Function of the Escherichia coli msbB Gene, a Multicopy Suppressor of htrB Knockouts, in the Acylation of Lipid A
1997; Elsevier BV; Volume: 272; Issue: 16 Linguagem: Inglês
10.1074/jbc.272.16.10353
ISSN1083-351X
AutoresTony Clementz, Zhimin Zhou, Christian R.H. Raetz,
Tópico(s)RNA and protein synthesis mechanisms
ResumoOverexpression of the Escherichia coli msbB gene on high copy plasmids suppresses the temperature-sensitive growth associated with mutations in thehtrB gene. htrB encodes the lauroyl transferase of lipid A biosynthesis that acylates the intermediate (Kdo)2-lipid IVA (Brozek, K. A., and Raetz, C. R. H. (1990) J. Biol. Chem.265, 15410–15417). SincemsbB displays 27.5% identity and 42.2% similarity tohtrB, we explored the possibility that msbBencodes a related acyltransferase. In contrast to htrB, extracts of strains with insertion mutations in msbB are not defective in transferring laurate from lauroyl acyl carrier protein to (Kdo)2-lipid IVA. However, extracts ofmsbB mutants do not efficiently acylate the product formed by HtrB, designated (Kdo)2-(lauroyl)-lipid IVA. Extracts of strains harboring msbB + bearing plasmids acylate (Kdo)2-(lauroyl)-lipid IVAvery rapidly compared with wild type. We solubilized and partially purified MsbB from an overproducing strain, lacking HtrB. MsbB transfers myristate or laurate, activated on ACP, to (Kdo)2-(lauroyl)-lipid IVA. Decanoyl, palmitoyl, palmitoleoyl, and (R)-3-hydroxymyristoyl-ACP are poor acyl donors. MsbB acylates (Kdo)2-(lauroyl)-lipid IVA about 100 times faster than (Kdo)2-lipid IVA. The slow, but measurable, rate whereby MsbB acts on (Kdo)2-lipid IVA may explain why overexpression of MsbB suppresses the temperature-sensitive phenotype ofhtrB mutations. Presumably, the acyloxyacyl group generated by excess MsbB substitutes for the one normally formed by HtrB. Overexpression of the Escherichia coli msbB gene on high copy plasmids suppresses the temperature-sensitive growth associated with mutations in thehtrB gene. htrB encodes the lauroyl transferase of lipid A biosynthesis that acylates the intermediate (Kdo)2-lipid IVA (Brozek, K. A., and Raetz, C. R. H. (1990) J. Biol. Chem.265, 15410–15417). SincemsbB displays 27.5% identity and 42.2% similarity tohtrB, we explored the possibility that msbBencodes a related acyltransferase. In contrast to htrB, extracts of strains with insertion mutations in msbB are not defective in transferring laurate from lauroyl acyl carrier protein to (Kdo)2-lipid IVA. However, extracts ofmsbB mutants do not efficiently acylate the product formed by HtrB, designated (Kdo)2-(lauroyl)-lipid IVA. Extracts of strains harboring msbB + bearing plasmids acylate (Kdo)2-(lauroyl)-lipid IVAvery rapidly compared with wild type. We solubilized and partially purified MsbB from an overproducing strain, lacking HtrB. MsbB transfers myristate or laurate, activated on ACP, to (Kdo)2-(lauroyl)-lipid IVA. Decanoyl, palmitoyl, palmitoleoyl, and (R)-3-hydroxymyristoyl-ACP are poor acyl donors. MsbB acylates (Kdo)2-(lauroyl)-lipid IVA about 100 times faster than (Kdo)2-lipid IVA. The slow, but measurable, rate whereby MsbB acts on (Kdo)2-lipid IVA may explain why overexpression of MsbB suppresses the temperature-sensitive phenotype ofhtrB mutations. Presumably, the acyloxyacyl group generated by excess MsbB substitutes for the one normally formed by HtrB. The htrB gene was first described by Karow and Georgopoulos (1Karow M. Fayet O. Cegielska A. Ziegelhoffer T. Georgopoulos C. J. Bacteriol. 1991; 173: 741-750Crossref PubMed Scopus (73) Google Scholar, 2Karow M. Georgopoulos C. Mol. Microbiol. 