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Chloramphenicol acetyltransferase specified by cat-86: relationship between the gene and the protein
Gene, 73 (1988) 209-214 Elsevier
209
GEN 02721
Chloramphenicol acetyltransferase specified by cat-&k relationship between the gene and the protein (Amino acid sequence; Bacillus subtilis; protein purification; trimeric enzyme)
James Laredo, Vicki L. Wolff and Paul S. Lovett Department of Biological Sciences, University of Maryland Baltimore County, Catonsville, MD 21228 (U.S.A.) Received 2 June 1988 Revised and accepted 8 August 1988 Received by publisher 24 August 1988
SUMMARY
Gene cat-86 is chloramphenicol (Cm)-inducible and specifies Cm acetyltransferase, CAT-86. The gene was previously cloned from the DNA of a strain of Bacilluspumilus. In the present study we report the construction of a constitutively expressed version of cat-86 that permits high-level expression of the gene on a plasmid in B. subtilis. A method is described that allows very rapid purification of CAT-86 protein to homogeneity. The sequence of 13 N-terminal amino acids of purified CAT-86, as well as the 26.6-kDa size of the subunit protein, agree with predictions made based on the nucleotide sequence of the gene. The M, of the native enzyme suggests that CAT-86 is a trimer consisting of three identical protein subunits. Our studies demonstrate that cat-86 provides a convenient system for analyzing relationships between a gene and a multimeric enzyme in the B. subtilis background.
INTRODUCTION
CAT is found commonly in Cm-resistant isolates of many species of bacteria (Shaw, 1983). CAT is an intracellular enzyme that catalyzes the acetylation of Cm, rendering the drug inactive as an antibiotic. The genes that specify CAT are designated cat, and the Correspondence to: Dr. P.S. Lovett, Department
of Biological Sciences, University ofMaryland Baltimore County, Catonsville, MD 21228 (U.S.A.) Tel. (301)445-2249. Abbreviations: aa, amino acid(s); CAT-86, Cm acetyltransferase specified by gene tat-86; Cm, chloramphenicol; HPLC, highpressure liquid chromatography; K,, substrate concentration at half-maximal velocity; nt, nucleotide(s); PMSF, phenylmethylsulfonic acid; RBS, ribosome-binding site; [ 1, designates plasmid-carrier state. 0378-I 119/88/$03.50 0 1988 Elsevier Science Publishers
B.V. (Biomedical
nucleotide sequences of five cat genes have been reported (Alton and Vapnek, 1979; Bruckner and Matzura, 1985; Harwood et al., 1983; Horinouchi and Weisblum, 1982; Shaw et al., 1985). Each is #edicted to specify a protein consisting of 215 to 220 aa, depending on the particular gene. Two of these cat genes, those residing on the Staphylococcus plasmids pC221 and pUB112, contain virtually the identical nucleotide sequence (Bruckner and Matzura, 1985; Shaw et al., 1985). The remaining three genes, cat-86 (from B. pumilus), Tn9 cat (of Gram-negative origin) and the cat resident on the Staphylococcus aureus plasmid pC 194 differ in nucleotide sequence both from each other and from the pC221/pUB112 cat gene. However, the proteins predicted from the sequences of the live genes contain several regions of amino acid identity or Division)
210
similarity. This is p~ic~~ly evident in the region of the proteins which is believed to contain the active site for catalysis. These data seem consistent with the view that the various cut genes are the result of divergence from a common ancestral gene (Lovett, 1985). The cat-86 gene and other cut genes from Grampositive bacteria are inducibly expressed and Cm is the inducer (Lovett, 1985). The cat-86, as well as the pUB112 and PC194 cut genes, have been used as models to explore the mech~ism through which an antibiotic inhibitor of protein synthesis can activate gene expression (Alexieva et al., 1988; Duvall and Lovett, 1986; Duvall et al., 1983; Bruckner and Matzura, 1985; Byeon and Weisblum, 1984). Nothing is known of enzymes specified by these genes although the proteins specified by several other cat genes of Gram-positive origin have been examined (Fitton and Shaw, 1977). However, most of our ~derst~d~g of CAT enzymes is derived from the extensive studies of CAT specified by the TnP cat gene. In the present report we describe several properties of the CAT enzyme specified by cat-86, hereafter referred to as CAT-86.
MATERIALS AND METHODS
that is not sequestered in RNA secondary structure (Ambulos et al., 1984; Nicholson et al., 1985). This can result from a deletion mutation that removes a portion of the inverted repeat sequences which span the cut-86 RBS, or from a duplication of the downstream inverted repeat which contains the RBS (Ambulos et al., 1985). We inserted a unique EcoRVsensitive site 10 nt 5’ to the cat-86 RBS by sitedirected mutagenesis (see Duvall et al., 1987; Taylor et al., 1985; Zoller and Smith, 1983). The EcoRV site is therefore located between the inverted-repeat sequences that dictate the fo~ation of an mRNA stem-loop that normally sequesters the cut-86 RBS. The resulting plasmid was cleaved with SalI, which cuts in the linker 144 bp 5’ to cat-86, and the SaiI ends were gap-filled. The plasmid was then cut with EcoRV and ligated. The resulting plasmid, pPL703C2, was consequently deleted for 122 bp of the upstream regulatory sequences. When cut-86 in pPL703C2 was tr~sc~ption~ly activated by inserting the Spat promoter the gene was constitutively expressed. The specific activity of CAT was about 60 in host B. subtih cells. To further facilitate the preparation of CAT-86 enzyme, pPL703C2-Spat was transformed into B. subtiiis BG2036 (Yang et al., 1984). This strain is a mutant derivative that lacks each of two genes which specify the major proteases in B. subtilis.
