 (Bartolomé et al., 1991
) (compatible with the pBluescript SK plasmid), yielding pTR78.
Nucleotide sequencing.
Double-strand plasmid DNA was prepared from deletion clones generated by nuclease ExoIII (Erase-a-base kit; Promega). Synthetic oligonucleotides were used to complete the sequence. The nucleotide sequence of the accA1 and accA2 region was determined by dideoxy sequencing (Sanger et al., 1977 ) using the Promega TaqTrack sequencing kit and double-stranded DNA templates.
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RESULTS |
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When single digests of S. coelicolor genomic DNA were probed with the 1·4 kb BamHI insert from pTR10 in Southern blots, two strongly hybridizing bands of similar intensities appeared in all digests (data not shown). None of the restriction enzymes used to cut the genomic DNA had a site within the 1·4 kb sequence, suggesting that the two hybridizing bands represented duplicate genes encoding this biotinylated protein. Moreover, when the same probe was used to screen the cosmid library of S. coelicolor M145 DNA (Redenbach et al., 1996 ), two unlinked cosmids gave positive signals (data not shown). One of the signals was eventually localized to cosmid K13 on AseI fragment K and the other to cosmid 2C4, recently mapped on AseI fragment B in the gap found between cosmids 2H4 and 10H5 (H. Kieser, personal communication).
The target sequences were cloned from two different size-enriched libraries as described in Methods. The nucleotide sequences of the two DNA fragments revealed two highly homologous loci that were named accA1, located in cosmid 2C4, and accA2, located in cosmid K13. The amino acid sequences of the two ORFs, deduced from the nucleotide sequences, were nearly identical (the two polypeptides differ in only 5 amino acids), and both were very similar to BcpA1 and BcpA2 of Saccharopolyspora erythraea (Donadio et al., 1996 ), presumed to be the
component of a PCCase.
Attempted disruption of accA1 and accA2
To understand the roles of AccA1 and AccA2 in the formation of the acyl-CoA carboxylase complexes, we attempted to disrupt the respective genes. Based on the remarkably high nucleotide sequence identity of accA1 and accA2 (99%), we designed an experiment that could generate insertion mutants in either of these two genes (Fig. 1a). An internal 802 bp BamHIBstEII segment of the accA2 coding region was blunt-ended and cloned into the SmaI site of pIJ2460 (Floriano & Bibb, 1996
), resulting in pTR36. Several ThR integrative transformants were obtained after transformation of M145 protoplasts with this plasmid. Total DNA of 10 independent transformants was isolated, digested with BamHI and analysed by Southern blot hybridization. In theory, the integration event should occur with comparable frequencies in both genes (the nucleotide sequence corresponding to the BamHIBstEII fragment of accA2 and accA1 differs in only 4 nucleotides). Nevertheless, the hybridization profiles of all 10 clones were identical and showed that the integration event had occurred exclusively within the accA1 locus. Fig. 1(b)
shows the result obtained with DNA from one of the accA1 mutants, MA4. Lane 1 shows the two hybridizing BamHI bands of M145 DNA; lane 2 shows that the 2·9 kb band containing the accA1 gene in the parental DNA is shifted to a larger-sized band in the MA4 mutant. The expected size of the BamHI fragment containing accA1 after integration of pTR36 was 8·1 kb; however, a band of approximately 20 kb was obtained (lane 2). When the same DNA was digested with BamHI and EcoRI (lane 3), the 20 kb band disappeared, yielding two new bands. The 5·2 kb hybridizing fragment corresponds to the size of pTR36 and, since this band is more intense than the 2·1 kb band, we suggest the integration of multimers of pTR36 in the chromosome at the accA1 locus.
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Analysis of the biotinylated proteins produced by M145 and the mutant strain MA4 was carried out using a modified Western blot procedure (Nikolau et al., 1985 ). In the accA1 mutant a biotinylated protein of 65 kDa was still observed, which was presumably the product of accA2 (data not shown).
