Biochemical and Molecular Analyses of the Streptococcus pneumoniae Acyl Carrier Protein Synthase, an Enzyme Essential for Fatty Acid Biosynthesis
2000; Elsevier BV; Volume: 275; Issue: 40 Linguagem: Inglês
10.1074/jbc.m004475200
ISSN1083-351X
AutoresKelly A. McAllister, Robert B. Peery, Timothy I. Meier, Anthony S. Fischl, Genshi Zhao,
Tópico(s)Antibiotic Resistance in Bacteria
ResumoAcyl carrier protein synthase (AcpS) is an essential enzyme in the biosynthesis of fatty acids in all bacteria. AcpS catalyzes the transfer of 4′-phosphopantetheine from coenzyme A (CoA) to apo-ACP, thus converting apo-ACP to holo-ACP that serves as an acyl carrier for the biosynthesis of fatty acids and lipids. To further understand the physiological role of AcpS, we identified, cloned, and expressed the acpS and acpP genes ofStreptococcus pneumoniae and purified both products to homogeneity. Both acpS and acpP form operons with the genes whose functions are required for other cellular metabolism. The acpS gene complements an Escherichia coli mutant defective in the production of AcpS and appears to be essential for the growth of S. pneumoniae. Gel filtration and cross-linking analyses establish that purified AcpS exists as a homotrimer. AcpS activity was significantly stimulated by apo-ACP at concentrations over 10 μm and slightly inhibited at concentrations of 5–10 μm. Double reciprocal analysis of initial velocities of AcpS at various concentrations of CoA or apo-ACP indicated a random or compulsory ordered bi bi type of reaction mechanism. Further analysis of the inhibition kinetics of the product (3′,5′-ADP) suggested that it is competitive with respect to CoA but mixed (competitive and noncompetitive) with respect to apo-ACP. Finally, apo-ACP bound tightly to AcpS in the absence of CoA, but CoA failed to do so in the absence of apo-ACP. Together, these results suggest that AcpS may be allosterically regulated by apo-ACP and probably proceeds by an ordered reaction mechanism with the first formation of the AcpS-apo-ACP complex and the subsequent transfer of 4′-phosphopantetheine to the apo-ACP of the complex. Acyl carrier protein synthase (AcpS) is an essential enzyme in the biosynthesis of fatty acids in all bacteria. AcpS catalyzes the transfer of 4′-phosphopantetheine from coenzyme A (CoA) to apo-ACP, thus converting apo-ACP to holo-ACP that serves as an acyl carrier for the biosynthesis of fatty acids and lipids. To further understand the physiological role of AcpS, we identified, cloned, and expressed the acpS and acpP genes ofStreptococcus pneumoniae and purified both products to homogeneity. Both acpS and acpP form operons with the genes whose functions are required for other cellular metabolism. The acpS gene complements an Escherichia coli mutant defective in the production of AcpS and appears to be essential for the growth of S. pneumoniae. Gel filtration and cross-linking analyses establish that purified AcpS exists as a homotrimer. AcpS activity was significantly stimulated by apo-ACP at concentrations over 10 μm and slightly inhibited at concentrations of 5–10 μm. Double reciprocal analysis of initial velocities of AcpS at various concentrations of CoA or apo-ACP indicated a random or compulsory ordered bi bi type of reaction mechanism. Further analysis of the inhibition kinetics of the product (3′,5′-ADP) suggested that it is competitive with respect to CoA but mixed (competitive and noncompetitive) with respect to apo-ACP. Finally, apo-ACP bound tightly to AcpS in