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• W2021853164 abstract "Bm3R1 is a helix-turn-helix transcriptional repressor from Bacillus megaterium whose binding to DNA is inhibited by fatty acids and a wide range of compounds that modulate lipid metabolism. The inactivation of Bm3R1/DNA binding activity results in the activation of transcription of the operon encoding a fatty acid hydroxylase, cytochrome P450 102. The metabolic role of this operon is unknown. It is possible that it is involved in the synthesis of modified fatty acids as part of normal cellular metabolism or may represent a protective mechanism by which B. megateriumdetoxifies harmful foreign lipids. In this report we demonstrate that polyunsaturated fatty acids (PUFA) activate the transcription of the CYP102 operon. These PUFA are the most potent activators of the CYP102 operon observed to date, and we show that their effects are due to binding directly to Bm3R1. In addition, cultures that have been treated with the CYP102 inducer, nafenopin, are protected against PUFA toxicity. Resistance to PUFA toxicity is also seen in a Bm3R1-deficient strain that constitutively expresses CYP102. The resistant phenotype of this Bm3R1 mutant strain is reversed by specific chemical inactivation of CYP102. These data demonstrate that Bm3R1 can act as a direct sensor of toxic fatty acids and, in addition, provide the first direct evidence of fatty acids binding to a prokaryotic transcription factor. Bm3R1 is a helix-turn-helix transcriptional repressor from Bacillus megaterium whose binding to DNA is inhibited by fatty acids and a wide range of compounds that modulate lipid metabolism. The inactivation of Bm3R1/DNA binding activity results in the activation of transcription of the operon encoding a fatty acid hydroxylase, cytochrome P450 102. The metabolic role of this operon is unknown. It is possible that it is involved in the synthesis of modified fatty acids as part of normal cellular metabolism or may represent a protective mechanism by which B. megateriumdetoxifies harmful foreign lipids. In this report we demonstrate that polyunsaturated fatty acids (PUFA) activate the transcription of the CYP102 operon. These PUFA are the most potent activators of the CYP102 operon observed to date, and we show that their effects are due to binding directly to Bm3R1. In addition, cultures that have been treated with the CYP102 inducer, nafenopin, are protected against PUFA toxicity. Resistance to PUFA toxicity is also seen in a Bm3R1-deficient strain that constitutively expresses CYP102. The resistant phenotype of this Bm3R1 mutant strain is reversed by specific chemical inactivation of CYP102. These data demonstrate that Bm3R1 can act as a direct sensor of toxic fatty acids and, in addition, provide the first direct evidence of fatty acids binding to a prokaryotic transcription factor. Unsaturated fatty acids are essential structural components of the cell membrane. They are also sophisticated signaling molecules that can mediate a myriad of processes involved in cellular communication, differentiation, and cell death (1Von Euler V.S. J. Physiol. ( Lond. ). 1936; 88: 208-218Google Scholar, 2Burr G.O. Burr M.M. J. Biol. Chem. 1930; 86: 587-621Abstract Full Text PDF Google Scholar, 3McPhail L.C. Clayton C.C. Snyderman R. Science. 1984; 224: 622-625Crossref PubMed Scopus (492) Google Scholar, 4Sagar P.S. Das U.N. Prostaglandins Leukot. Essent. Fatty Acids. 