Plasmid-directed Expression of Staphylococcus aureus /I-Lactamase by Bacillus subtilis in Vitro *

A plasmid carrying the Gram-positive Staphylococ- cus aureus PC1 8-lactamase gene is active in directing a cell-free transcription and translation system from Bacillus subtilis. The major protein synthesized has been identified as the S. aureus 8-lactamase on the basis of peptide mapping. The protein is larger than the extracellular enzyme by about M, = 3100. Significant in uitro translation of the 8-lactamase mRNA occurs in the absence of the initiation factor fraction as is characteristic of translation of mRNAs of Gram-positive origin. The 1250 base transcript that encodes the p-lactamase and leader sequence has been mapped on the plasmid molecule.

Ribosomes from the Gram-positive bacterium, Bacillus subtilis, are extremely inefficient in translating mRNAs isolated from Escherichia coli and other Gram-negative bacteria (1-7). These ribosomes are as active as those from E. coli, however, in translating mRNAs of Gram-positive origin (1,2). The lack of RNA phages with a Gram-positive host range (8) has hampered attempts to analyze specific Gram-positive mRNA-ribosome complexes. The development of an in uitro transcription system from B. subtilis (9) has enabled us to synthesize a defined population of mRNAs from the small bacillus phage $29 DNA (10).
Utilizing the phage (p29 DNA-directed cell-free transcription and translation system, we were able to show that the B. subtilis translation system incorporates labeled amino acids into discrete protein products at an efficiency similar to the E. coli system. The most striking result of this study was that in both E. coli and B. subtilis translation systems, significant amounts (20-40%) of the same proteins are synthesized in the absence of the initiation factor fraction as are made in its presence (11). Previous studies indicated that this reduced dependence on the initiation factor fraction is not peculiar to $29 mRNA but is a property of less well defined Gram-positive mRNA populations as well (3,12). In marked contrast, translation of most mRNAs of Gram-negative origin by E. coli ribosomes is strictly dependent on the initiation factor fraction (3, [13][14][15]. Thus, there is some feature of mRNAs derived from Gram-positive bacteria that facilitates protein synthesis in the absence of initiation factors and which may be, at least in part, responsible for the efficient mRNA recognition by ribosomes from Gram-positive bacteria.
This observation suggests that a structural difference between mRNAs isolated from Gram-positive and Gram-negative bacteria is involved in species specific translation. It seemed valuable, therefore, to characterize a specific mRNA * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. from a Gram-positive source with regard to the initiation site for protein synthesis. Since none of the $29 proteins has been identified by its NHp-terminal amino acid sequence or by function, we chose instead to characterize the Gram-positive Staphylococcus aureus PC1 p-lactamase gene.
The S, aureus PC1 p-lactamase gene is carried on a variant of the naturally occurring Staphylococcus plasmid pI258 (pen 1-443) and is expressed constitutively (16)(17)(18). An Eco RI fragment of this plasmid has been cloned in pSClOl and confers ampicillin resistance upon E. coli hosts (19). Strain PC1 was chosen for the main sequence studies because it produced the highest proportion of extracellular enzyme. Not only is the amino acid sequence of the secreted p-lactamase known (ZO), but the activity of the enzyme is easily assayed by its hydrolysis of the p-lactam bond of penicillin-like substrates (21,22).
We report here the transfer of the staphylococcal fragment encoding ampicillin resistance to a pMB9 vector. The hybrid plasmid, designated pJM13, directs the in uitro synthesis of large amounts of S. aureus p-lactamase in a B. subtilis transcription and translation system. The p-lactamase formed in uitro has been identified by peptide mapping, It is larger than the extracellular enzyme by about M, = 3100, suggesting the presence of a signal (leader) peptide sequence in the in vitro product. Furthermore, significant in vitro synthesis of the /3lactamase by both B. subtilis and E . coli systems occurs in the absence of the initiation factor fraction. The transcript encoding S. aureus p-lactamase has been identified and mapped on the plasmid molecule as have the other major transcripts specified by this plasmid. This specific mRNA, which is recognized efEciently by B. subtilis ribosomes, has enabled us to determine possible differences in initiation site structure that are important for recognition by Gram-positive ribosomes.