1991; 5: 2285-2292Crossref PubMed Scopus (32) Google Scholar) as essential for rapid growth of Escherichia coli on nutrient broth above 33 °C. At elevated temperatures, peculiar morphological changes are observed inhtrB-deficient strains, including bulging of the cell surface and filamentation (1Karow M. Fayet O. Cegielska A. Ziegelhoffer T. Georgopoulos C. J. Bacteriol. 1991; 173: 741-750Crossref PubMed Scopus (73) Google Scholar, 2Karow M. Georgopoulos C. Mol. Microbiol. 1991; 5: 2285-2292Crossref PubMed Scopus (32) Google Scholar, 3Karow M. Georgopoulos C. J. Bacteriol. 1992; 174: 702-710Crossref PubMed Scopus (97) Google Scholar). At permissive temperatures,htrB mutants display increased resistance to bile salts (3Karow M. Georgopoulos C. J. Bacteriol. 1992; 174: 702-710Crossref PubMed Scopus (97) Google Scholar). Based on these phenotypes, Karow and Georgopoulos (1Karow M. Fayet O. Cegielska A. Ziegelhoffer T. Georgopoulos C. J. Bacteriol. 1991; 173: 741-750Crossref PubMed Scopus (73) Google Scholar, 3Karow M. Georgopoulos C. J. Bacteriol. 1992; 174: 702-710Crossref PubMed Scopus (97) Google Scholar, 4Karow M. Fayet O. Georgopoulos C. J. Bacteriol. 1992; 174: 7407-7418Crossref PubMed Google Scholar, 5Karow M. Georgopoulos C. Mol. Microbiol. 1993; 7: 69-79Crossref PubMed Scopus (125) Google Scholar) suggested function(s) for htrB in cell envelope assembly, including possible roles in peptidoglycan, lipopolysaccharide, and fatty acid biosynthesis. We have recently demonstrated (6Clementz T. Bednarski J.J. Raetz C.R.H. J. Biol. Chem. 1996; 271: 12095-12102Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar) that the htrB gene ofE. coli encodes the lauroyl transferase that acylates the key lipid A biosynthesis intermediate (Kdo 1The abbreviation used is: Kdo, 3-deoxy-d-manno-octulosonic acid. )2-lipid IVA(7Brozek K.A. Raetz C.R.H. J. Biol. Chem. 1990; 265: 15410-15417Abstract Full Text PDF PubMed Google Scholar) (Fig. 1). This conclusion is based on assays of extracts prepared from htrB-deficient mutants, in which lauroyl transferase activity is undetectable (6Clementz T. Bednarski J.J. Raetz C.R.H. J. Biol. Chem. 1996; 271: 12095-12102Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). Conversely, cells overexpressing thehtrB gene on hybrid plasmids overproduce the transferase several hundredfold (6Clementz T. Bednarski J.J. Raetz C.R.H. J. Biol. Chem. 1996; 271: 12095-12102Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). We have purified the overproduced enzyme to homogeneity and have shown by N-terminal sequencing thathtrB is indeed the structural gene for the lauroyl transferase. 2S. Carty and C. R. H. Raetz, manuscript in preparation. Identification of htrBas the lauroyl transferase is further supported by fatty acid analyses of lipopolysaccharide isolated from htrB-deficient strains of E. coli, in which the amount of laurate is reduced (4Karow M. Fayet O. Georgopoulos C. J. Bacteriol. 1992; 174: 7407-7418Crossref PubMed Google Scholar). Lipid A from htrB-deficient Hemophilus influenzaeis also under-acylated (8Lee N.-G. Sunshine M.G. Engstrom J.J. Gibson B.W. Apicella M.A. J. Biol. Chem. 1995; 270: 27151-27159Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Karow and Georgopoulos (3Karow M. Georgopoulos C. J. Bacteriol. 1992; 174: 702-710Crossref PubMed Scopus (97) Google Scholar, 5Karow M. Georgopoulos C. Mol. Microbiol. 1993; 7: 69-79Crossref PubMed Scopus (125) Google Scholar) identified several genes that, when introduced on hybrid plasmids, suppress the temperature-sensitive growth associated with htrB mutations. The msbAsuppressor encodes a putative transport protein with a remarkable similarity to the mammalian mdr genes (5Karow M. Georgopoulos C. Mol. Microbiol. 