1995; 53: 287-299Abstract Full Text PDF PubMed Scopus (62) Google Scholar, 5Kliewer S.A. Lenhard J.M. Willson T.M. Patel I. Morris D.C. Lehmann J.M. Cell. 1995; 83: 813-819Abstract Full Text PDF PubMed Scopus (1849) Google Scholar). It is for these reasons that all organisms require tight regulation of the lipid composition of the cell. Perturbations in the levels of different types of lipid may be fatal due to disruption of membrane structure and metabolic or regulatory processes (2Burr G.O. Burr M.M. J. Biol. Chem. 1930; 86: 587-621Abstract Full Text PDF Google Scholar). The mammalian liver responds to perturbations in lipid homeostasis by the induction of cytochrome P450 fatty acid hydroxylases and the enzymes for peroxisomal β-oxidation (6Roman L.J. Palmer C.N.A. Clark J.E. Muerhoff A.S. Griffin K.J. Johnson E.F. Masters B.S. Arch. Biochem. Biophys. 1993; 307: 57-65Crossref PubMed Scopus (79) Google Scholar, 7Bell D.R. Bars R.G. Elcombe C.R. Eur. J. Biochem. 1992; 206: 979-986Crossref PubMed Scopus (26) Google Scholar). Perturbations in lipid homeostasis may take the form of high fat diet, diabetes, or treatment with fatty acid mimetics such as peroxisome proliferators or non-steroidal anti-inflammatory drugs (8). The regulation of lipid metabolism under such conditions has been shown to be a direct genomic response where a transcription factor responds directly to free fatty acids and acts as a molecular switch for the regulated transcription of genes encoding fatty acid-metabolizing enzymes (9Lee S.S. Pineau T. Drago J. Lee E.J. Owens J.W. Kroetz D.L. Fernandez S.P. Westphal H. Gonzalez F.J. Mol. Cell. Biol. 1995; 15: 3012-3022Crossref PubMed Scopus (1487) Google Scholar, 10Dreyer C. Krey G. Keller H. Givel F. Helftenbein G. Wahli W. Cell. 1992; 68: 879-887Abstract Full Text PDF PubMed Scopus (1191) Google Scholar, 11Kliewer S.A. Sundseth S.S. Jones S.A. Brown P.J. Wisely G.B. Koble C.S. Devchand P. Wahli W. Willson T.M. Lenhard J.M. Lehmann J.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4312-4317Crossref PubMed Scopus (1859) Google Scholar, 12Gottlicher M. Widmark E. Li Q. Gustafsson J.A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4653-4657Crossref PubMed Scopus (795) Google Scholar, 13Palmer C.N.A. Hsu M.H. Muerhoff A.S. Griffin K.J. Johnson E.F. J. Biol. Chem. 1994; 269: 18083-18089Abstract Full Text PDF PubMed Google Scholar); however, the role of mammalian fatty acid hydroxylases in the clearance of fatty acids is not well defined. The cytochrome P450 fatty acid hydroxylases are regulated by peroxisome proliferators in many other types of organisms including plants andBacillus megaterium (14English N. Hughes V. Wolf C.R. Biochem. J. 1996; 316: 279-283Crossref PubMed Google Scholar, 15Weissbart D. Salaun J.P. Durst F. Pflieger P. Mioskowski C. Biochim. Biophys. Acta. 1992; 1124: 135-142Crossref PubMed Scopus (25) Google Scholar). It could therefore be hypothesized that inducible fatty acid hydroxylation represents an ancient regulatory metabolic response to lipid overload. The simplest of these organisms, B. megaterium, is a soil-living Gram-positive bacterium that utilizes branched chain fatty acids rather than straight chain fatty acids as its main membrane phospholipid (16Kaneda T. Bacteriol. Rev. 1977; 41: 391-418Crossref PubMed Google Scholar). B. megaterium only synthesizes small amounts of unsaturated fatty acids as a transient response to cold (17Fulco A.J. Biochim. Biophys. Acta. 1967; 144: 701-703Crossref PubMed Scopus (26) Google Scholar), and exogenously applied unsaturated fatty acids are toxic (18English N. Hughes V. Wolf C.R. J. Biol. Chem. 1994; 269: 26836-26841Abstract Full Text PDF PubMed Google Scholar). This raises the intriguing question, how does B. megateriumcope with these toxic yet highly abundant carbon sources,i.e. plant-derived unsaturated fatty acids? It has been known for some time that B. megaterium has the capacity to hydroxylate and epoxygenate unsaturated fatty acids (19Buchanan J.F. Fulco A.J. Biochem. Biophys. Res. Commun. 