(a) Plasmids and bacteria
(b) CAT-86 purification
Plasmid pPL703 is a promoter-clo~g plasmid previously constructed by inserting the promoterless cat-86 gene, cloned from 3. pumilus, between the EcoRI and BumHI sites of the high-copy, neomycinresistance plasmid pUBll0 (Williams et al., 1981; Mongkolsuk et al., 1983). Promoters cloned 5’ to cat-86 activate transcription of the gene. In promoter-containing versions of pPL703, cat-86 specifies Cm-inducible CAT. The maximum CAT-specific activity obtained in B. subtilis by use of pPL703 is about 10 when the cat-86 gene is activated by a strong promoter, Spat (Yansura and Henner, 1984) and is induced with Cm. Constitutively expressed versions of cat-86 in mutant derivatives of pPL703 specify higher levels of CAT than do inducible versions of the gene. Constitutive expression of inducible cat genes results from mutations that provide the cut coding sequence with an RB S sequence
Two liters of BG2036 (pPL703C2-Spat) were grown to late log phase in penassay broth. Cells were harvested and washed with cold buffer (1 M KCi, 10 mM Tris * HCl, pH 8.0, 5% PMSF). The pellet was frozen and stored at -80°C for up to two months with no loss of CAT-86 specific activity. Pellets were thawed on ice in 20 ml of 50 mM Tris * HCl, pH 7.8, containing 50 PM of /I-mercaptoethanol. Lysozyme was added to 200 pg/ml and alter 30 min at 37”C, the lysate was passed through a French press twice at 16000 psi at 4°C. The lysate was i~ediately sonicated for 20 s with glass powder, and centrifuged at 30 000 xg for 20 mm. The supematant fraction was then centrituged at 15OOOOxg for 1.5 h. The resulting supernatant fraction was 19.5 ml. This was loaded onto an HPLC BioGel TSK DEAE-5PW column (150 x 2 1.5 mm) pre-equilibrated with 50 mM
211
Tris * HCl, pH 7.8, and 50 ,uM /?-mercaptoethanol. Flow rate was 3 ml/m& A gradient of NaCl (0 M + 1 M) in running buffer was passed through the column in 60 min. Fractions (5 ml) were collected and a 5-4 sample of each was assayed for CAT in a microtiter plate. The calorimetric CAT assay produces a yellow color as an indicator of enzyme activity. Thus, the location of CAT in the fractions was initially determined by eye and fractions spanning the CAT peak were then assayed by the standard enzyme assay. CAT eluted from the column at 0.3 M NaCl. Two CAT-containing fractions (total volume of 10.3 ml) from the DEAE column were made 1 M with respect to (NH&SO, and were centrifuged at 1OOOOxgfor 10 min. The supematant fraction was applied to an HPLC BioGel TSK Phenyl-5PW column equilibrated with 50 mM Tris * HCl, pH 7.8,50 PM /I-mercaptoethanol in 1 M
(NH&SO,. CAT was eluted with a linear gradient of (NH&SO, (1 M + 0 M) over 60 min. CAT eluted at 0.3 M NH,S04. The CAT-containing fractions, assayed as above, were dialyzed against 50 mM Tris * HCl, pH 7.8, and 50 PM j%mercaptoethanol in the cold for 2 h and the 10.3~ml sample was passed over the HPLC-DEAE column for a second time. CAT eluted from the column at 0.2 M NaCl. Two fractions (10 ml total volume) were pooled. These contained CAT-86 at a specific activity of 1760. Recovery of active enzyme by this procedure was 60%. (c) Enzyme assays CAT was assayed by the calorimetric procedure of Shaw (1975). Protein was measured by the method of Lowry et al. (195 1). Specific activity is the number of pmol of Cm acetylated/min/mg protein at 25 ‘C. (d) Sodium dodecyl electrophoresis
sulfate-polyacrylamide-gel
Proteins were analyzed by electrophoresis through 0.1% SDS-12.5% polyacrylamide gels (Laemmli, 1975).
RESULTS
(a) CAT-86 purification, subunit M,, and aminoterminal sequencing CAT-86 was purified from 2000 ml of log phase cells, as described in MATERIALS AND METHODS section b. Purity at each stage of the procedure was analyzed by electrophoresis of samples through SDS-polyacrylamide gels (Fig. 1). In this gel system the purified CAT-86 subunit protein migrated as a 26.6-kDa species. This Mr value of the CAT-86 subunit protein is consistent with the value predicted from the nucleotide sequence of the gene, which is 26 061. The nucleotide sequence of cat-86 predicts that the start codon for the gene is TTG rather than ATG (Harwood et al., 1983). The use of TTG as an alternative start codon in Gram-positive bacteria has been previously shown by others (e.g., McLaughlin et al., 1981). TTG is normally a leucine codon, but B. subtilis [pPL703C2Spac]
Fig. 1. SDS-polyacrylamide gel electrophoresis of CAT-86 containing extracts at various stages of purification. CAT-86 was purified through the S-150 step, the first DEAE peak (DEAE-l), the phenyl peak and the two peak fractions from the second DEAE column (DEAE-2). Samples of each were held at lOO”C, mixed with an equal volume of loading dye and aliquots of 3 @l (S-150), 5 ~1 (DEAE-I), 50 ~1 (phenyl peak), and 50 ~1 each of the two DEAE-2 peak fractions were applied to a 12% polyacrylamide gel. Electrophoresis was at 4 mA for 18 h at room temperature. CAT-86 subunit protein was determined to have a mass of 26.6 kDa relative to the migration of the following standards (in kDa): lysozyme (14.4), soybean trypsin inhibitor (21.5), carbonic anhydrase (31), ovalbumin (42.7). Gels were stained with Coomassie blue.