1993; 7: 69-79Crossref PubMed Scopus (125) Google Scholar). Most Mdr proteins pump hydrophobic drugs out of animal cells, but some are involved in secretion of phospholipids into bile (9Oude-Elferink R.P.J. Ottenhoff R. van Wijland M. Smit J.J.M. Schinkel A.H. Groen A.K. J. Clin. Invest. 1995; 95: 31-38Crossref PubMed Google Scholar). Although the biochemical function of msbA is unknown, the sequence similarity ofmsbA to mdr suggests that MsbA might be involved in a translocation process, such as the movement of newly made lipid A from the cytoplasmic surface of the inner membrane to the periplasm (10Osborn M.J. Inouye M. Bacterial Outer Membranes. Wiley Interscience, New York1979: 15-34Google Scholar, 11Raetz C.R.H. Annu. Rev. Biochem. 1990; 59: 129-170Crossref PubMed Scopus (1041) Google Scholar, 12Raetz C.R.H. Neidhardt F.C.E.A. Escherichia coli and Salmonella: Cellular and Molecular Biology. 2nd Ed. 1. American Society for Microbiology, Washington, D. C.1996: 1035-1063Google Scholar). Whatever its role, the msbA gene is essential for growth (5Karow M. Georgopoulos C. Mol. Microbiol. 1993; 7: 69-79Crossref PubMed Scopus (125) Google Scholar). A second suppressor gene, designated msbB, was found to have 27.5% sequence identity and 42.2% similarity to htrB (3Karow M. Georgopoulos C. J. Bacteriol. 1992; 174: 702-710Crossref PubMed Scopus (97) Google Scholar) suggesting a biochemical function related to htrB. MsbBreverses the temperature sensitivity associated with htrBmutations when introduced on plasmids that are maintained at high copy number (3Karow M. Georgopoulos C. J. Bacteriol. 1992; 174: 702-710Crossref PubMed Scopus (97) Google Scholar). The msbB gene itself is not essential for growth (3Karow M. Georgopoulos C. J. Bacteriol. 1992; 174: 702-710Crossref PubMed Scopus (97) Google Scholar). MsbB knockouts greatly reduce the amount of myristate attached lipid A, but they do not affect the laurate content (13Somerville Jr., J.E. Cassiano L. Bainbridge B. Cunningham M.D. Darveau R.P. J. Clin. Invest. 1996; 97: 359-365Crossref PubMed Scopus (242) Google Scholar). Lipopolysaccharide isolated from msbB mutants contains penta-acylated lipid A (designated (Kdo)2-(lauroyl)-lipid IVA in Fig. 1) (13Somerville Jr., J.E. Cassiano L. Bainbridge B. Cunningham M.D. Darveau R.P. J. Clin. Invest. 1996; 97: 359-365Crossref PubMed Scopus (242) Google Scholar). MsbB was discovered independently by Somerville et al. (13Somerville Jr., J.E. Cassiano L. Bainbridge B. Cunningham M.D. Darveau R.P. J. Clin. Invest. 1996; 97: 359-365Crossref PubMed Scopus (242) Google Scholar), who found that whole E. coli cells harboring msbB insertions are orders of magnitude less immunostimulatory than are wild-type cells. Using direct enzymatic assays, we now demonstrate that themsbB gene of E. coli encodes a distinct, late-functioning acyltransferase of lipid A assembly (7Brozek K.A. Raetz C.R.H. J. Biol. Chem. 1990; 265: 15410-15417Abstract Full Text PDF PubMed Google Scholar). MsbB functions optimally after laurate incorporation by HtrB has taken place (Fig. 1). The slow, but significant, rate at which MsbB acylates (Kdo)2-lipid IVA explains whymsbB + works only as a high multi-copy suppressor of htrB mutations (3Karow M. Georgopoulos C. J. Bacteriol. 1992; 174: 702-710Crossref PubMed Scopus (97) Google Scholar). Our findings support the view that at least one acyloxyacyl residue must be present on a significant fraction of the lipid A moieties of E. coli to allow rapid growth above 33 °C. A preliminary abstract of our findings has appeared (14Clementz T. Bednarski J. Raetz C.R.H. FASEB J. 1995; 9: 1311Crossref PubMed Scopus (271) Google Scholar). [γ-32P]ATP was obtained from DuPont NEN. Pyridine, chloroform, methanol, and 88% formic acid were from Fisher. All detergents were of high quality grade (peroxide- and