1978; 85: 1254-1260Crossref PubMed Scopus (23) Google Scholar). The enzyme responsible for these activities is a soluble cytochrome P450, designated CYP102 1The abbreviations used are: CYP, cytochrome P450; EMSA, electrophoretic mobility shift assay; PUFA, polyunsaturated fatty acids; PBS, phosphate-buffered saline; 12-AO, 12-anthracene oleic acid; 17-ODA, 17-octadecynoic acid. 1The abbreviations used are: CYP, cytochrome P450; EMSA, electrophoretic mobility shift assay; PUFA, polyunsaturated fatty acids; PBS, phosphate-buffered saline; 12-AO, 12-anthracene oleic acid; 17-ODA, 17-octadecynoic acid. or cytochrome P450BM-3 (20Wen L.P. Fulco A.J. J. Biol. Chem. 1987; 262: 6676-6682Abstract Full Text PDF PubMed Google Scholar). Intriguingly, the repressor that controls the transcription of the CYP102 operon in response to a wide range of xenobiotic compounds is a helix-turn-helix DNA-binding protein known as Bm3R1 (21Shaw G.C. Fulco A.J. J. Biol. Chem. 1993; 268: 2997-3004Abstract Full Text PDF PubMed Google Scholar). This protein was originally characterized as mediating the induction of CYP102 by barbiturates. It has been shown that barbiturates abolish the binding of Bm3R1 to its operator DNA sequence and that this allows transcription to proceed through the Bm3R1 and CYP102 coding sequences. We have since shown that DNA binding by Bm3R1 is inhibited, and cytochrome CYP102 is induced, by a wide range of compounds that are known to perturb lipid metabolism in mammals (14English N. Hughes V. Wolf C.R. Biochem. J. 1996; 316: 279-283Crossref PubMed Google Scholar, 18English N. Hughes V. Wolf C.R. J. Biol. Chem. 1994; 269: 26836-26841Abstract Full Text PDF PubMed Google Scholar). These compounds include hypolipidemic drugs and non-steroidal anti-inflammatory drugs that all appear to act as fatty acid mimetics. We have also shown that the chlorophyll metabolite, phytanic acid, induces CYP102 and is metabolized to a less potent inducing form by CYP102 (22English N. Palmer C.N.A. Alworth W.L. Kang L. Hughes V. Wolf C.R. Biochem. J. 1997; 316: 279-283Crossref Scopus (28) Google Scholar). These findings raised the possibility that one function of CYP102 was to detoxify foreign lipids. In support of this concept was the observation that the most potent inhibitors of Bm3R1 DNA binding in vitro are polyunsaturated fatty acids (18English N. Hughes V. Wolf C.R. J. Biol. Chem. 1994; 269: 26836-26841Abstract Full Text PDF PubMed Google Scholar). In these studies induction of CYP102 was not observed due to the toxicity of these compounds at the high concentrations used. In this report we provide the first evidence that unsaturated fatty acids are activators of transcription of the CYP102 operon over a very narrow range of concentrations and that these unsaturated fatty acids bind directly to Bm3R1. The narrow range of concentrations that are required for induction by unsaturated fatty acids is due to toxicity at concentrations immediately above the binding constants for Bm3R1. We have observed that treating cultures with appropriate concentrations of unsaturated fatty acids produces a very transient induction of the levels of CYP102 and that pretreatment of cultures with the peroxisome proliferator, nafenopin, allows growth in normally toxic concentrations of unsaturated fatty acids. This work demonstrates that the CYP102 operon encodes a finely tuned sensor for unsaturated fatty acids and a fatty acid hydroxylase that can mediate the detoxification of these compounds. This is the first demonstration of this cytoprotective response to toxic fatty acids. Fatty acids were purchased from Sigma and Cayman Research Laboratories. Nafenopin was a generous gift from Zeneca Central Toxicology Laboratories, Macclesfield, UK, and from Dr. Brian Lake at Bibra, Carshalton, Surrey, UK. Antibodies and oligonucleotides