212
Initiation Codorl
cat-86 nt Sequence Amino Acid Sequence of N-terminus of CAT-86
Met Phe Lys Gln ile
Asp
Glu Asn Tyr Leu Arg
Lys
Glu
Fig. 2. Correlation of the nucleotide sequence of the first 39 bp of cat-86 with the N-terminal 13 aa of CAT-86.
when ‘ITG serves as an start codon it specifies methionine. The frst 13 aa of the puril?ed CAT-86 protein were determined by automated Edman degradation. This sequence is in agreement with that which is predicted by the nucleotide sequence of the gene (Fig. 2). Moreover, these data confirm that when TTG is the start codon, the corresponding amino acid inserted is methionine. (b) M, of nativeCAT-86 Initial studies of CAT enzymes lead to the conclusion that the native enzymes were tetramers of a single subunit protein (Shaw et al,, 1983). More recent data from the analysis of the crystal structure of a CAT enzyme suggest that the native enzyme may be trimer (Leslie et al., 1986)+ We infer that CAT-86 consists of only one species of subunit pro-
\
c
tein because purified preparations of the enzyme reveal only a single M, species on denaturing gels. Moreover, the N-terminal sequence data of CAT-86 are also consistent with the existence of a single species of subunit protein, Lastly, both the size of the CAT-86 subunit protein (26.6 kDa) and the N-terminal sequence data for this protein demonstrate that it is the product of the cat-86 gene. If native CAT-86 were a trimer or a tetramer the predicted &f, for the enzyme is either 79800 or 106400, respectively, assuming the M, of the subunit protein is 26600. If the M, of the subunit protein is 26 061, as calculated from the nucleotide sequence of the gene, then the M,s of a trimer and tetramer are 78 183 and 104244, respectively. Native CAT-86 was therefore analyzed on a Sephadex G-150 gel filtration column. The average M, of the enzyme was determined to be 75 700 relative to standard proteins (Fig. 3). This apparent M, is consistent with the trimeric form of CAT-86 and is ~consistent with the tetrameric ~~gement.
-I
I
LO
50
Log Molecular
loo
Weight ( x 103)
Fig. 3. Mobility of native CAT-86 in a Sephadex G-150 column relative to protein standards. Puritied CAT-86 (30 units in 100 pl of 50 mM Tris *HCl, pH 7.8 and 50 PM /I-mercaptoethanol) was applied to a Sephadex G-150 column (1.6x 70 cm) equilibrated with the same buffer. Flow was 6 ml/h. 47 fractions of 2-ml were collected and assayed for CAT activity. Protein standards (shown in the figure) were run separately, and each was monitored by absorbance at 280nm. The calculated M, of CAT-86 was 75 700.
I
I
I
2
4 v/s
I
I
6
x 104
Fig. 4. Determination ofthe Km for the CAT-86 enzyme. CAT-86 (2 units) was incubated with varying concentrations of Cm (Segal, 1976). Initial rates ofacetylation were measured at 25°C and the data are shown in the form of a Woo~pIot (Segal, 1976). v, velocity, S, substrate ~on~ntration.
213
0030
40
50
Temperature
60
70
00
90
(“C)
Fig. 5. Heat inactivation of CAT-86. Purified CAT-86 (0.19 units in 200 ~1 of 50 mM Tris *HCl, pH 7.8, and 50 PM p-mercaptoethanol) was kept at the indicated temperatures for 5 min and placed on ice. Each was then assayed for CAT activity at 25 “C.
(c) K,,, and thermal inactivation of CAT-86
The K,.,,for CAT-86 was determined to be 7.4 PM by use of a Woolf plot (Fig. 4; Segal, 1976). This value is within the range of K,,,‘s determined previously for other CAT enzymes (Shaw, 1983). Gene cat-86 originated in a mesophile, B. pumihs, and we were interested in the thermostability of the corresponding native enzyme. A fared concentration of CAT-86 (0.19 units in 200 ~1) was exposed to various temperatures for 5 min, chilled and assayed for remaining CAT activity at 25°C. Temperatures up to 45 “C had no measurable influence on the enzyme activity, whereas temperatures above 60’ C rapidly inactivated the enzyme (Fig. 5). We calculated that exposure of purified CAT-86 to 55 “C inactivated 50% of the initial activity in 5 min.