carbonyl-free). Triton X-100 was from Pierce, and Thesit was from Sigma. Acyl carrier protein was purchased from Sigma. Other items were obtained from the following companies: 0.25-mm glass-backed silica gel 60 thin layer chromatography plates (E. Merck), yeast extract and Tryptone (Difco), and DEAE-Sepharose CL-6B (Pharmacia Biotech Inc.). Strains used in this study are derivatives of E. coli K12, and their genotypes are listed in Table I. Cultures were grown in Luria broth, consisting of 5 g of NaCl, 5 g of yeast extract, and 10 g of Tryptone per liter (15Miller J.R. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1972Google Scholar). Antibiotics were added, when required, at 100 μg/ml for ampicillin, 12 μg/ml for tetracycline, and 10 μg/ml for chloramphenicol.Table IPlasmids and E. coli K12 strains used in this studyStrainRelevant genotypeSourceW3110Wild-type (htrB + msbB +), F +, λ−E. coli Genetic Stock Center, Yale UniversityXL1-BluehtrB + msbB +StratageneMLK1067W3110msbB1::ΩCamRef. 3Karow M. Georgopoulos C. J. Bacteriol. 1992; 174: 702-710Crossref PubMed Scopus (97) Google ScholarMLK53W3110htrB1::Tn10Ref. 1Karow M. Fayet O. Cegielska A. Ziegelhoffer T. Georgopoulos C. J. Bacteriol. 1991; 173: 741-750Crossref PubMed Scopus (73) Google ScholarMLK986MLK53msbB1::ΩCamRef.3Karow M. Georgopoulos C. J. Bacteriol. 1992; 174: 702-710Crossref PubMed Scopus (97) Google ScholarpKS12pBluescript carrying htrB +Ref. 1Karow M. Fayet O. Cegielska A. Ziegelhoffer T. Georgopoulos C. J. Bacteriol. 1991; 173: 741-750Crossref PubMed Scopus (73) Google ScholarpBS233pBluescript carrying msbB +Ref.3Karow M. Georgopoulos C. J. Bacteriol. 1992; 174: 702-710Crossref PubMed Scopus (97) Google Scholar Open table in a new tab Plasmid DNAs were isolated using the Wizard miniprep kit (Promega). Other recombinant DNA techniques were performed as described previously (16Ausubel F.M. Brent R Kingston R.E Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. John Wiley & Sons, Inc., , New York1989Google Scholar). Lipid IVA (17Raetz C.R.H. Purcell S. Meyer M.V. Qureshi N. Takayama K. J. Biol. Chem. 1985; 260: 16080-16088Abstract Full Text PDF PubMed Google Scholar), (Kdo)2lipid IVA (18Brozek K.A. Hosaka K. Robertson A.D. Raetz C.R.H. J. Biol. Chem. 1989; 264: 6956-6966Abstract Full Text PDF PubMed Google Scholar, 19Belunis C.J. Raetz C.R.H. J. Biol. Chem. 1992; 267: 9988-9997Abstract Full Text PDF PubMed Google Scholar), [4′-32P]lipid IVA (20Brozek K.A. Bulawa C.E. Raetz C.R.H. J. Biol. Chem. 1987; 262: 5170-5179Abstract Full Text PDF PubMed Google Scholar, 21Hampton R.Y. Raetz C.R.H. Methods Enzymol. 1992; 209: 466-475Crossref PubMed Scopus (12) Google Scholar), and (Kdo)2-[4′-32P]lipid IVA (18Brozek K.A. Hosaka K. Robertson A.D. Raetz C.R.H. J. Biol. Chem. 1989; 264: 6956-6966Abstract Full Text PDF PubMed Google Scholar, 19Belunis C.J. Raetz C.R.H. J. Biol. Chem. 1992; 267: 9988-9997Abstract Full Text PDF PubMed Google Scholar) were prepared as described previously. (Kdo)2-(lauroyl)-[4′-32P]lipid IVA was synthesized from lauroyl-ACP and (Kdo)2-[4′-32P]lipid IVA using a DEAE-Sepharose purified HtrB that was isolated from anmsbB-deficient strain (MLK1067) containing thehtrB overexpressing plasmid, pKS12 (6Clementz T. Bednarski J.J. Raetz C.R.H. J. Biol. Chem. 1996; 271: 12095-12102Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). The preparative reaction contained 50 mm HEPES, pH 7.5, 0.2% Triton X-100, 50 μm (Kdo)2-[4′-32P]lipid IVA (2 × 103 dpm/nmol), 50 μm lauroyl-ACP, 5 mm MgCl2, 50 mm NaCl, and 8 μg of lauroyl transferase (specific activity, 2200 nmol × min−1 × mg−1) per ml. The total reaction volume was 150 μl. After incubation at 30 °C for 30 min, the reaction mixture was applied as a single line onto a 20 × 