were produced at the Imperial Cancer Research Fund laboratories at Clare Hall, Herts, UK. Mouse anti-rabbit secondary antiserum was purchased from Sigma. B. megaterium ATCC 14581 was purchased from the American Type Culture Collection. G39E mutant strain was obtained from Prof. A. Fulco, UCLA. Histidine tagged-Bm3R1 was expressed from the pET15b plasmid in Escherichia colistrain BL21(DE3) as described previously (18English N. Hughes V. Wolf C.R. J. Biol. Chem. 1994; 269: 26836-26841Abstract Full Text PDF PubMed Google Scholar), with the following modifications. Induction of a 10-liter culture with 1 mmisopropyl-1-thio-β-d-galactopyranoside was performed at 30 °C for 4.5 h. The cells were then harvested by centrifugation and resuspended in 600 ml of PBS containing 0.1 mg/ml lysozyme. The suspension was incubated on ice for 15 min and then the spheroplasts were harvested by centrifugation. The spheroplasts were then resuspended in 600-ml culture volume of PBS and frozen at −20 °C overnight. The spheroplasts were then thawed in cold water, and β-mercaptoethanol was added to 5 mm. The cells were lysed by sonication, and the cell debris was removed by centrifugation at 100,000 × g for 45 min. The supernatant was made up to 10% glycerol, applied to a 25-ml nickel-agarose column, washed with 250 ml of loading buffer (PBS, 5 mm β-mercaptoethanol, 10% glycerol) and then with 500 ml of loading buffer containing 125 mm imidazole, pH 8.8. The Bm3R1 was eluted in loading buffer containing 250 mm imidazole, pH 8.8. The eluted protein was concentrated and desalted into loading buffer using a Centriprep concentrator (Amicon). Protein concentrations were determined by the Bio-Rad protein assay. The Bm3R1 was stored as a working stock of 250 ng/μl in 50% glycerol, PBS at −20 °C. The protein was greater than 95% pure as determined by scanning densitometry of the preparation visualized on a Coomassie Blue-stained, 12% SDS-polyacrylamide gel (data not shown). Purified recombinant Bm3R1 was combined with 300 nm 12-anthracene oleic acid (Molecular Probes) in 1 ml of 25 mm Tris-HCl, pH 7.5. Fluorescence was measured using a excitation wavelength of 383 nm and emission wavelength of 460 nm using a Perkin-Elmer LS-3 fluorescence spectrophotometer. The background fluorescence resulting from the protein alone and the 12-AO alone were combined and deducted from each experimental value. Kd values were obtained using Ultrafit for Macintosh. Growth of bacterial cultures was as described previously (18English N. Hughes V. Wolf C.R. J. Biol. Chem. 1994; 269: 26836-26841Abstract Full Text PDF PubMed Google Scholar). B. megaterium were grown at 37 °C with aeration to an optical density of 0.2 at 600 nm. Fatty acids and drugs were prepared in dimethyl sulfoxide (Me2SO) and added as required. In the case of controls, Me2SO alone was added. The final Me2SO concentration did not exceed 0.5%(v/v) in any incubation. Genomic fragments corresponding to the CYP102 regulatory sequences were isolated by polymerase chain reaction. Fragment C143 corresponds to positions 62 to 1573 and fragment A45 corresponds to positions 62 to 949 of the CYP102 operon, GenBankTM accession number J04832 (23Ruettinger R.T. Wen L.P. Fulco A.J. J. Biol. Chem. 1989; 264: 10987-10995Abstract Full Text PDF PubMed Google Scholar). These were subcloned into the EcoRI site of the Bacillus subtilis/E. coli shuttle vector pSB355. The luciferase cDNA of pSB355 was excised by digestion with KpnI andSacI, and a KpnI/SacI fragment containing the cDNA encoding β-galactosidase from pSVβ-gal was ligated downstream of the CYP102 sequences. These constructs were used to transform a lacZ mutant strain of B. megaterium, PV586. The resulting strains were designated PV586/C143 and PV586/A45. Cells were harvested from 1 ml of culture by centrifugation and resuspended in 200 μl of PBS containing 0.2 mg/ml lysozyme. This suspension was incubated on ice for 30 min and then 37 °C for 5 min. The resulting spheroplasts were lysed by freeze-thawing (three times) and then sonication (25% power for 5 s). The cell debris was removed by centrifugation in a microcentrifuge at 4 °C for 10 min. The protein concentration of the supernatant was assayed using the Bio-Rad protein assay. 