the absence of CoA, but CoA failed to do so in the absence of apo-ACP. Together, these results suggest that AcpS may be allosterically regulated by apo-ACP and probably proceeds by an ordered reaction mechanism with the first formation of the AcpS-apo-ACP complex and the subsequent transfer of 4′-phosphopantetheine to the apo-ACP of the complex. acyl carrier protein acyl carrier protein synthase coenzyme A polyacrylamide gel electrophoresis high performance liquid chromatography polymerase chain reaction 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid ethylene glycolbis(succinimidylsuccinate) N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine The biosynthesis of fatty acids is known to be required for the growth of bacteria as fatty acids are essential components of bacterial membrane lipids and lipopolysacharides (1Cronan Jr., J.E. Rock C.O. Neidhardt F.C. Curtiss III, R. Ingramham J.L. Lin E.C.C. Low K.B. Magasanik B. Reznikoff W.S. Riley M. Schaechter M. Umbarger H.E. Escherichia coli and Salmonella: Cellular and Molecular Biology. American Society for Microbiology Press, Washington, D. C.1996: 612-636Google Scholar, 2Rock C.O. Cronan Jr., J.E. Biochem. Biophys. Acta. 1996; 1302: 1-16Crossref PubMed Scopus (291) Google Scholar). The fatty acid biosynthetic pathway in bacteria is well characterized (1Cronan Jr., J.E. Rock C.O. Neidhardt F.C. Curtiss III, R. Ingramham J.L. Lin E.C.C. Low K.B. Magasanik B. Reznikoff W.S. Riley M. Schaechter M. Umbarger H.E. Escherichia coli and Salmonella: Cellular and Molecular Biology. American Society for Microbiology Press, Washington, D. C.1996: 612-636Google Scholar, 2Rock C.O. Cronan Jr., J.E. Biochem. Biophys. Acta. 1996; 1302: 1-16Crossref PubMed Scopus (291) Google Scholar). Bacteria utilize the type II or dissociated, fatty acid synthase system for fatty acid synthesis (1Cronan Jr., J.E. Rock C.O. Neidhardt F.C. Curtiss III, R. Ingramham J.L. Lin E.C.C. Low K.B. Magasanik B. Reznikoff W.S. Riley M. Schaechter M. Umbarger H.E. Escherichia coli and Salmonella: Cellular and Molecular Biology. American Society for Microbiology Press, Washington, D. C.1996: 612-636Google Scholar, 2Rock C.O. Cronan Jr., J.E. Biochem. Biophys. Acta. 1996; 1302: 1-16Crossref PubMed Scopus (291) Google Scholar). The type II fatty acid synthase system consists of individual enzymes that are encoded by separate genes (1Cronan Jr., J.E. Rock C.O. Neidhardt F.C. Curtiss III, R. Ingramham J.L. Lin E.C.C. Low K.B. Magasanik B. Reznikoff W.S. Riley M. Schaechter M. Umbarger H.E. Escherichia coli and Salmonella: Cellular and Molecular Biology. American Society for Microbiology Press, Washington, D. C.1996: 612-636Google Scholar,2Rock C.O. Cronan Jr., J.E. Biochem. Biophys. Acta. 1996; 1302: 1-16Crossref PubMed Scopus (291) Google Scholar). On the other hand, the type I fatty acid synthase system, almost exclusively present in eukaryotes, is characterized by the presence of a multifunctional protein that possesses all the catalytic activities required for fatty acid synthesis (3Smith S. FASEB J. 1994; 8: 1248-1259Crossref PubMed Scopus (524) Google Scholar). In both systems, fatty acids are synthesized by using a repeated cycle of condensation, reduction, dehydration, and reduction reactions (1Cronan Jr., J.E. Rock C.O. Neidhardt F.C. Curtiss III, R. Ingramham J.L. Lin E.C.C. Low K.B. Magasanik B. Reznikoff W.S. Riley M. Schaechter M. Umbarger H.E. Escherichia coli and Salmonella: Cellular and Molecular Biology. American Society for Microbiology