20-cm Silica Gel 60 thin layer chromatography plate. After air drying, the plate was developed to the top in the solvent chloroform, pyridine, 88% formic acid, water (30:70:16:10, v/v). The product was detected by a brief autoradiography, and the region of the silica plate containing the desired, more rapidly migrating product was scraped off into a scintered glass funnel. The silica chips were washed once with 4 ml of chloroform. The product, (Kdo)2-(lauroyl)-[4′-32P]lipid IVA, was then eluted with three 3.8-ml portions of a single phase, acidic Bligh and Dyer solvent mixture consisting of chloroform, methanol, 0.1 m HCl (1:2:0.8, v/v) (22Nishijima M. Raetz C.R.H. J. Biol. Chem. 1979; 254: 7837-7844Abstract Full Text PDF PubMed Google Scholar). The eluted material (12 ml) was converted to a two-phase, acidic Bligh and Dyer system by the addition of 3 ml each of chloroform and 0.1 mHCl. The tube was mixed vigorously, and the phases were separated by a brief centrifugation. The lower phase was transferred to a new glass tube, and 18 drops of pyridine were added before the solvent was evaporated under a stream of nitrogen. The dried lipid was resuspended in 10 mm Tris chloride, pH 7.5, containing 1 mmEDTA. It was stored at −80 °C and was resuspended by brief (1 min) sonic irradiation prior to use. Acyl-ACPs containing various acyl chains were synthesized from the corresponding free fatty acids and acyl carrier protein using solubilized membranes from the acyl-ACP synthase overproducing strain,E. coli LCH109/pLCH5/pGP1-2, as described previously (6Clementz T. Bednarski J.J. Raetz C.R.H. J. Biol. Chem. 1996; 271: 12095-12102Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). Crude cell-free extracts were made from 1–2 liters of logarithmically growing cultures. After harvesting by low speed centrifugation at 2 °C, cells were washed once in 30 mmHEPES, pH 7.5, containing 1 mm EDTA and 1 mmEGTA (half the volume of the original culture). The washed cell pellet was resuspended in 30 mm HEPES, pH 7.5, containing 1 mm EDTA and 1 mm EGTA (a volume approximately equal to the volume of the cell pellet). Cells were broken using an ice-cold French pressure cell (SLM Instruments, Urbana, IL) at 20,000 psi. The broken cell suspension was adjusted to 10 mmMgSO4, and DNase I was added to 1 μg/ml. After a brief sonic irradiation on an ice water bath to decrease the viscosity, the suspension was incubated for 30 min at 30 °C. Unbroken cells were removed by centrifugation at 1,000 × g for 10 min. Membranes and soluble fractions were separated by centrifugation at 150,000 × g for 60 min. The supernatant was centrifuged a second time to remove residual contaminating membranes. The membrane pellet was resuspended in 25 ml of 30 mmHEPES, pH 7.5, containing 1 mm EDTA and 1 mmEGTA, and it was centrifuged again as above to generate the final, washed membrane fraction (∼40 mg/ml). The membrane suspension was stored at −80 °C. Protein concentrations were determined with the bicinchoninic assay (Pierce), using bovine serum albumin as the standard (23Smith P.K. Krohn R.I. Hermanson G.T. Mallia A.K. Gartner F.H. Provenzano M.D. Fujimoto E.K. Goeke N.M. Olson B.J. Klenk D.C. Anal. Biochem. 