40 μl of the lysate was added to 160 μl of 5-bromo-4-chloro-3-indolyl β-d-galactopyranoside solution (1 mg/ml 5-bromo-4-chloro-3-indolyl β-d-galactopyranoside, 2 mm magnesium chloride, 0.02% Nonidet P-40, 0.01% sodium deoxycholate, 5 mm potassium ferricyanide, and 5 mm potassium ferricyanate in PBS) and incubated at 37 °C on a microtiter plate. Optical density was measured at 600 nm at hourly intervals. Lysates were prepared as described for the measurement of β-galactosidase activity, and proteins within these lysates (30 μg) were resolved using SDS-polyacrylamide gel electrophoresis. A 7.5% acrylamide gel was used to resolve CYP102. Following separation by SDS-polyacrylamide gel electrophoresis, the proteins were transferred to nitrocellulose. CYP102 was identified using a polyclonal rabbit antiserum (1/2000), raised to the reductase domain of CYP102 (English et al. (18English N. Hughes V. Wolf C.R. J. Biol. Chem. 1994; 269: 26836-26841Abstract Full Text PDF PubMed Google Scholar)), followed by mouse anti-rabbit serum (1/2000) conjugated with horseradish peroxidase (Sigma). Following development with ECL reagent (Amersham Pharmacia Biotech), the bands were visualized by autoradiography. EMSAs were carried out as described previously (18English N. Hughes V. Wolf C.R. J. Biol. Chem. 1994; 269: 26836-26841Abstract Full Text PDF PubMed Google Scholar). A double-stranded oligonucleotide, encompassing the high affinity binding site of Bm3R1 designated OIII (5′-CGGAATGAACGTTCATTCCG-3′) (21Shaw G.C. Fulco A.J. J. Biol. Chem. 1993; 268: 2997-3004Abstract Full Text PDF PubMed Google Scholar), was incubated with purified recombinant Bm3R1. His-tagged Bm3R1 has a dissociation constant of 1.8 nm for this DNA sequence (24Shaw G.C. Sun C.H. Chiang A. Biochem. Mol. Biol. Int. 1995; 37: 1197-1205PubMed Google Scholar). All assays were carried out in 30 μl final volume, 60 mm KCl, 12 mm Hepes, 1 mm EDTA, 1 mmdithiothreitol, and glycerol (10% v/v) (EMSA buffer) containing 1 μg of carrier DNA poly(dI-dC) on ice for 15 min. 10 fmol of radioactive oligonucleotide was then added, and the sample was incubated for a further 15 min on ice. Drugs, diluted in EMSA buffer, were added to the incubations prior to the addition of the olignucleotide. The fatty acids used in this study do not disrupt the binding of mammalian nuclear proteins to DNA. 2E. Axen, unpublished data. Following incubation, 4 μl was loaded onto a 4% non-denaturing polyacrylamide gel, electrophoresed at 16 mA constant current, dried, and autoradiographed. The relative radioactivity present in the free and protein bound oligonucleotide fractions was determined using a Bio-Rad PhosphorImager. Ki estimates were obtained using Ultrafit for Macintosh. Recombinant Bm3R1 (500 ng) was combined with 14C-labeled linoleic acid (Amersham Pharmacia Biotech) in a total volume of 100 μl of binding buffer (50 mm Hepes, pH 8.0, 100 mm KCl, 1 mmdithiothreitol, 20% glycerol) and incubated on ice for 4 h. The specific activity of the linoleic acid was 59 mCi/mmol. The sample was applied to a 0.45-μm HAWP filter (Millipore) under vacuum and then washed twice with 5 ml of binding buffer. The filters were then dried, and the radioactivity was determined by scintillation counting. Each sample was performed in triplicate and the mean value calculated. The