Cloning and disruption of pccB
An ORF with high similarity to PccB from Saccharopolyspora erythraea was found in a BamHI fragment of 5 kb containing the cya (cyclic-AMP synthase) gene (A. Danchin, personal communication), which is located in cosmid K13 of the S. coelicolor cosmid library (Redenbach et al., 1996 ). Using the sequence data kindly sent to us by Dr Danchin, we cloned the pccB gene as a 4·5 kb PstI fragment from a size-enriched library. Analysis of the predicted amino acid sequence of the major ORF contained within this fragment revealed that the best matching sequence in the GenBank database was the PccB homologue from Saccharopolyspora erythraea (77% amino acid sequence identity) (Donadio et al., 1996
); it was somewhat less similar to AccD5 of M. tuberculosis (68% identity) and PccB of M. leprae (67% identity) (Cole et al., 1998
). We therefore followed the same nomenclature used for Saccharopolyspora erythraea and named the gene pccB. Interestingly, the putative N-terminal sequence of S. coelicolor PccB does not match the N-terminal sequence of the 66 kDa protein reported as the carboxyl transferase subunit of the PCCase complex purified from this micro-organism (Bramwell et al., 1996
). From these data we could not define whether we had cloned the carboxyl transferase subunit of a new PCCase complex or the ß subunit of a yet unidentified acyl-CoA carboxylase complex of S. coelicolor.
To determine whether PccB was part of an acyl-CoA carboxylase complex, a pccB mutant was constructed by gene replacement (Fig. 2a). An Am resistance cassette was cloned in the unique BglII site present in the coding sequence of pccB contained in pTR58, and the PstI fragment containing the mutated allele was cloned in the conjugative vector pSET151. The resulting plasmid, pTR61, was introduced into the E. coli donor strain ET12567/pUZ8002 and transferred by conjugation into M145, yielding ThR AmR exconjugants. Fig. 2(b)
shows the Southern blot of one of the purified ThR AmR exconjugants and one ThS AmR segregant derived from it after three rounds of sporulation in a medium lacking Th. The hybridizing bands correspond to the expected sizes for integration of pTR61 in one of the pccB flanking regions (lane 3) and for the replacement of the wild-type pccB by the AmR mutant allele after a second crossover (lane 2). The resulting mutant, MTC21, was studied by Western blotting using antibodies raised against PccB. Fig. 2(c)
shows a strong band in M145 (pccB+) that is absent in the pccB mutant MTC21.
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In vitro reconstitution of a PCCase complex
Disruption of pccB in S. coelicolor and enzyme assays of the mutant MTC21 suggested that this gene encodes the ß subunit of a PCCase. In order to study the substrate specificity of this subunit, and at the same time to identify the component of the enzyme complex, we attempted in vitro reconstitution of the enzyme activity by mixing E. coli crude extracts containing each of the two available biotinylated proteins with the putative carboxyl transferase subunit PccB. It is important to recall that E. coli does not contain a PCCase enzyme and that ACCase activity cannot be assayed directly by carboxylation of acetyl-CoA in this organism (Polakis et al., 1972
). Cell-free extracts of RG4 containing the putative ß subunit were mixed with cell-free extracts of the E. coli strains containing each of the biotinylated proteins AccA1 or AccA2. After incubation for 1, 24 or 72 h at 4 °C, the mixtures were assayed for ACCase and PCCase activity (Table 3
). A complex with PCCase activity was successfully reconstituted when the carboxyl transferase subunit, PccB, was incubated in the presence of comparable amounts of either AccA1 or AccA2. The reconstituted enzyme complex specifically catalyses the carboxylation of propionyl-CoA; therefore, we propose that the enzyme formed with these
and ß components is a PCCase rather than an acyl-CoA carboxylase with dual substrate specificity as has been described for other actinomycetes (Erfle, 1973
; Henrikson & Allen, 1979
; Huanaiti & Kolattukudy, 1982).
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PCCase activity was readily measured after 1 h of incubation of the cell-free extracts containing the individual subunits, and remained stable for as long as 72 h. Interestingly, the levels of PCCase activity in the crude extracts prepared from S. coelicolor increased (approx. 50% after 72 h) with the time of incubation at 4 °C. This effect could be accounted for by the inactivation of an interfering activity present in the crude extracts.