EXPERIMENTAL PROCEDURES
Materials-The sources of many materials have been cited previously (IO). In addition, [a-"PIATP and ~- [4,5-"H]lysine were purchased from ICN. Ethidium bromide was purchased from Calbiochem. Cesium chloride (special biochemical grade) and sodium dodecyl sulfate were purchased from Gallard Schlesinger. Dowex AG 50W-X8 (200-400 mesh) and polyacrylamide gel reagents were from Bio-Rad. Ampicillin, tetracycline, and chloramphenicol were purchased from Sigma. TPCK-trypsin (223 unit.s/mg) and DNase I (2029 units/mg) were obtained from Worthington. Nitrocefin (compound 87/312) was extract, I0 g of NaCI, 1 NaOH pellet/liter) in the presence of 10 pg/ ml of tetracycline and/or 30 pg/mI of ampicillin. S. aureus PC1 was obtained from the National Collection of Industrial Bacteria (No.

11195).
Nucleases-The sources of exoIII' and S1 nuclease were previously cited (10). The restriction endonucleases Eco RI, Xba I, and Sma I were purchased from Bethesda Research Labs. HindIII was prepared by a published procedure (24) by Warren Gish working i n thjs laboratory.
Purification of Plasmid DNA-Bacteria were grown to A,, = 0.5 in L broth and were treated 18-20 h with 25 pg/ml ofchloramphenicol in order to amplify the plasmid DNA. Cleared lysates were prepared as described (25) and then precipiated with 0.6 volumes of isopropanol. Pellets were resuspended in 4.8 ml of 50 mM Na2-EDTA (pH 8.0). Solid CsCl (4.75 g) and 0.2 ml of ethidium bromide (10 mg/ml) were added. The refractive index was generally 1.392. Samples were overlaid with paraffin oil (Fisher) and spun at 20 "C and 34,000 rpm for 40 h in a Spinco 50 Ti rotor. The plasmid band (visualized by UV illumination ifnecessary) was removed with a I-ml disposable syringe and 18-gauge needle. Ethidium bromide was removed by passing the DNA over a 4-5 mI Dowex AG 50W-X8 column (1.1 X 4.5 cm) in 10 mM Tris-KC1 (pH 8.0) and 1 mM EDTA buffer. The Dowex, 100 g, had been washed with 1 liter each of 1 N HCI and I N NaOH prior to autoclaving in Hz0 at pH 6. Dowex stored in this manner was washed 3 times with 1 M Tris-HC1 (pH 8) prior to use in a column. CsCl in the pooled DNA was removed by overnight dialysis at 4 "C against buffer containing 20 mM Tris-HC1 (pH 8), 20 mM KC], 1 mM EDTA.
IsoZation of the S. aureus DNA Fragment-Fifty pg of supercoiled pJM13 was digested with Eco RI and Sma I endonucleases and phenol extracted. The phenol-purified DNA was divided into 15O-,ul aliquots which were layered on each of two 4.4-1711 linear 10-30' 36 sucrose gradients containing 20 mM Tris-HCI (pH 8.0), 12 mM MgC12, 10 mM EDTA, 150 mM KCI. Samples were spun at 5 "C and 35,000 rnm for 21 h in the Spinco SW60 rotor. Fractions were collected from t le bottom of the tube and anaIyzed by agarose gel electrophoresis prior to pooling the largest fragment which was identified as the staph.ylococcal DNA.
Nuclease Digestion-Cleavages of pJMI3 DNA with restriction endonucleases Eco HI, HindIII, Sma I, and Xba I were carried out at 37 "C according to the conditions described by Bethesda Research Labs. For use in transcription reactions, endonuclease-treated DNA was generally deproteinized by phenol extraction and dialyzed overnight at 4 "C against IO mM Tris-HC1 (pH 8), 1 mM EDTA.
Plasmid Construction and Selection-One pg of Eco RI-digested pSCI22 and 1 pg of Eco RI-digested pMB9 were ligated at 4 "C in ligase buffer (66 mM Tris-HCI (pH 7.6), 6.6 mM MgCl2. 10 mM dithiothreitol, 0.4 mM ATP) with 0.8 unit of TI ligase. After 1 h, the reaction was diluted 2-fold with an additional 0.8 unit of ligase in ligase buffer and the reaction was allowed to continue for about 16 h.
This DNA was used to transform E. coli RRI as described.' Transformants were selected from fresh L agar plates containing 10 pg/ml of tetracycline and 30 pg/ml of ampicillin (antibiotics added after agar had cooled to 50 "C). Individual colonies were screened as described (26) except for decreasing volumes 10-fold so that 1.5-ml microfuge tubes could be used. Putative recombinant plasmids were treated with restriction endonucleases and analyzed on agarose gels.