level of nonspecific binding of linoleic acid to the filters was subtracted from the resulting value. The Kd andKi values were estimated using Ultrafit for Macintosh. Initial studies were directed toward establishing the role of fatty acids in regulating the CYP102 operon. In previous work we demonstrated that polyunsaturated fatty acids (PUFA) are the most potent of the compounds tested in dissociating the Bm3R1·DNA complex in vitro(18English N. Hughes V. Wolf C.R. J. Biol. Chem. 1994; 269: 26836-26841Abstract Full Text PDF PubMed Google Scholar). The finding that these compounds disrupt the binding of purified recombinant Bm3R1 to DNA provides strong evidence that the fatty acids bind to the repressor; however, this evidence is indirect. In order to demonstrate a direct interaction between unsaturated fatty acids and Bm3R1, we carried out binding experiments using a fluorescent fatty acid probe, 12-anthracene oleic acid (12-AO). This fatty acid has only a low fluorescence in aqueous solution; however, 12-AO becomes highly fluorescent when bound to the hydrophobic lipid binding sites of proteins. This interaction has been directly visualized by the crystallization of 12-AO in the lipid binding pocket of the adipocyte lipid-binding protein (25Sha R.S. Kane C.D. Xu Z. Banaszak L.J. Bernlohr D.A. J. Biol. Chem. 1993; 268: 7885-7892Abstract Full Text PDF PubMed Google Scholar, 26Xu Z. Bernlohr D.A. Banaszak L.J. J. Biol. Chem. 1993; 268: 7874-7884Abstract Full Text PDF PubMed Google Scholar). Binding studies were carried out using purified recombinant His-tagged Bm3R1. In DNA binding experiments, 12-AO inhibited the formation of the Bm3R1·DNA complex with aKi of 1.05 μm, as previously observed for other unsaturated fatty acids (18English N. Hughes V. Wolf C.R. J. Biol. Chem. 1994; 269: 26836-26841Abstract Full Text PDF PubMed Google Scholar) (Fig. 1, A and B,open squares). This was accompanied by a dose-dependent increase in fluorescence that was saturable at around 2 μm, indicating that 12-AO binds directly to Bm3R1 (Fig. 1 B, filled squares). The apparentKd of Bm3R1 as judged by the fluorescence activation is 645 nm. These values are comparable with those obtained using the adipocyte lipid-binding protein which has aKd for 12-AO of 2 μm (25Sha R.S. Kane C.D. Xu Z. Banaszak L.J. Bernlohr D.A. J. Biol. Chem. 1993; 268: 7885-7892Abstract Full Text PDF PubMed Google Scholar, 26Xu Z. Bernlohr D.A. Banaszak L.J. J. Biol. Chem. 1993; 268: 7874-7884Abstract Full Text PDF PubMed Google Scholar). A non-lipid binding protein, trypsinogen, displayed no detectable activation of fluorescence in similar experiments (data not shown). Incubation of increasing concentrations of recombinant Bm3R1 with 3 μm 12-AO (above Kd) showed linear binding to 3 μm protein and a sharp saturation of binding at higher concentrations (Fig. 1 C). This experiment demonstrated that the protein preparation is largely active and binds one molecule of 12-AO per monomer unit of Bm3R1. In addition to these experiments we also studied fatty acid binding to recombinant Bm3R1 using a rapid filtration binding assay with14C-labeled linoleic acid. Linoleic acid disrupts the Bm3R1·DNA complex with a Ki of 648 nm(Fig. 2 A). The saturation curve for the binding (Fig. 2 B, open squares) was similar to that observed for the disruption of the Bm3R1·DNA complex (Fig. 2 B, filled squares) with a Kd of 1.8 μm. Peroxisome proliferators, such as nafenopin, also induce CYP102 and disrupt Bm3R1·DNA binding in vitro. We therefore investigated whether nafenopin would displace linoleic acid from Bm3R1. Increasing concentrations of nafenopin were added to the binding reactions. A dose-dependent displacement of linoleic acid from Bm3R1 was observed (Fig. 2 C). Nafenopin displaced