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DISCUSSION |
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In order to establish the role of the accA1 and accA2 genes in the formation of acyl-CoA carboxylase complexes in S. coelicolor, we tried to isolate mutants in each of these genes and assay them for ACCase and PCCase activities. Cell-free extracts of the accA1 mutant showed the same enzyme activity levels as the parental strain (Table 2), suggesting that the product of this gene was not involved in the formation of any of these enzyme complexes. However, the high similarity between accA1 and accA2, and the fact that we could not generate a mutant in the latter gene because of its apparent essentiality, leave open the possibility that AccA2 could substitute for AccA1 in vivo.
Mutants in pccB showed reduced PCCase activity, suggesting that PccB is the carboxyl transferase subunit of a PCCase complex. This result agrees with that obtained with the pccB homologue of Saccharopolyspora erythraea (Donadio et al., 1996 ), although in this micro-organism only one PCCase complex seems to be present (less than 5% of the PCCase activity remained in the pccB mutant). In contrast, in S. coelicolor, 50% of the original PCCase activity is still present in the pccB mutant, in accordance with the results of Bramwell et al. (1996)
who purified a complex with affinity for propionyl-CoA and with a carboxyl transferase component different from PccB.
Heterologous expression of accA1, accA2 and pccB in E. coli and in vitro reconstitution experiments established that either AccA2 or AccA1 could function as the component of a PCCase in S. coelicolor and confirmed that PccB was the ß component of this enzyme complex (Table 3
).
Based on the apparent inviability of accA2 mutants in S. coelicolor, and on the fact that we successfully assayed ACCase activity in crude extracts of M145 and in the accA1 mutant (Table 2), we hypothesize that accA2 could also encode the
subunit of an essential ACCase whose ß subunit has not yet been identified.
In the genus Streptomyces, several pathways leading to mmCoA have been described, and in S. coelicolor many of these pathways may exist (Bramwell et al., 1993 ; Redenbach et al., 1996
). Our results and those of Bramwell et al. (1996)
demonstrate that S. coelicolor contains at least two PCCase complexes, indicating that this enzyme activity may play a major role in the metabolism of this micro-organism. In Streptomyces, mmCoA is generally related to the production of secondary metabolites (Hopwood & Sherman, 1990
). S. coelicolor is not known to make a natural secondary metabolite derived from mmCoA, although it must presumably be able to make the precursor since it has been shown to be able to synthesize significant quantities of the cyclic macrolactone of erythromycin after introduction of the ery polyketide synthase genes (Kao et al., 1994
). Thus, the physiological role of mmCoA in S. coelicolor remains to be established; it might perhaps be used to feed the TCA cycle, after its conversion into succinyl-CoA catalysed by the mmCoA mutase (Birch et al., 1993
), rather than in the biosynthesis of secondary metabolites.
As we have shown here, heterologous expression of the individual components of the S. coelicolor PCCase and in vitro reconstitution of a functional complex have proved to be successful. This approach will be very useful for the characterization of the and ß components of other acyl-CoA carboxylases, not only in S. coelicolor, but also in other actinomycetes such as M. tuberculosis, where several acyl-CoA carboxylases seem to be present (Cole et al., 1998
).
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Bartolomé, B., Jubete, Y., Martínez, E. & de la Cruz, F. (1991). Construction and properties of a family of pACYC184-derived cloning vectors compatible with pBR322 and its derivatives. Gene 102, 75-78.[Medline]
Behal, V., Jechova, V., Vanek, Z. & Hostalek, Z. (1977). Alternative pathways of malonyl-CoA formation in Streptomyces aureofaciens. Phytochemistry 16, 347-350.