DISCUSSION
Gene cat-86 has heretofore been used exclusively as a genetic tool. A promoter-deficient version of cat-86 serves as the reporter gene in the extensively used promoter-cloning plasmid pPL703 (Lovett and Mongkolsuk, 1987). Promoters cloned into a multicloning site-linker located 5’ to the gene activate transcription of the gene, which in turn confers the
Cm-resistant phenotype on resulting transformants of B. subtilis. In addition, the regulation of cat-86 has been studied because expression of the gene is induced by chloramphenicol, which is typical of cat genes of Gram-positive origin. The induction has been shown to result from the activation of the translation of cat-86 mRNA by drug-modified ribosomes in a process that has been termed translational attenuation (Alexieva et al., 1988). Because of the extensive use of cat-86 in various contexts it is important to establish key features of the corresponding protein which are predicted from the nucleotide sequence of the structural gene. The biochemical studies reported in the present study demonstrate the size of the CAT-86 subunit protein as well as the N-terminal sequence of that protein are in complete agreement with that which is predicted from the gene sequence. Furthermore, the mass of the native CAT-86 enzyme from B. subtih is consistent with the interpretation that the enzyme is a trimer of a single protein subunit, the cat-86 product. The reported K, and heat-inactivation data suggest that the CAT-86 enzyme is not grossly dissimilar from previously studied CAT enzymes (Shaw, 1983). We have constructed a version of cat-86 which is constitutively expressed at very high levels in B. subtilis. Moreover, the assay for CAT is simple and quantitative, and the CAT-86 enzyme can be rapidly purified with a high recovery of active enzyme. Thus, the cat-86 system provides an ideal system to begin to examine several fundamental aspects of geneenzyme relationships in B. subtilis.
ACKNOWLEDGEMENTS
We thank Dennis Henner for the Spat promoter and the protease-deficient strain of B. subtilis, Walter Mulbry and Jeff Kams of the United States Department of Agriculture for their gracious aid in the purification of CAT-86, and Beth Lovett and Tim Smith for constructing pPL703C2. We are indebted to Joe Crouse for his initial studies some years ago which first suggested that CAT-86 was probably a trimer. Ralph Pollack of the UMBC Chemistry Department provided valuable advice on enzyme kinetics. Lastly, we thank Clark Riley and Van Vogel
214
of the Howard Hughes Medical Institutes at The Johns Hopkins University. School of Medicine for determiniig the N-terminal sequence of CAT-86 This investigation was supported by Public Health Service Research Grant AI-21350.
REFERENCES
Alexieva, Z., Duvall, E.J., Ambulos Jr., N.P., Kim, U.J. and Loveti, P.S.: Chloramphenicol induction of cat-86 requires ribosome stalliug at a specific site in the regulatory leader. Proc. Natl. Acad. Sci. USA 85 (1988) 3057-3061. Alton, N.K. and Vapnek, D.: Nucleotide sequence analysis ofthe c~or~ph~col resistance transposon Tn9. Nature 282 (1979) 864-866. Ambulos Jr., N.P., Chow, J.H., Mon~olsuk, S., Preis, L.H., Vollmar II, W.R., and Lovett, P.S.: ~nstitotive variants of the pC194 cut gene exhibit DNA alterations in the vicmity of the r&some-bmding site sequence. Gene 28 (1984) 171-176. Ambulos Jr., N.P., Mongkolsuk, S., Kaufman, J.D. and Lovett, P.S.: Chloramphenicol-induced translation of cat-86 mRNA requires two c&acting regulatory regions. J. Bacterial. 164 (1985) 696-703. Bruckner, R. and Matzura, H.: Regulation of the inducible chlor~p~e~col acetyltransferase gene of the StapkyZoc~cc~ aweus plasmid pUBt 12. EMBO. Y.4 (1985) 2295-2300. Byeon, W.-H. and Weisblum, B.~Post~~ansc~ption~ regulation of c~or~phenicol acetyltr~sfer~e. J. Bacterial. 158 (1984) 543-550. Duvall, E.J. and Lovett, P.S.: ~loramphe~col induces translation ofthe mRNA for a chior~phenicol-resistance gene in Bacilli su&t&s.Proc. Natl. Acad. Sci. USA 83 (1986) 3939-3943. Duvall, E.J., Williams, D.M., Lovett, P.S., Rudolph, C., Vasantha, N. and Guyer, M.: Chloramphenicol-inducible gene expression in Bad&s mbtih. Gene 24 (1983) 171-177. Duvall, E.J., Ambulos Jr., N.P. and Lovett, P.S.: Drug-free induction of a c~or~pheniwl a~tyltr~sferase gene in Bacillus subtilisby stalling ribosomes in a regulate leader. J. Bacterial. 169 (1987) 4235-4241. Fitton, J.E. and Shaw, W.V.: Comp~son of c~or~ph~col acetyltr~sfer~e variants in staphylococci. Purification, inhibitor studies and N-terminal sequences. Biochem. J. 177 (1977) 575-582. Harwood, CR., Williams, D.M., and Lovett, P.S.: Nucleotide sequence of a Bacihs pumiko gene specifying chloramphenico1 acetyltransferase. Gene 24 (1983) 163-169. Horinouchi, S. and Weisbhun, B.: Nucleotide sequence and functional map of pC194, a plasmid that specifies inducible c~or~phe~col resistance. J. Bacterial. IS0 (1982) 815-825. Laemmli, U.K.: Cleavage of structural proteins during the assembly of the head of bacte~opha8e T4. Nature 227 (1970) 680-685.