linoleic acid with a Ki of 86 μm, and this value agrees well with the concentrations required to induce CYP102 activity in vivo and displace Bm3R1 from DNA in vitro (18English N. Hughes V. Wolf C.R. J. Biol. Chem. 1994; 269: 26836-26841Abstract Full Text PDF PubMed Google Scholar). Another peroxisome proliferator, Wy 14,643, and the non-steroidal anti-inflammatory drug, indomethacin, were also able to displace linoleic acid from Bm3R1 at concentrations that resulted in the induction of CYP102 (data not shown). Stearic acid (100 μm) failed to significantly displace 0.5 μm linoleic acid (data not shown), thus demonstrating the specificity of Bm3R1 for unsaturated fatty acids. The above data showed that unsaturated fatty acids bind directly to Bm3R1 and dissociate it from DNA. The consequence of this effect in vivo should be the induction of CYP102. However, this was inconsistent with our previous findings that unsaturated fatty acids did not induce CYP102. One reason for this discrepancy was the extreme toxicity of these fatty acids at the doses used. We therefore made a more detailed analysis of the effects of fatty acids on the transcription of the CYP102 operon. For these experiments we employed a reporter system where the regulatory regions of the CYP102 operon were ligated to the coding sequence for β-galactosidase (Fig. 3). Initial experiments using B. megaterium cultures transfected with these constructs and treated with the peroxisome proliferator, nafenopin, confirmed that the regulatory sequences required for tight regulation of the reporter plasmid included the entire coding sequence for Bm3R1. The plasmid-encoded expression of Bm3R1 is required for tight regulation as endogenous levels of Bm3R1 are not sufficient for the full repression of the multicopy reporter plasmid (27Shaw G.C. Fulco A.J. J. Biol. Chem. 1992; 267: 5515-5526Abstract Full Text PDF PubMed Google Scholar). In order to study the effects of fatty acids on the transcription of CYP102, cultures were treated with three polyunsaturated fatty acids at a concentration (5 μm) where significant displacement of Bm3R1 from its operator DNA is observed in vitro (Fig. 2 B, data not shown). Stearic acid was included at 50 μm, a concentration below that required for binding to Bm3R1 to demonstrate the specificity of the induction. Saturated fatty acids such as stearic acid and palmitic acid have been shown to induce CYP102 only at concentrations greater than 200 μm (18English N. Hughes V. Wolf C.R. J. Biol. Chem. 1994; 269: 26836-26841Abstract Full Text PDF PubMed Google Scholar, 28Shaw G.C. Sung C.H. Chiang A. Curr. Microbiol. 1996; 32: 124-128Crossref Scopus (4) Google Scholar). Cell lysates were prepared at several time points after treatment and then assayed for β-galactosidase activity (Fig. 4 A). Stearic acid treatment did not increase reporter activity at any time point; however, the three polyunsaturated fatty acids tested all activated transcription at 1 h post-treatment. The transient nature of this response would be consistent with the hypothesis that CYP102 will metabolize and attenuate the fatty acid signal. In order to confirm that reporter activity reflected the accumulation of CYP102 protein, CYP102 levels inB. megaterium treated with γ-linolenic acid were determined by Western blot analysis. A transient increase in signal was observed which was maximal at 1 h, returning to background at 2 h (Fig. 4 B). This correlated well with the transcriptional activation of the CYP102 gene.Figure 4A, polyunsaturated fatty acids transiently activate CYP102 transcription. Cultures were treated with 5 μm linoleic acid (hatched bars), linolenic acid (cross-hatched bars), γ-linolenic acid (open bars), and 50 μm stearic acid (dotted bars) Cell lysates were prepared