Best, E. & Knauf, V. (1993). Organization and nucleotide sequences of the genes encoding the biotin carboxyl carrier protein and biotin carboxylase protein of Pseudomonas aeruginosa acetyl coenzyme A carboxylase. J Bacteriol 175, 6881-6889.[Abstract]
Bierman, M., Logan, R., OBrien, K., Seno, E. T., Nagaranja Rao, R. & Schoner, B. E. (1992). Plasmid cloning vectors for the conjugal transfer of DNA from Escherichia coli to Streptomyces spp. Gene 116, 43-49.[Medline]
Birch, A., Leiser, A. & Robinson, J. (1993). Cloning, sequencing, and expression of the gene encoding methylmalonyl-CoA mutase from Streptomyces cinnamonensis. J Bacteriol 175, 3511-3519.[Abstract]
Bloch, K. & Vance, D. (1977). Control mechanisms in the synthesis of saturated fatty acids. Annu Rev Biochem 46, 263-298.[Medline]
Bradford, M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of proteindye binding. Anal Biochem 72, 248-254.[Medline]
Bramwell, H., Nimmo, H. G., Hunter, I. S. & Coggins, J. R. (1993). Phosphoenolpyruvate carboxylase from Streptomyces coelicolor A3(2): purification of the enzyme, cloning of the ppc gene and over-expression of the protein in a streptomycete. Biochem J 293, 131-136.[Medline]
Bramwell, H., Hunter, I. S., Coggins, J. R. & Nimmo, H. G. (1996). Propionyl-CoA carboxylase from Streptomyces coelicolor A3(2): cloning of the gene encoding the biotin-containing subunit. Microbiology 142, 649-655.[Abstract]
Browner, M., Taroni, F., Stzul, E. & Rosenberg, L. (1989). Sequence analysis, biogenesis and mitochondrial import of the alpha subunit of rat liver propionyl-CoA carboxylase. J Biol Chem 264, 1280-1285.
Burnette, W. N. (1981). Western blotting: electrophoretic transfer of proteins from sodium dodecyl sulfatepolyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem 112, 195-203.[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.[Medline]
Cronan, J. E.Jr (1990). Biotination of proteins in vivo. J Biol Chem 265, 10327-10333.
Donadio, S., Staver, M. & Katz, L. (1996). Erythromycin production in Saccharopolyspora erythraea does not require a functional propionyl-CoA carboxylase Mol Microbiol 19, 977-984.[Medline]
Erfle, J. D. (1973). Acetyl-CoA and propionyl-CoA carboxylation by Mycobacterium phlei. Partial purification and some properties of the enzyme. Biochim Biophys Acta 316, 143-155.[Medline]
Floriano, B. & Bibb, M. J. (1996). afsR is a pleiotropic but conditionally required regulatory gene for antibiotic production in Streptomyces coelicolor A3(2). Mol Microbiol 21, 385-396.[Medline]
Gorst-Allman, C. P., Rudd, B. A. M., Chang, C. J. & Floss, H. G. (1981). Biosynthesis of actinorhodin. Point of dimerization. J Org Chem 46, 455-456.
Gottschalk, G. (1986). Bacterial metabolism, 2nd edn. New York: Springer.
Hanahan, D. (1983). Studies of transformation of Escherichia coli with plasmids. J Mol Biol 166, 557-580.[Medline]
Harder, M. E., Beacham, I., Cronan, J.Jr, Beachan, K., Honegger, J. & Silbert, D. (1972). Temperature-sensitive mutants of Escherichia coli requiring saturated and unsaturated fatty acids for growth: isolation and properties. Proc Natl Acad Sci USA 69, 3105-3109.[Abstract]
Harwood, J. L. (1988). Fatty acid metabolism. Annu Rev Plant Physiol 39, 101-138.
Henrikson, K. P. & Allen, S. H. G. (1979). Purification and subunit structure of propionyl coenzyme A carboxylase of Mycobacterium smegmatis J Biol Chem 254, 5888-5891.[Abstract]
Hopwood, D. A. & Sherman, D. H. (1990). Molecular genetics of polyketides and its comparison to fatty acid biosynthesis. Annu Rev Genet 24, 37-66.[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.
Hopwood, D., Chater, K. & Bibb, M. J. (1994). Genetics of antibiotic production in Streptomyces coelicolor A3(2), a model streptomycete. In Genetics and Biochemistry of Antibiotic Production, pp. 65-102. Edited by L. C. Vining & C. Studdard. Wenheim: Butterworth-Heinemann.
Hunaiti, A. R. & Kolattukudy, P. E. (1982). Isolation and characterisation of an acyl-coenzyme A carboxylase from an erythromycin-producing Streptomyces erythyreus. Arch Biochem Biophys 216, 362-371.[Medline]
Janssen, G. R. & Bibb, M. J. (1993). Derivatives of pUC18 that have BglII sites flanking a modified multiple cloning site and that retain the ability to identify recombinant clones by visual screening of Escherichia coli colonies. Gene 124, 133-134.[Medline]
Kao, C., Luo, G., Katz, L., Cane, D. & Khosla, C. (1994). Engineered biosynthesis of a complete macrolactone in a heterologous host. J Am Chem Soc 116, 11612-11613.