Leslie, A.G.W., Liddell, J.M. and Shaw, W.V.: Crystallization of a type III chlorampheuicol acetyltransferase. J. Mol. Biol. 188 (1986) 283-285. Lovett, P.S.: Antibiotic inducible regulation of a plasmid gene encoding chlor~phe~col acetyltr~sfer~e in Buci#~ sub&r. In Schlessinger, D. (Ed.), Mi~obiolo~-1985, American Society for Microbiolo~, Wanton, DC, 1985, pp. 397-400. Lovett, P.S. and Mo~olsuk, S.: Promoter probe plasmids for Gr~-~sitive bacteria In Rodriguez, R.L. and Denhardt, D.T. (Eds.), Vectors: A Survey of Molecular Cloning Vectors and Their Uses. Butterworth, Boston, MA, 1987, pp. 363-384. Lowry, O.H., Rosebrough, N.J., Fat-r, A.L. and Randall, R.J.: Protein measurement with the Folin phenol reagent. J. Biol. Chetn. 193 (1951) 265-215. McLaughlin, J.R., Murray, C.L. and Rabinowitz, J.C: Unique features in the ribosome binding site sequence of the Gtampositive St~kylo~~~ atom p-lactamase gene. J. Biol. Chem. 256 (1981) 11283-I 1291. Mo~ols~, S., Chiang,Y.-W., Reynolds, R.B. and Lovett, P.S.: Restriction fragments that exert promoter activity during postex~nenti~ growth of Bacillus sub&s. J. Bacterial. 155 (1983) 1399-1406. Nicholson, W.L., Chambliss, G.H., Buckbmder, L., Ambulos Jr,, N.P. and Lovett, P.S.: Isolation and expression of a constitutive variant of the chloramphenicol-inducible plasmid gene c&6 under control of the Bacillus s&r&r 168 amylase promoter. Gene 35 (1985) 113-120. Segal, I.H.: Biochemical Calculations. Wiley, New York, 1976. Shaw, W.V.: ~or~phenicol acetyltr~sferase from chloramphenicol-resis~t bacteria. Methods Enzymol. 43 (1975) 737-75s. Shaw, W.V.: Chlor~phe~col acetyl~~sfer~e: e~~olo~ and molecular biology. Crit. Rev. B&hem. 4 (1983) 4-43. Shaw, W.V., Breuner, D.G., I.&rice, S.F.J., Skinner, S.E. and Hawkins, A.R.: Chloramphenicol acetyltransferase gene of staphylococcal plasmid pC221. FEBS Lett. 179 (1985) 101-106. Taylor, J.W., Ott, J. and Eckstein, F.: The generation of oligonucleotide-directed mutations at high-frequency using phosphorothioate-rn~~ed DNA. Nucleic Acids Res. 13 (1985) 8765-8785. Willies, D.M., Duvall, E.J. and Lovett, P.S.: Cloning restriction fragments that promote expression of a gene in ~~c~~u~ subti~. J. Bacterial. 146 (1981) 1162-116.5. Yang, M.Y., Ferrari, E. and Hermer, D.J.: Cloning of the neutral protease gene of3ffc~~~~bti~ and the use ofthe cloned gene to create an in vitro-derived mutation. J. Bacterial. 160 (1984) 15-21. Yansura, D.G. and Henner, D.J.: Use of the Exkeri?kiu colilac repressor and operator to control gene expression in Bacillus subtilis.Proc. Natl. Acad. Sci. USA 81 (1984) 439-443. Zoller, M.J. and Smith, M.: Oligonucleotide-directed mutagenesis of DNA fragments cloned into Ml3 vectors, Methods Enzymol. 100 (1983) 468-500. Communicated by R.E. Yasbin.
209
GEN 02721
Chloramphenicol acetyltransferase specified by cat-&k relationship between the gene and the protein (Amino acid sequence; Bacillus subtilis; protein purification; trimeric enzyme)
James Laredo, Vicki L. Wolff and Paul S. Lovett Department of Biological Sciences, University of Maryland Baltimore County, Catonsville, MD 21228 (U.S.A.) Received 2 June 1988 Revised and accepted 8 August 1988 Received by publisher 24 August 1988
SUMMARY
Gene cat-86 is chloramphenicol (Cm)-inducible and specifies Cm acetyltransferase, CAT-86. The gene was previously cloned from the DNA of a strain of Bacilluspumilus. In the present study we report the construction of a constitutively expressed version of cat-86 that permits high-level expression of the gene on a plasmid in B. subtilis. A method is described that allows very rapid purification of CAT-86 protein to homogeneity. The sequence of 13 N-terminal amino acids of purified CAT-86, as well as the 26.6-kDa size of the subunit protein, agree with predictions made based on the nucleotide sequence of the gene. The M, of the native enzyme suggests that CAT-86 is a trimer consisting of three identical protein subunits. Our studies demonstrate that cat-86 provides a convenient system for analyzing relationships between a gene and a multimeric enzyme in the B. subtilis background.