from cultures at different time points after treatment and then assayed for β-galactosidase activity and protein content as described under “Experimental Procedures.” Shown is the β-galactosidase activity expressed relative to the cultures treated with solvent (Me2SO) alone (Fold induction). The values are from a single representative of three independent experiments. B, γ-linolenic acid gives a transient induction of CYP102 protein. Cultures of B. megaterium ATCC 14581 were treated with 10 μmγ-linolenic acid and samples taken at 1, 2, and 3 h. The samples were lysed, and 30 μg of cellular protein was subjected to electrophoresis through a 7.5% SDS-polyacrylamide gel. The proteins were then transferred onto nitrocellulose, and the CYP102 was visualized by immunostaining as described under “Experimental Procedures.” C, linoleic, γ-linolenic, and linolenic acid activate CYP102 transcription over a narrow range of concentrations. Increasing concentrations of fatty acids were added to cultures of PV586/C143, and growth was continued for 1 h. Cell lysates were assayed for β-galactosidase activity and protein content. Shown is the β-galactosidase activity expressed relative to the cultures treated with 100 μm nafenopin. Cultures were treated with increasing concentrations of linoleic acid (open squares), linolenic acid (filled squares), and γ-linolenic acid (open circles). The values are from a single representative of three independent experiments. Also shown are the structures of the three polyunsaturated fatty acids, linoleic acid, linolenic acid, and γ-linolenic acid.View Large Image Figure ViewerDownload (PPT) We then investigated the relationship between fatty acid concentration and CYP102 transcriptional activation. The regulation of the reporter was studied over a range of concentrations of several different unsaturated fatty acids. The dienoic fatty acid, linoleic acid, was induced over a very narrow range of concentrations between 2 and 7 μm (Fig. 4 C), whereas the trienoic, γ-linolenic and linolenic acids were effective inducers between 2 and 20 μm (Fig. 4 C). At concentrations above 20 μm significant toxicity was observed with all three unsaturated fatty acids. The above experiments demonstrated that polyunsaturated fatty acids bind with high affinity to Bm3R1 and activate the CYP102 gene. In order to establish the consequences of this induction we examined the relationship between induction and the ability to inhibit Bm3R1 binding to DNA and fatty acid toxicity (Fig. 5). Two closely related fatty acids were chosen for these experiments, linoleic acid and ricinoleic acid. Ricinoleic acid was chosen as it only differs from linoleic acid by the loss of a double bond and the addition of a single hydroxyl group at carbon 12 (Fig. 5 A). The concentrations of these fatty acids required to abolish Bm3R1 binding to DNA (Fig. 5, A andB) correlated closely with those required to induce transcription in vivo, with linoleic acid (Ki = 648 nm) being about 30-fold more potent than ricinoleic acid (Ki = 21 μm). These data further demonstrate that subtle differences in fatty acid structure can have profound effects on Bm3R1 binding affinity. At higher concentrations there was a rapid loss of CYP102 induction. This was accompanied by a profound inhibition of cell growth, with the transcriptional activation being abolished at 8 μm linoleic acid (Fig. 5 C), and above 300 μm in the case of ricinoleic acid" @default.
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• W2021853164 date "1998-07-01" @default.
• W2021853164 modified "2023-10-15" @default.
• W2021853164 title "The Repressor Protein, Bm3R1, Mediates an Adaptive Response to Toxic Fatty Acids in Bacillus megaterium" @default.
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