1985; 150: 76-85Crossref PubMed Scopus (18713) Google Scholar). HtrB-catalyzed acylation was assayed using Method I described earlier (6Clementz T. Bednarski J.J. Raetz C.R.H. J. Biol. Chem. 1996; 271: 12095-12102Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). In most cases, the reaction mixture contained 50 mm HEPES, pH 7.5, 0.1% Triton X-100, 25 μm (Kdo)2-[4′-32P]lipid IVA (∼2 × 103 dpm/nmol), 25 μm lauroyl-ACP, 0.1 mg/ml bovine serum albumin, and 0.1–1000 μg/ml enzyme at 30 °C in a final volume of 10–20 μl. With highly purified preparations of HtrB, enzyme stability is increased by including 50 mm NaCl and 5 mmMgCl2 in the assay mixture (6Clementz T. Bednarski J.J. Raetz C.R.H. J. Biol. Chem. 1996; 271: 12095-12102Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). The MsbB-catalyzed acylation of the product generated by HtrB, designated (Kdo)2-(lauroyl)-[4′-32P]-lipid IVA, was assayed in a reaction mixture containing 50 mm HEPES, pH 7.5, 0.1% Triton X-100, and 0.1 mg/ml bovine serum albumin. Unless otherwise indicated, 25 μm(Kdo)2-(lauroyl)-[4′-32P]lipid IVA (∼2 × 103 dpm/nmol) was used as the acceptor, and 25 μm myristoyl-ACP was the donor. Under these conditions, MsbB displays a slight kinetic preference for myristoyl-ACP over lauroyl-ACP. MsbB functions about 100-fold more rapidly with (Kdo)2-(lauroyl)-[4′-32P]lipid IVA than with (Kdo)2-[4′-32P]lipid IVA as the acceptor. Since studies using partially purified MsbB showed a stabilizing effect of sodium chloride, 50 mm NaCl was also included in the reaction in some experiments. MgCl2inhibited MsbB slightly. Reactions were stopped by spotting a 4–5-μl sample onto a Silica Gel 60 thin layer chromatography plate. After air drying and developing the thin layer chromatography plate in the solvent chloroform, pyridine, 88% formic acid, water (30:70:16:10, v/v), the plates were exposed to a PhosphorImager screen. The amount of radioactivity present in the labeled spots corresponding to substrate and product were quantitated using a Molecular Dynamics Imager. The specific activity of both HtrB and MsbB was expressed in terms of nmol/min/mg protein. Cells were grown and labeled by the method of Galloway and Raetz (24Galloway S.M. Raetz C.R.H. J. Biol. Chem. 1990; 265: 6394-6402Abstract Full Text PDF PubMed Google Scholar), with several modifications. Briefly, bacteria from 30 °C overnight cultures were inoculated into 20 ml of fresh LB broth and grown for several hours at 30 °C until A 600 had reached ∼0.1. Next, two 5.0-ml portions of each culture were transferred into two new culture tubes, and 32Pi (5 μCi/ml) was added to both. One was grown for 3 h at 30 °C, and the other was grown for 3 h at 42 °C. 32P-Labeled cells were washed twice with 5.0 ml of phosphate-buffered saline, pH 7.4, and resuspended in 0.8 ml of the same buffer. Washed cells were extracted at room temperature for 60 min with a single phase Bligh and Dyer mixture, formed by the addition of 2 ml of methanol and 1 ml of chloroform. After centrifugation in a clinical centrifuge at top speed for 20 min, the pellets were recovered and washed once with 5.0 ml of a single phase of Bligh and Dyer mixture, consisting of chloroform/methanol/water (1:2:0.8, v/v). The pellets were resuspended in 3.6 ml of 0.2 m HCl by sonic irradiation in a bath. The resuspended pellets were incubated at 100 °C for 90 min. The acid-hydrolyzed material was converted to a two-phase Bligh and Dyer system by addition of 4 ml of chloroform and 4 ml of methanol. After centrifugation at low speed, the upper phases were removed, and the lower phases were washed twice with 4.0 ml of pre-equilibrated acidic upper phase of a two-phase Bligh and Dyer system, generated by mixing chloroform, methanol, 0.2 m HCl (2:2:1.8, v/v). The washed lower phases were dried under a nitrogen stream. The lipid A 4′-monophosphates were redissolved in 100 μl of chloroform/methanol (4:1, v/v), and approximately 1000 cpm of the samples was applied to each lane of a Silica Gel 60 thin layer chromatography plate. The plate was developed in the solvent of chloroform, pyridine, 88% formic acid, water (50:50:16:5, v/v). The plate was dried and exposed overnight to a PhosphorImager screen. Extracts of strains bearinghtrB mutations do not catalyze