Press, Washington, D. C.1996: 612-636Google Scholar, 2Rock C.O. Cronan Jr., J.E. Biochem. Biophys. Acta. 1996; 1302: 1-16Crossref PubMed Scopus (291) Google Scholar, 3Smith S. FASEB J. 1994; 8: 1248-1259Crossref PubMed Scopus (524) Google Scholar). In these reactions, holo-acyl carrier protein (holo-ACP)1 plays an essential role as an acyl carrier for fatty acid precursors, growing acyl intermediates, and nascent fatty acid products (1Cronan Jr., J.E. Rock C.O. Neidhardt F.C. Curtiss III, R. Ingramham J.L. Lin E.C.C. Low K.B. Magasanik B. Reznikoff W.S. Riley M. Schaechter M. Umbarger H.E. Escherichia coli and Salmonella: Cellular and Molecular Biology. American Society for Microbiology Press, Washington, D. C.1996: 612-636Google Scholar, 2Rock C.O. Cronan Jr., J.E. Biochem. Biophys. Acta. 1996; 1302: 1-16Crossref PubMed Scopus (291) Google Scholar, 3Smith S. FASEB J. 1994; 8: 1248-1259Crossref PubMed Scopus (524) Google Scholar, 4Elovson J. Vagelos P.R. J. Biol. Chem. 1968; 243: 3603-3611Abstract Full Text PDF PubMed Google Scholar, 5Lambalot R.H. Walsh C.T. J. Biol. Chem. 1995; 270: 24658-24661Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar). ACP is a small acidic protein in bacteria (1Cronan Jr., J.E. Rock C.O. Neidhardt F.C. Curtiss III, R. Ingramham J.L. Lin E.C.C. Low K.B. Magasanik B. Reznikoff W.S. Riley M. Schaechter M. Umbarger H.E. Escherichia coli and Salmonella: Cellular and Molecular Biology. American Society for Microbiology Press, Washington, D. C.1996: 612-636Google Scholar, 2Rock C.O. Cronan Jr., J.E. Biochem. Biophys. Acta. 1996; 1302: 1-16Crossref PubMed Scopus (291) Google Scholar) or a small domain of the type I fatty acid synthase in eukaryotes (3Smith S. FASEB J. 1994; 8: 1248-1259Crossref PubMed Scopus (524) Google Scholar). ACP inEscherichia coli is encoded by the acpP gene (1Cronan Jr., J.E. Rock C.O. Neidhardt F.C. Curtiss III, R. Ingramham J.L. Lin E.C.C. Low K.B. Magasanik B. Reznikoff W.S. Riley M. Schaechter M. Umbarger H.E. Escherichia coli and Salmonella: Cellular and Molecular Biology. American Society for Microbiology Press, Washington, D. C.1996: 612-636Google Scholar,2Rock C.O. Cronan Jr., J.E. Biochem. Biophys. Acta. 1996; 1302: 1-16Crossref PubMed Scopus (291) Google Scholar). The newly synthesized ACP, or apo-ACP, is not functional in fatty acid synthesis. The conversion of apo-ACP to holo-ACP by ACP synthase (AcpS) is required for its functionality (1Cronan Jr., J.E. Rock C.O. Neidhardt F.C. Curtiss III, R. Ingramham J.L. Lin E.C.C. Low K.B. Magasanik B. Reznikoff W.S. Riley M. Schaechter M. Umbarger H.E. Escherichia coli and Salmonella: Cellular and Molecular Biology. American Society for Microbiology Press, Washington, D. C.1996: 612-636Google Scholar, 2Rock C.O. Cronan Jr., J.E. Biochem. Biophys. Acta. 1996; 1302: 1-16Crossref PubMed Scopus (291) Google Scholar, 4Elovson J. Vagelos P.R. J. Biol. Chem. 1968; 243: 3603-3611Abstract Full Text PDF PubMed Google Scholar, 5Lambalot R.H. Walsh C.T. J. Biol. Chem. 1995; 270: 24658-24661Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar). AcpS catalyzes the transfer of the 4′-phosphopantetheine moiety from coenzyme A (CoA) onto a serine residue of apo-ACP, thereby converting apo-ACP to holo-ACP (1Cronan Jr., J.E. Rock C.O. Neidhardt F.C. Curtiss III, R. Ingramham J.L. Lin E.C.C. Low K.B. Magasanik B. Reznikoff W.S. Riley M. Schaechter M. Umbarger H.E. Escherichia coli and Salmonella: Cellular and Molecular Biology. American Society for Microbiology Press, Washington, D. C.1996: 