Katz, L. & Donadio, S. (1993). Polyketide synthesis: prospects for hybrid antibiotics. Annu Rev Microbiol 47, 875-912.[Medline]
Kondo, H., Shiratsuchi, K., Yoshimoto, T., Masuda, T., Kitazono, A., Tsuru, D., Anai, M., Sekiguchi, M. & Tanabe, T. (1991). Acetyl-CoA carboxylase from Escherichia coli: gene organization and nucleotide sequence of the biotin carboxylase subunit. Proc Natl Acad Sci USA 88, 9730-9733.[Abstract]
Laakel, M., Lebrihi, A., Khaoua, S., Schneider, F., Lefebvre, G. & Germain, P. (1994). A link between primary and secondary metabolism: malonyl-CoA formation in Streptomyces ambofaciens growing on ammonium ions or valine. Microbiology 140, 1451-1456.
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685.[Medline]
Li, S. J. & Cronan, J. E.Jr (1992). The gene encoding the biotin carboxylase subunit of Escherichia coli acetyl-CoA carboxylase. J Biol Chem 267, 16841-16847.
MacNeil, D. J., Gewain, K. M., Ruby, C. L., Dezeny, G., Gibbons, P. H. & MacNeil, T. (1992). Analysis of Streptomyces avermitilis genes required for avermectin biosynthesis utilizing a novel integration vector. Gene 111, 61-68.[Medline]
Martín, J. F. & Liras, P. (1989). Organization and expression of genes involved in the biosynthesis of antibiotics and other secondary metabolites. Annu Rev Microbiol 43, 173-206.[Medline]
Nikolau, B. J., Wurtele, E. S. & Stumpf, P. K. (1985). Acetyl-coenzyme A carboxylase in maize leaves. Anal Biochem 149, 448-453.[Medline]
Norman, E., De Smet, K. A. L., Stoker, N. G., Ratledge, C., Wheeler, P. R. & Dale, J. W. (1994). Lipid synthesis in Mycobacteria: characterization of the biotin carboxyl carrier protein genes from Mycobacterium leprae and Mycobacterium tuberculosis. J Bacteriol 176, 2525-2531.[Abstract]
Paget, M. S. B., Chamberlin, L., Atrih, A., Foster, S. J. & Buttner, M. J. (1999). Evidence that the extracytoplasmic function sigma factor E is required for normal cell wall structure in Streptomyces coelicolor A3(2). J. Bacteriol 181, 204-211.
Polakis, S., Guchhait, R. & Lane, M. (1972). On the possible involvement of a carbonyl phosphate group intermediate in the adenosine triphosphate-dependent carboxylation of biotin. J Biol Chem 247, 1335-1337.
Redenbach, M., Kieser, H. M., Denapaite, D., Eichner, A., Cullum, J., Kinashi, H. & Hopwood, D. (1996). A set of ordered cosmids and a detailed genetic and physical map for the 8 Mb Streptomyces coelicolor A3(2) chromosome. Mol Microbiol 21, 77-95.[Medline]
Sambrook, J., Fritsch, E. F. & 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 USA 74, 5463-5467.[Abstract]
Studier, F. W. & Moffatt, B. A. (1986). Use of the bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol 189, 113-130.[Medline]
Wasserman, H., Rodgers, G. & Keith, D. (1976). Undecylprodigiosin. Tetrahedron 32, 1851-1854.
Wright, F. & Bibb, M. J. (1992). Codon usage in the G+C-rich Streptomyces genome. Gene 113, 55-65.[Medline]
Zerbe-Burkhardt, K., Ratnatilleke, A., Phillipon, N., Birch, A., Leiser, A., Vrijbloed, J., Hess, D., Hunziker, P. & Robinson, J. (1998). Cloning, sequencing, expression, and insertional inactivation of the gene for the large subunit of the coenzyme B12-dependent isobutyryl-CoA mutase from Streptomyces cinnamonensis. J Biol Chem 273, 6508-6517.
Received 13 April 1999;
revised 21 June 1999;
accepted 14 July 1999.