lauroyl-ACP-dependent acylation of (Kdo)2-[4′-32P]lipid IVA, as shown in Fig. 2 by the absence of (Kdo)2-(lauroyl)-lipid IVA formation (producta in lane 2). The transfer of laurate from lauroyl-ACP to (Kdo)2-[4′-32P]lipid IVA was efficient in extracts of strains harboring an insertion in msbB (Fig. 2, lane 6), as in the wild type (Fig. 2, lane 4). A similar pattern was observed when myristoyl-ACP was substituted for lauroyl-ACP (not shown), except that the initial rates of (Kdo)2-[4′-32P]lipid IVAacylation were 5–10-fold slower with myristoyl-ACP, given the selectivity of HtrB for laurate (6Clementz T. Bednarski J.J. Raetz C.R.H. J. Biol. Chem. 1996; 271: 12095-12102Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). In crude extracts or membrane preparations from wild-type cells one observes two distinct acylations of (Kdo)2-[4′-32P]lipid IVA (7Brozek K.A. Raetz C.R.H. J. Biol. Chem. 1990; 265: 15410-15417Abstract Full Text PDF PubMed Google Scholar). With lauroyl-ACP as the donor, the first acylation is relatively rapid compared with the second (7Brozek K.A. Raetz C.R.H. J. Biol. Chem. 1990; 265: 15410-15417Abstract Full Text PDF PubMed Google Scholar). With myristoyl-ACP, the first acylation is 5–10-fold slower than with lauroyl-ACP, but the second acylation is slightly faster than with lauroyl-ACP (7Brozek K.A. Raetz C.R.H. J. Biol. Chem. 1990; 265: 15410-15417Abstract Full Text PDF PubMed Google Scholar). Given that purified HtrB catalyzes only one rapid acylation of (Kdo)2-[4′-32P]lipid IVA (6Clementz T. Bednarski J.J. Raetz C.R.H. J. Biol. Chem. 1996; 271: 12095-12102Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar), it seemed plausible that MsbB might encode the second acyltransferase (Fig. 1) that we previously observed in cell extracts (7Brozek K.A. Raetz C.R.H. J. Biol. Chem. 1990; 265: 15410-15417Abstract Full Text PDF PubMed Google Scholar). To approach this problem, we initially incubated membranes of wild-type cells that overexpress either htrB + ormsbB + with (Kdo)2-[4′-32P]lipid IVA and lauroyl-ACP. As shown in Fig. 3, panel B,overexpression of htrB + in the presence ofmsbB + on the chromosome resulted in very rapid formation and accumulation of a monoacylated product at the relatively low extract concentrations employed (4 μg/ml). Alternatively, overexpression of msbB + in the presence ofhtrB + on the chromosome (Fig. 3, panel A) did not increase the overall extent of (Kdo)2-[4′-32P]lipid IVAacylation compared with wild-type (hence the need to use 100 μg/ml membranes), but it did result in the accumulation of the diacylated material (product b) at the expense of the monoacylated product a. The difference between HtrB and MsbB overproduction in a wild-type background is especially apparent when comparing the product distribution at the 40-min time point inpanel A of Fig. 3 (excess MsbB) to the 10-min time point inpanel B of Fig. 3 (excess HtrB). In both cases the conversion of substrate to product is similar (∼30%), but diacylated material is generated in strains overproducing MsbB, whereas monoacylated product is formed when HtrB is overproduced. The results of Fig. 3 provide support for the idea that MsbB catalyzes the second acylation and can use lauroyl-ACP as the donor under the in vitro assay conditions employed. To evaluate further the function of MsbB and its relationship to HtrB, we prepared membranes from strains of msbB orhtrB-deficient cells overexpressing htrB andmsbB, respectively (6Clementz T. Bednarski J.J. Raetz C.R.H. J. Biol. Chem. 1996; 271: 12095-12102Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). In this way, we could study cell extracts that contained only one or the other enzyme. As shown in Fig. 4, panel A, membranes of