INTRODUCTION
CAT is found commonly in Cm-resistant isolates of many species of bacteria (Shaw, 1983). CAT is an intracellular enzyme that catalyzes the acetylation of Cm, rendering the drug inactive as an antibiotic. The genes that specify CAT are designated cat, and the Correspondence to: Dr. P.S. Lovett, Department
of Biological Sciences, University ofMaryland Baltimore County, Catonsville, MD 21228 (U.S.A.) Tel. (301)445-2249. Abbreviations: aa, amino acid(s); CAT-86, Cm acetyltransferase specified by gene tat-86; Cm, chloramphenicol; HPLC, highpressure liquid chromatography; K,, substrate concentration at half-maximal velocity; nt, nucleotide(s); PMSF, phenylmethylsulfonic acid; RBS, ribosome-binding site; [ 1, designates plasmid-carrier state. 0378-I 119/88/$03.50 0 1988 Elsevier Science Publishers
B.V. (Biomedical
nucleotide sequences of five cat genes have been reported (Alton and Vapnek, 1979; Bruckner and Matzura, 1985; Harwood et al., 1983; Horinouchi and Weisblum, 1982; Shaw et al., 1985). Each is #edicted to specify a protein consisting of 215 to 220 aa, depending on the particular gene. Two of these cat genes, those residing on the Staphylococcus plasmids pC221 and pUB112, contain virtually the identical nucleotide sequence (Bruckner and Matzura, 1985; Shaw et al., 1985). The remaining three genes, cat-86 (from B. pumilus), Tn9 cat (of Gram-negative origin) and the cat resident on the Staphylococcus aureus plasmid pC 194 differ in nucleotide sequence both from each other and from the pC221/pUB112 cat gene. However, the proteins predicted from the sequences of the live genes contain several regions of amino acid identity or Division)
210
similarity. This is p~ic~~ly evident in the region of the proteins which is believed to contain the active site for catalysis. These data seem consistent with the view that the various cut genes are the result of divergence from a common ancestral gene (Lovett, 1985). The cat-86 gene and other cut genes from Grampositive bacteria are inducibly expressed and Cm is the inducer (Lovett, 1985). The cat-86, as well as the pUB112 and PC194 cut genes, have been used as models to explore the mech~ism through which an antibiotic inhibitor of protein synthesis can activate gene expression (Alexieva et al., 1988; Duvall and Lovett, 1986; Duvall et al., 1983; Bruckner and Matzura, 1985; Byeon and Weisblum, 1984). Nothing is known of enzymes specified by these genes although the proteins specified by several other cat genes of Gram-positive origin have been examined (Fitton and Shaw, 1977). However, most of our ~derst~d~g of CAT enzymes is derived from the extensive studies of CAT specified by the TnP cat gene. In the present report we describe several properties of the CAT enzyme specified by cat-86, hereafter referred to as CAT-86.
MATERIALS AND METHODS
that is not sequestered in RNA secondary structure (Ambulos et al., 1984; Nicholson et al., 1985). This can result from a deletion mutation that removes a portion of the inverted repeat sequences which span the cut-86 RBS, or from a duplication of the downstream inverted repeat which contains the RBS (Ambulos et al., 1985). We inserted a unique EcoRVsensitive site 10 nt 5’ to the cat-86 RBS by sitedirected mutagenesis (see Duvall et al., 1987; Taylor et al., 1985; Zoller and Smith, 1983). The EcoRV site is therefore located between the inverted-repeat sequences that dictate the fo~ation of an mRNA stem-loop that normally sequesters the cut-86 RBS. The resulting plasmid was cleaved with SalI, which cuts in the linker 144 bp 5’ to cat-86, and the SaiI ends were gap-filled. The plasmid was then cut with EcoRV and ligated. The resulting plasmid, pPL703C2, was consequently deleted for 122 bp of the upstream regulatory sequences. When cut-86 in pPL703C2 was tr~sc~ption~ly activated by inserting the Spat promoter the gene was constitutively expressed. The specific activity of CAT was about 60 in host B. subtih cells. To further facilitate the preparation of CAT-86 enzyme, pPL703C2-Spat was transformed into B. subtiiis BG2036 (Yang et al., 1984). This strain is a mutant derivative that lacks each of two genes which specify the major proteases in B. subtilis.
(a) Plasmids and bacteria
(b) CAT-86 purification
Plasmid pPL703 is a promoter-clo~g plasmid previously constructed by inserting the promoterless cat-86 gene, cloned from 3. pumilus, between the EcoRI and BumHI sites of the high-copy, neomycinresistance plasmid pUBll0 (Williams et al., 1981; Mongkolsuk et al., 1983). Promoters cloned 5’ to cat-86 activate transcription of the gene. In promoter-containing versions of pPL703, cat-86 specifies Cm-inducible CAT. The maximum CAT-specific activity obtained in B. subtilis by use of pPL703 is about 10 when the cat-86 gene is activated by a strong promoter, Spat (Yansura and Henner, 1984) and is induced with Cm. Constitutively expressed versions of cat-86 in mutant derivatives of pPL703 specify higher levels of CAT than do inducible versions of the gene. Constitutive expression of inducible cat genes results from mutations that provide the cut coding sequence with an RB S sequence
Two liters of BG2036 (pPL703C2-Spat) were grown to late log phase in penassay broth. Cells were harvested and washed with cold buffer (1 M KCi, 10 mM Tris * HCl, pH 8.0, 5% PMSF). The pellet was frozen and stored at -80°C for up to two months with no loss of CAT-86 specific activity. Pellets were thawed on ice in 20 ml of 50 mM Tris * HCl, pH 7.8, containing 50 PM of /I-mercaptoethanol. Lysozyme was added to 200 pg/ml and alter 30 min at 37”C, the lysate was passed through a French press twice at 16000 psi at 4°C. The lysate was i~ediately sonicated for 20 s with glass powder, and centrifuged at 30 000 xg for 20 mm. The supematant fraction was then centrituged at 15OOOOxg for 1.5 h. The resulting supernatant fraction was 19.5 ml. This was loaded onto an HPLC BioGel TSK DEAE-5PW column (150 x 2 1.5 mm) pre-equilibrated with 50 mM