612-636Google Scholar, 2Rock C.O. Cronan Jr., J.E. Biochem. Biophys. Acta. 1996; 1302: 1-16Crossref PubMed Scopus (291) Google Scholar, 4Elovson J. Vagelos P.R. J. Biol. Chem. 1968; 243: 3603-3611Abstract Full Text PDF PubMed Google Scholar, 5Lambalot R.H. Walsh C.T. J. Biol. Chem. 1995; 270: 24658-24661Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, 6Flugel R.S. Hwangbo Y. Lambalot R.H. Cronan Jr., J.E. Walsh C.T. J. Biol. Chem. 2000; 275: 959-968Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 7Lambalot R.H. Gehring A.M. Flugel R.S. Zuber P. LaCelle M. Marahiel M.A. Reid R. Khosla C. Walsh C.T. Curr. Biol. 1996; 3: 923-936Scopus (725) Google Scholar). The holo-ACP formed then mediates the transfer of acyl intermediates by the covalent attachment of all acyl intermediates via their carboxyl group to the thiol group of the 4′-phosphopantetheine group of holo-ACP (1Cronan Jr., J.E. Rock C.O. Neidhardt F.C. Curtiss III, R. Ingramham J.L. Lin E.C.C. Low K.B. Magasanik B. Reznikoff W.S. Riley M. Schaechter M. Umbarger H.E. Escherichia coli and Salmonella: Cellular and Molecular Biology. American Society for Microbiology Press, Washington, D. C.1996: 612-636Google Scholar, 2Rock C.O. Cronan Jr., J.E. Biochem. Biophys. Acta. 1996; 1302: 1-16Crossref PubMed Scopus (291) Google Scholar, 3Smith S. FASEB J. 1994; 8: 1248-1259Crossref PubMed Scopus (524) Google Scholar, 4Elovson J. Vagelos P.R. 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Brignell S.C. et al.Nature. 1997; 390: 249-256Crossref PubMed Scopus (3134) Google Scholar, 14Tomb J.-F. White O Kerlavage A.R. Clayton R.A. Sutton G.G. Fleischmann R.D. Ketchum K.A. Klenk H.P. Gill S. Dougherty B.A. Nelson K. Quackenbush J. Zhou L. Kirkness E.F. Peterson S. et al.Nature. 1997; 388: 539-547Crossref PubMed Scopus (3028) Google Scholar). E. coli AcpS has been well studied (4Elovson J. Vagelos P.R. J. Biol. Chem. 1968; 243: 3603-3611Abstract Full Text PDF PubMed Google Scholar, 5Lambalot R.H. Walsh C.T. J. Biol. Chem. 1995; 270: 24658-24661Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, 6Flugel R.S. Hwangbo Y. Lambalot R.H. Cronan Jr., J.E. Walsh C.T. J. Biol. Chem. 2000; 275: 959-968Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 7Lambalot R.H. Gehring A.M. Flugel R.S. Zuber P. LaCelle M. Marahiel M.A. Reid R. Khosla C. Walsh C.T. Curr. Biol. 1996; 3: 923-936Scopus (725) Google Scholar, 8Majerus P.W. Alberts A.W. Vagelos P.R. Proc. Natl. Acad. Sci. U. S. A. 1965; 53: 410-417Crossref PubMed Scopus (64) Google Scholar, 15Gehring A.M. Lambalot R.H. Vogel K.W. Drueckhammer D.G. Walsh C.T. Chem. Biol. 1997; 4: 17-24Abstract Full Text PDF PubMed Scopus (103) Google Scholar). The acpS gene from E. coli forms an operon with the upstream gene, pdxJ, whose function is required for vitamin B6 biosynthesis (16Lam H.-M. Tancula E. Dempsey W.B. Winkler M.E. J. Bacteriol. 1992; 174: 1554-1567Crossref PubMed Google Scholar,17Takiff J.E. Baker T. Copeland T. Chen S-M. Court D.L. J. Bacteriol. 1992; 174: 1544-1553Crossref PubMed Google Scholar). The acpS gene was originally identified asdpj (downstream of pdxJ) whose function, although unknown, was required for the growth of E. coli (16Lam H.-M. Tancula E. Dempsey W.B. Winkler M.E. J. Bacteriol. 1992; 174: 1554-1567Crossref PubMed Google Scholar, 17Takiff J.E. Baker T. Copeland T. Chen S-M. Court D.L. J. Bacteriol. 