strain MLK53/pBS233 (containing only msbB +) did not catalyze any measurable acylation of (Kdo)2-[4′-32P]lipid IVA under the conditions employed. Membranes of cells containing only HtrB (Fig.4, panel B) catalyzed mainly the formation of producta. In Fig. 4, panel A, the (Kdo)2-[4′-32P]lipid IVA and acyl-ACP were first preincubated for 60 min with 10 μg/ml membranes of MLK53/pBS233. At time 0, a second 60-min incubation was started. Samples were then withdrawn during the second incubation period for product analysis at the indicated times. In Fig. 4, panels Band C, membranes of strain MLK1067/pKS12 (containing onlyhtrB +) were preincubated for 60 min with (Kdo)2-[4′-32P]lipid IVA and acyl-ACP to generate significant amounts of (Kdo)2-(lauroyl)-[4′-32P]lipid IVA (product a). At time 0, only a small volume of buffer was added to the reaction mixture in panel B. Inpanel C, a 10 μg/ml portion of membranes of MLK53/pBS233 (containing only msbB +) was added at time 0. The incubations were continued for another 60 min and analyzed as inpanel A. The system containing only HtrB (panel B) catalyzed the formation of more product a, whereas the system containing both HtrB and MsbB (panel C) resulted in very rapid accumulation of the diacylated product bderived from product a. These findings show that MsbB functions efficiently only after HtrB-catalyzed acylation of (Kdo)2-[4′-32P]lipid IVA has taken place (Fig. 4, panel A compared with panel C). A direct assay for MsbB was developed using 25 μm(Kdo)2-(lauroyl)-[4′-32P]lipid IVA as the acceptor and 25 μm acyl-ACP as the donor (see "Experimental Procedures"). Acylation was detected by thin layer chromatography and PhosphorImager analysis as in Fig. 4. With the direct assay, the specific activity of MsbB in extracts of wild-type cells was ∼0.3 nmol/min/mg, but in extracts ofmsbB-deficient mutants, the activity was below the limits of detection (∼0.03 nmol/min/mg). Overexpression ofmsbB + on multi-copy hybrid plasmids resulted in ∼100-fold higher specific activity of MsbB in cell extracts or membranes compared with wild-type, as shown in Table II. The large size of the observed effects on MsbB-specific activity supports the proposal that msbB is the structural gene encoding the MsbB acyltransferase.Table IIPurification of MsbB from E. coli MLK53/pBS233Total volumeTotal proteinSpecific activityYieldmlmgnmol × min−1 × mg−1%Membranes5.020827100Triton X-100 extract24846596Triton X-100-insoluble5.0903.66DEAE-Sepharose1.55.164057Myristoyl transferase activities were assayed in 50 mmHEPES, pH 7.5, 0.1% Triton X-100, 50 mm NaCl, 25 μm myristoyl-ACP, and 25 μm(Kdo)2-[lauroyl]-[4′-32P]-lipid IVA (2 × 103 dpm/nmol) at 30 °C. Open table in a new tab Myristoyl transferase activities were assayed in 50 mmHEPES, pH 7.5, 0.1% Triton X-100, 50 mm NaCl, 25 μm myristoyl-ACP, and 25 μm(Kdo)2-[lauroyl]-[4′-32P]-lipid IVA (2 × 103 dpm/nmol) at 30 °C. Washed membranes (5 ml) from E. coli MLK53/pBS233 were diluted in 20 mm Tris chloride, pH 7.8, containing 1 mmEDTA and 1 mm EGTA to a final protein concentration of ∼17 mg/ml protein (208 mg of protein in a total volume of 12 ml). The membrane suspension was mixed with an equal volume of solubilization buffer, giving final concentrations of 2.5% Triton X-100, 20 mm Tris chloride, pH 7.8, 1 mm EDTA, 1 mm EGTA, 100 mm sodium phosphate, 150 mm NaCl, and 10% glycerol. The solubilization mixture was incubated with slow stirring at 5 °C for 2 h. Insoluble material was removed by centrifugation for 60 min at 170,000 ×g at 5 °C. The supernatant was transferred to a new tube and diluted to a final volume of 72 ml with 20 mm Tris chloride, pH 7.8, 1 mm EDTA, and 1 mm EGTA to reduce
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