211
Tris * HCl, pH 7.8, and 50 ,uM /?-mercaptoethanol. Flow rate was 3 ml/m& A gradient of NaCl (0 M + 1 M) in running buffer was passed through the column in 60 min. Fractions (5 ml) were collected and a 5-4 sample of each was assayed for CAT in a microtiter plate. The calorimetric CAT assay produces a yellow color as an indicator of enzyme activity. Thus, the location of CAT in the fractions was initially determined by eye and fractions spanning the CAT peak were then assayed by the standard enzyme assay. CAT eluted from the column at 0.3 M NaCl. Two CAT-containing fractions (total volume of 10.3 ml) from the DEAE column were made 1 M with respect to (NH&SO, and were centrifuged at 1OOOOxgfor 10 min. The supematant fraction was applied to an HPLC BioGel TSK Phenyl-5PW column equilibrated with 50 mM Tris * HCl, pH 7.8,50 PM /I-mercaptoethanol in 1 M
(NH&SO,. CAT was eluted with a linear gradient of (NH&SO, (1 M + 0 M) over 60 min. CAT eluted at 0.3 M NH,S04. The CAT-containing fractions, assayed as above, were dialyzed against 50 mM Tris * HCl, pH 7.8, and 50 PM j%mercaptoethanol in the cold for 2 h and the 10.3~ml sample was passed over the HPLC-DEAE column for a second time. CAT eluted from the column at 0.2 M NaCl. Two fractions (10 ml total volume) were pooled. These contained CAT-86 at a specific activity of 1760. Recovery of active enzyme by this procedure was 60%. (c) Enzyme assays CAT was assayed by the calorimetric procedure of Shaw (1975). Protein was measured by the method of Lowry et al. (195 1). Specific activity is the number of pmol of Cm acetylated/min/mg protein at 25 ‘C. (d) Sodium dodecyl electrophoresis
sulfate-polyacrylamide-gel
Proteins were analyzed by electrophoresis through 0.1% SDS-12.5% polyacrylamide gels (Laemmli, 1975).
RESULTS
(a) CAT-86 purification, subunit M,, and aminoterminal sequencing CAT-86 was purified from 2000 ml of log phase cells, as described in MATERIALS AND METHODS section b. Purity at each stage of the procedure was analyzed by electrophoresis of samples through SDS-polyacrylamide gels (Fig. 1). In this gel system the purified CAT-86 subunit protein migrated as a 26.6-kDa species. This Mr value of the CAT-86 subunit protein is consistent with the value predicted from the nucleotide sequence of the gene, which is 26 061. The nucleotide sequence of cat-86 predicts that the start codon for the gene is TTG rather than ATG (Harwood et al., 1983). The use of TTG as an alternative start codon in Gram-positive bacteria has been previously shown by others (e.g., McLaughlin et al., 1981). TTG is normally a leucine codon, but B. subtilis [pPL703C2Spac]
Fig. 1. SDS-polyacrylamide gel electrophoresis of CAT-86 containing extracts at various stages of purification. CAT-86 was purified through the S-150 step, the first DEAE peak (DEAE-l), the phenyl peak and the two peak fractions from the second DEAE column (DEAE-2). Samples of each were held at lOO”C, mixed with an equal volume of loading dye and aliquots of 3 @l (S-150), 5 ~1 (DEAE-I), 50 ~1 (phenyl peak), and 50 ~1 each of the two DEAE-2 peak fractions were applied to a 12% polyacrylamide gel. Electrophoresis was at 4 mA for 18 h at room temperature. CAT-86 subunit protein was determined to have a mass of 26.6 kDa relative to the migration of the following standards (in kDa): lysozyme (14.4), soybean trypsin inhibitor (21.5), carbonic anhydrase (31), ovalbumin (42.7). Gels were stained with Coomassie blue.
212
Initiation Codorl
cat-86 nt Sequence Amino Acid Sequence of N-terminus of CAT-86
Met Phe Lys Gln ile
Asp
Glu Asn Tyr Leu Arg
Lys
Glu
Fig. 2. Correlation of the nucleotide sequence of the first 39 bp of cat-86 with the N-terminal 13 aa of CAT-86.
when ‘ITG serves as an start codon it specifies methionine. The frst 13 aa of the puril?ed CAT-86 protein were determined by automated Edman degradation. This sequence is in agreement with that which is predicted by the nucleotide sequence of the gene (Fig. 2). Moreover, these data confirm that when TTG is the start codon, the corresponding amino acid inserted is methionine. (b) M, of nativeCAT-86 Initial studies of CAT enzymes lead to the conclusion that the native enzymes were tetramers of a single subunit protein (Shaw et al,, 1983). More recent data from the analysis of the crystal structure of a CAT enzyme suggest that the native enzyme may be trimer (Leslie et al., 1986)+ We infer that CAT-86 consists of only one species of subunit pro-
\
c
tein because purified preparations of the enzyme reveal only a single M, species on denaturing gels. Moreover, the N-terminal sequence data of CAT-86 are also consistent with the existence of a single species of subunit protein, Lastly, both the size of the CAT-86 subunit protein (26.6 kDa) and the N-terminal sequence data for this protein demonstrate that it is the product of the cat-86 gene. If native CAT-86 were a trimer or a tetramer the predicted &f, for the enzyme is either 79800 or 106400, respectively, assuming the M, of the subunit protein is 26600. If the M, of the subunit protein is 26 061, as calculated from the nucleotide sequence of the gene, then the M,s of a trimer and tetramer are 78 183 and 104244, respectively. Native CAT-86 was therefore analyzed on a Sephadex G-150 gel filtration column. The average M, of the enzyme was determined to be 75 700 relative to standard proteins (Fig. 3). This apparent M, is consistent with the trimeric form of CAT-86 and is ~consistent with the tetrameric ~~gement.