1992; 174: 1544-1553Crossref PubMed Google Scholar). Later, the landmark biochemical study by Lambalot and Walsh (5Lambalot R.H. Walsh C.T. J. Biol. Chem. 1995; 270: 24658-24661Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar) led to the identification of Dpj as AcpS. E. coli AcpS is a small, highly basic protein of about 14 kDa (5Lambalot R.H. Walsh C.T. J. Biol. Chem. 1995; 270: 24658-24661Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar). The E. coli enzyme has been purified and characterized (5Lambalot R.H. Walsh C.T. J. Biol. Chem. 1995; 270: 24658-24661Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar). The purified AcpS appears to be a homodimer (5Lambalot R.H. Walsh C.T. J. Biol. Chem. 1995; 270: 24658-24661Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar). The enzyme exhibits a broad substrate specificity and can utilize a variety of ACPs that are required for many diverse aspects of cellular metabolism (5Lambalot R.H. Walsh C.T. J. Biol. Chem. 1995; 270: 24658-24661Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, 6Flugel R.S. Hwangbo Y. Lambalot R.H. Cronan Jr., J.E. Walsh C.T. J. Biol. Chem. 2000; 275: 959-968Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 7Lambalot R.H. Gehring A.M. Flugel R.S. Zuber P. LaCelle M. Marahiel M.A. Reid R. Khosla C. Walsh C.T. Curr. Biol. 1996; 3: 923-936Scopus (725) Google Scholar, 8Majerus P.W. Alberts A.W. Vagelos P.R. Proc. Natl. Acad. Sci. U. S. A. 1965; 53: 410-417Crossref PubMed Scopus (64) Google Scholar, 15Gehring A.M. Lambalot R.H. Vogel K.W. Drueckhammer D.G. Walsh C.T. Chem. Biol. 1997; 4: 17-24Abstract Full Text PDF PubMed Scopus (103) Google Scholar,18Carreras C.W. Gehring A.M. Walsh C.T. Khosla C. Biochemistry. 1997; 36: 11757-11761Crossref PubMed Scopus (42) Google Scholar, 19Crosby J. Sherman D.H. Bibb M.J. Revill W.P. Hopwood D.A. Simpson T.J. Biochim. Biophys. Acta. 1995; 1251: 32-42Crossref PubMed Scopus (57) Google Scholar, 20Kutchma A.J. Hoang T.T. Schweizer H.P. J. Bacteriol. 1999; 181: 5498-5504Crossref PubMed Google Scholar, 21Tropf S. Revill W.P. Bibb M.J. Howood D.A. Schweizer M. Chem. Biol. 1998; 5: 135-146Abstract Full Text PDF PubMed Scopus (21) Google Scholar, 22Zhou P. Florova G. Reynolds K.A. Chem. Biol. 1999; 6: 577-584Abstract Full Text PDF PubMed Scopus (39) Google Scholar). These results indicate that AcpS may be able to participate in other metabolism besides fatty acid biosynthesis in the cell. Purified AcpS also exhibits activity with a number of CoA derivatives (15Gehring A.M. Lambalot R.H. Vogel K.W. Drueckhammer D.G. Walsh C.T. Chem. Biol. 1997; 4: 17-24Abstract Full Text PDF PubMed Scopus (103) Google Scholar). Finally, AcpS is a very low abundance protein in E. coli (4Elovson J. Vagelos P.R. J. Biol. Chem. 1968; 243: 3603-3611Abstract Full Text PDF PubMed Google Scholar, 5Lambalot R.H. Walsh C.T. J. Biol. Chem. 1995; 270: 24658-24661Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar). In contrast, ACP is a very abundant protein that was estimated to be present at 25,000–60,000 molecules/cell (1Cronan Jr., J.E. Rock C.O. Neidhardt F.C. Curtiss III, R. Ingramham J.L. Lin E.C.C. Low K.B. Magasanik B. Reznikoff W.S. Riley M. Schaechter M. Umbarger H.E. Escherichia coli and Salmonella: Cellular and Molecular Biology. American Society for Microbiology Press, Washington, D. C.1996: 612-636Google Scholar, 2Rock C.O. Cronan Jr., J.E. Biochem. Biophys. Acta. 1996; 1302: 1-16Crossref PubMed Scopus (291) Google Scholar, 23Jackowski S. Rock C.O. J. Bacteriol. 1984; 158: 115-120Crossref PubMed Google Scholar,24Vallari D.S. Jackowski S. J. Bacteriol. 