-I
I
LO
50
Log Molecular
loo
Weight ( x 103)
Fig. 3. Mobility of native CAT-86 in a Sephadex G-150 column relative to protein standards. Puritied CAT-86 (30 units in 100 pl of 50 mM Tris *HCl, pH 7.8 and 50 PM /I-mercaptoethanol) was applied to a Sephadex G-150 column (1.6x 70 cm) equilibrated with the same buffer. Flow was 6 ml/h. 47 fractions of 2-ml were collected and assayed for CAT activity. Protein standards (shown in the figure) were run separately, and each was monitored by absorbance at 280nm. The calculated M, of CAT-86 was 75 700.
I
I
I
2
4 v/s
I
I
6
x 104
Fig. 4. Determination ofthe Km for the CAT-86 enzyme. CAT-86 (2 units) was incubated with varying concentrations of Cm (Segal, 1976). Initial rates ofacetylation were measured at 25°C and the data are shown in the form of a Woo~pIot (Segal, 1976). v, velocity, S, substrate ~on~ntration.
213
0030
40
50
Temperature
60
70
00
90
(“C)
Fig. 5. Heat inactivation of CAT-86. Purified CAT-86 (0.19 units in 200 ~1 of 50 mM Tris *HCl, pH 7.8, and 50 PM p-mercaptoethanol) was kept at the indicated temperatures for 5 min and placed on ice. Each was then assayed for CAT activity at 25 “C.
(c) K,,, and thermal inactivation of CAT-86
The K,.,,for CAT-86 was determined to be 7.4 PM by use of a Woolf plot (Fig. 4; Segal, 1976). This value is within the range of K,,,‘s determined previously for other CAT enzymes (Shaw, 1983). Gene cat-86 originated in a mesophile, B. pumihs, and we were interested in the thermostability of the corresponding native enzyme. A fared concentration of CAT-86 (0.19 units in 200 ~1) was exposed to various temperatures for 5 min, chilled and assayed for remaining CAT activity at 25°C. Temperatures up to 45 “C had no measurable influence on the enzyme activity, whereas temperatures above 60’ C rapidly inactivated the enzyme (Fig. 5). We calculated that exposure of purified CAT-86 to 55 “C inactivated 50% of the initial activity in 5 min.
DISCUSSION
Gene cat-86 has heretofore been used exclusively as a genetic tool. A promoter-deficient version of cat-86 serves as the reporter gene in the extensively used promoter-cloning plasmid pPL703 (Lovett and Mongkolsuk, 1987). Promoters cloned into a multicloning site-linker located 5’ to the gene activate transcription of the gene, which in turn confers the
Cm-resistant phenotype on resulting transformants of B. subtilis. In addition, the regulation of cat-86 has been studied because expression of the gene is induced by chloramphenicol, which is typical of cat genes of Gram-positive origin. The induction has been shown to result from the activation of the translation of cat-86 mRNA by drug-modified ribosomes in a process that has been termed translational attenuation (Alexieva et al., 1988). Because of the extensive use of cat-86 in various contexts it is important to establish key features of the corresponding protein which are predicted from the nucleotide sequence of the structural gene. The biochemical studies reported in the present study demonstrate the size of the CAT-86 subunit protein as well as the N-terminal sequence of that protein are in complete agreement with that which is predicted from the gene sequence. Furthermore, the mass of the native CAT-86 enzyme from B. subtih is consistent with the interpretation that the enzyme is a trimer of a single protein subunit, the cat-86 product. The reported K, and heat-inactivation data suggest that the CAT-86 enzyme is not grossly dissimilar from previously studied CAT enzymes (Shaw, 1983). We have constructed a version of cat-86 which is constitutively expressed at very high levels in B. subtilis. Moreover, the assay for CAT is simple and quantitative, and the CAT-86 enzyme can be rapidly purified with a high recovery of active enzyme. Thus, the cat-86 system provides an ideal system to begin to examine several fundamental aspects of geneenzyme relationships in B. subtilis.
ACKNOWLEDGEMENTS
We thank Dennis Henner for the Spat promoter and the protease-deficient strain of B. subtilis, Walter Mulbry and Jeff Kams of the United States Department of Agriculture for their gracious aid in the purification of CAT-86, and Beth Lovett and Tim Smith for constructing pPL703C2. We are indebted to Joe Crouse for his initial studies some years ago which first suggested that CAT-86 was probably a trimer. Ralph Pollack of the UMBC Chemistry Department provided valuable advice on enzyme kinetics. Lastly, we thank Clark Riley and Van Vogel
214
of the Howard Hughes Medical Institutes at The Johns Hopkins University. School of Medicine for determiniig the N-terminal sequence of CAT-86 This investigation was supported by Public Health Service Research Grant AI-21350.
REFERENCES
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