1988; 170: 3961-3966Crossref PubMed Google Scholar). The majority of ACPs present in the cell are found to be holo-ACPs (1Cronan Jr., J.E. Rock C.O. Neidhardt F.C. Curtiss III, R. Ingramham J.L. Lin E.C.C. Low K.B. Magasanik B. Reznikoff W.S. Riley M. Schaechter M. Umbarger H.E. Escherichia coli and Salmonella: Cellular and Molecular Biology. American Society for Microbiology Press, Washington, D. C.1996: 612-636Google Scholar, 2Rock C.O. Cronan Jr., J.E. Biochem. Biophys. Acta. 1996; 1302: 1-16Crossref PubMed Scopus (291) Google Scholar, 25Jackowski S. Rock C.O. J. Biol. Chem. 1983; 258: 15186-15191Abstract Full Text PDF PubMed Google Scholar, 26Heath R.J. Rock C.O. J. Biol. Chem. 1996; 271: 1833-1836Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar). Although E. coli AcpS is well studied, the reaction mechanism of AcpS remains unknown. In addition, only the AcpS fromE. coli, a rod-shaped, Gram-negative bacterium, has been thoroughly characterized to date. It still remains to be determined whether AcpS from Gram-positive bacteria would play the same physiological role. Finally, AcpS appears to possess all of the features necessary for a good antibacterial target, such as its essential nature, widespread existence in bacteria, and unique catalytic position in a pathway (fatty acid biosynthesis). Thus, AcpS might be a valuable antibacterial target for identifying novel antimicrobial agents. To better understand the function of AcpS inStreptococcus pneumoniae, a sphere-shaped, Gram-positive bacterium and also a major human pathogen of the upper respiratory tract, and to explore AcpS as an antibacterial target, we first cloned and expressed the acpS and acpP genes of S. pneumoniae and characterized both gene products. The results of this study show that S. pneumoniae AcpS shares many biochemical properties with E. coli AcpS but also exhibits major differences with respect to their native structures and substrate regulations. In addition, the results of this study suggest that AcpS proceeds by an ordered reaction mechanism with the first formation of the enzyme-apo-ACP intermediate from apo-ACP followed by the transfer of 4′-phosphopantetheine from CoA to the apo-ACP of the complex. Finally, both acpS and acpP form complex operons with the genes whose functions are not required for fatty acid biosynthesis. Unless specified otherwise, all fine chemicals were purchased from Sigma. All fast protein liquid chromatography resins and columns used for protein purification and strains and reagents for construction, expression, and purification of GST-fused proteins were obtained from Amersham Pharmacia Biotech. Luria Bertani (LB) broth medium was purchased from Bio 101, Inc. (Vista, CA). All polyacrylamide gels and reagents were purchased from Novex (San Diego, CA). SYPRO Orange and Bradford protein assay reagents were purchased from Bio-Rad, and ethylene glycolbis(succinimidylsuccinate) (sulfo-EGS) was obtained from Pierce. [3H]CoA (specific activity, 1.5 Ci/mmol) was custom-synthesized by NEN Life Science Products. The acpP andacpS genes were cloned from S. pneumoniae by PCR as described before (27Zhao G. Meier T.I. Peery R.B Matsushima P. Skatrud P.L. Microbiology. 1999; 145: 791-800Crossref PubMed Scopus (15) Google Scholar). All of the reagents, plasmids, and cell lines used for cloning and expression were the same as those described before (27Zhao G. Meier T.I. Peery R.B Matsushima P. Skatrud P.L. Microbiology. 1999; 145: 791-800Crossref PubMed Scopus (15) Google Scholar). Based on the initial sequence of acpS, it was thought that the acpS open reading frame started at the second Met codon (see GenBankTM accession numberAF276617). Thus, the primers corresponding to this sequence were designed and used for cloning the S. pneumoniae acpS gene (see below). However, we have recently realized that the open reading frame of the acpS gene probably starts at the first Met codon rather than the second codon (GenBankTMaccession number AF276617). Thus, the acpS gene cloned and expressed might lack the first two codons encoding the amino acid residues of Met-Arg. To clone the acpS gene, the following PCR primers were designed and used to amplify the acpS gene for cloning into E. coli expression systems. The 5′ PCR primer (5′-CGCGGATCCCATATGATAGTTGGACACGGAATTG-3′) was designed at the ATG start codon of acpS and contains BamHI andNdeI sites for cloning purposes. The 3′ PCR primer (5′-CGCGGATCCCTAGCTTTCATGAATTTCCTCC-3′) was designed at the stop codon of acpS and contains a BamHI site after the stop codon. Using these primers, acpS was PCR-amplified from S. pneumoniae for 25 cycles under the conditions as described before (27Zhao G. Meier T.I. Peery R.B Matsushima P. Skatrud P.L. Microbiology. 1999; 145: 791-800Crossref PubMed Scopus (15) Google Scholar). Five PCR products were combined, and a portion of the pooled PCR products was digested withBamHI. The BamHI-digested PCR fragment was cloned into pCZA342, a low copy number plasmid (28Baltz, R. H., Matsushima, P., Peery, R. B., Hoskins, J., Young, M., Norris, F. H., DeHoff, B. S., Rockey, P., Porter, G., Burgett, S. G., Rosteck, P. R., Skatrud, P. L., and Jaskunas, S. R. (1997) Abstracts of the 37th ICAAC, p. 391(S-46), Interscience Conference on Antimicrobial Agents and Chemotherapy, Toronto, Canada.Google Scholar) that had been digested with BamHI and dephosphorylated with calf intestinal alkaline phosphatase. acpS from several pCZA342 clones was sequenced, and a clone containing the consensus acpS gene sequence was used for constructing expression systems. This pCZA342 clone was digested with NdeI and BamHI. TheNdeI–BamHI DNA fragment containingacpS was subcloned into pET-11a (Novagen). The resulting construct was designated as pRBP-19. The pCZA342 clone was also digested with BamHI, and the BamHI fragment ofacpS was subcloned into pGEX-2T, resulting in pRBP-20. To clone the acpP gene, the following PCR primers were used for amplification: the 5′ PCR primer (5′-CGCGGATCCCATATGACAGAAAAAGAAATTTTTGACCGTATTG -3′) and the 3′ PCR primer (5′-CGCGGATCCGAATTCCTATTTTCCTTGAATGATTTTAACCACATC-3′). Using these primers, acpP was PCR-amplified from S. pneumoniae as described above. The PCR products were digested withBamHI. The BamHI-digested PCR fragment was cloned into pCZA342. The pCZA342 clone was digested with NdeI andBamHI. The NdeI–BamHI DNA fragment containing acpP was subcloned into pET-11a (Novagen), resulting in pRBP-16. LY128 (E. coli BL21 (pLysS)/pRBP-19) was first grown at 35 °C overnight in LB broth medium supplemented with 100 μg/ml ampicillin. The overnight culture (40 ml) was then inoculated into 1000 ml of LB medium supplemented with ampicillin and grown at 33 °C with shaking at 250 rpm until an A 590 of 0.5–0.6 was reached. The culture was induced with 1 mmisopropyl-1-thio-β-d-galactopyranoside for 3 h. Cells were harvested by centrifugation at 4500 × g at 4 °C for 8 min, washed twice in phosphate-buffered saline (PBS), resuspended in 50 mm citrate phosphate, pH 6.0, and disrupted by passing twice through a French pressure cell. The resulting cell extract was centrifuged at 160,000 × gfor 40 min at 4 °C. The supernatant fraction was collected and applied to a 15S Source S column (2.5 × 8 cm) that had been equilibrated with 50 mm citrate phosphate, pH 6.0 (buffer A). The column was washed with buffer A and eluted with a linear gradient of 0–1.0 m KCl in buffer A. Fractions (7 ml each) were collected. The presence of AcpS in the fractions was detected by SDS-PAGE analysis (16% Tricine gels) (29Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar). The fractions containing AcpS were pooled and applied to a S-100 Sepharose preparative gel filtration fast protein liquid chromatography column (5.0 × 60 cm) equilibrated with 50 mm Tris-HCl, pH 7.0, 100 mm KCl. The fractions containing AcpS were collected, adjusted with glycerol to a final concentration of 15% (v/v), and stored in small aliquots at −70 °C. Protein concentration was determined using a protein assay kit (Bio-Rad) with bovine serum albumin as a standard (30Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217544) Google Scholar). LY135 (E. coli XL1 Blue (mRF′)/pRBP-20) was grown, induced, harvested, disrupted, and centrifuged as above. The supernatant fraction was applied to a glutathione-Sepharose 4B column (10 ml) that had been equilibrated with 100 ml of PBS. The column was washed with 100 ml of PBS, and the GST-AcpS fusion protein was eluted with 10 mm glutathione in PBS. Fractions were analyzed by SDS-PAGE (12% glycine), and those fractions containing GST-AcpS were pooled, dialyzed against 50 mm Tris-HCl, pH 7.0 (4 liter), adjusted with glycerol to a final concentration of 15% (v/v), and stored at −70 °C as described above. LY140 (E. coli BL21 (pLysS)/pRBP-16) was grown, induced, harvested, and disrupted as described above. The resulting cell extract was centrifuged as described above. The supernatant fraction was collected and applied to a 15S Source Q column (2.5 × 8 cm) that had been equilibrated with 50 mm Tris-HCl, pH 8.0, 100 mm KCl (buffer C). The column was washed with 100 ml of buffer C and eluted with a linear gradient of 0.0–1.0 mKCl in buffer C. Fractions (7 ml each) were collected, and the presence of apo-ACP in the fractions was detected by SDS-PAGE as described above (16% tricine gels). The fractions containing apo-ACP were pooled and applied to a S-100 Sepharose gel filtration column (5 × 60 cm) equilibrated with 50 mm Tris-HCl, pH 7.0, 100 mm KCl. The column was eluted with the same buffer. Fractions (10 ml each) containing apo-ACP were collected, analyzed by electrospray mass spectrometry, and stored at −70 °C as described above. To determine the native structure of AcpS, a purified AcpS preparation (375 μg) was applied to a S-75 Superdex gel filtration column (HR 1.0 × 30 cm), equilibrated with 50 mm Tris-HCl, pH 7.0, 50 mm KCl, 10 mm MgCl2. The column was calibrated with the protein molecular weight standards (Sigma). The effect of detergent or salt on the native structure of AcpS was analyzed by treating AcpS with 6 mm CHAPS or 50–500 mm KCl before and during column chromatography. Sedimentation centrifugation analysis of AcpS was carried out using an XLA ultracentrifuge (Beckman Instruments, Fullerton, CA). A purified AcpS preparation (adjusted to 0.2 and 0.4 mg/ml) was centrifuged at 16,000 rpm for 24 h at 22 °C. The absorbance at 280 nm as a function of radius after the system reaches equilibrium was analyzed using XL-A/XL-1, a nonlinear le
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