In Vitro RNA Synthesis-RNA synthesis for gel analysis was performed as described (9). Reactions contained in a volume of 50 or IOOpl, 100 mM Tris-HC1 (pH 8  cell-free systems DNA-directed translation assays were performed as described under "Experimental Procedures." Assays contained either no DNA, 0.32 pmol of pJMl3 DNA (3 pg), 0.6 pmol of $29 DNA (7.5 pg), or 0.23 pmol of T7 DNA (6,ug). r3H)Lysine was 240 cpm/pmol. Reactions containing pMB9 DNA resulted in incorporation that was not significantly above background (-DNA).  (29), 250 pg of B. subtilis S150 protein (2) (prepared as described except for the omission of the DEAE-cellulose column as the last step), or 210 pg of E. coli S150T protein (2). and 55 pg of salt wash protein from B. subtilis ribosomes or 42 pg of salt wash protein from E. coli ribosomes (29) when included. Assays were incubated at 37 "C for 25 min, then cooled on ice. A 10-pl aliquot of each was counted on a filter paper disc as described previously (1).
S. aureus P-Lactamase Purifiration and Assay-Extracellular fllactamase was purified as described by Richmond (30). Activity was determined with Nitrocefin (compound 87/312) according to UCallaghan et al. (22). Milliunits are defined as micromoles of substrate destroyed/min/ml of enzyme X at 37 "C and pH 7.0. Gel Blectrophoresis-DNA was analyzed by electrophoresis in agarose slab gels (0.3 X 14 X 12 cm") ranging from 0.7 to 1.0% with the Tris-acetate buffer system of Dugaiczyk et al. (31) or the Trisglycine buffer system of McDonell et al. (32). Hpa I1 and Eco RI restriction fragments of 429 DNA were used as molecular weight standards? (33).
For analysis of proteins, polyacrylamide gel electrophoresis in the presence of SDS was performed in slab gels (36) with a discontinuous buffer system (37) and an acryIamide/bisacrylamide ratio of 300.8. Treatment in Autofluor (National Diagnostics) allowed detection of "H on Kodak Blue Band fdm.
Gel Elution and Peptide Mapping-In vitro pJM13-directed protein synthesis products from the homologous B. subtilis system were separated on a 1596 SDS-polyacrylamide gel. The gel was dried and the proteins were visualized by autoradiography with LKB "H Ultrofilm. The 32-kilodalton protein band was excised from two identical lanes of the gel (400,000 cpm total), removed from the paper backing.
the rehydrated with 60 Gg of TPCK-treated trypsin (I mg/ml). Two rnl (20 mg) of purified S. aureus p-lactamase in 1% ammonium bicarbonate were added and the mixture was incubated at 37 "C for 10.5 h. An additional 60 pg of TPCK-treated trypsin were added and incubation was continued for 5.5 h. The sample was then lyophilized to dryness and stored at -20 "C. The peptides were separated on Aminex A-5 (38). One-half of each 4-ml fraction was dried at 85 "C under vacuum, redissolved in 1.0 ml of distilled water, and counted in 10 ml of Triton-toluene-Omnifluor (38) in a Beckman LS-8100 P liquid scintillation counter. One ml of each fraction was assayed for ninhydrin reactivity (39).

Construction ofpJMl3
Plasmid-The plasmid pSC122 (19) is composed of the E . coli tetracycline resistance plasmid C. I , . Murray, B. L. Davison, and J. C. Rabinowitz, unpublished observations. pSC101 and a 4.6 X 106-dalton Eco RI restriction fragment of a derivative of a naturally occurring S. aureus plasmid $258 (17,18). This staphylococcal plasmid DNA fragment carries genetic information for constitutive penicillin-ampicillin resistance and confers resistance on both E. coli and B. subtilis hosts in vivo (19,40). In order to increase yields of plasmid DNA, it was desirable to transfer the staphylococcal ampicillin resistance fragment from the stringently controlled pSClOl replicon to the relaxed replicon, pMB9. Illustration of this recombination is shown in Fig. 1. Transformants resistant to both tetracycline and ampicillin were selected and characterized by restriction analysis. The 8.6 X 10'"dalton plasmid pJM13 was further shown to consist of pMB9 (3.5 X 10' daltons), the 4.6 X 10'"dalton staphylococcal plasmid DNA, and an additional 0.5 X 10'"dalton Eco RI fragment of unknown origin. E. coli (RRI) containing pJM13 are resistant to 50 p g / d of tetracycline (minimum inhibitory concentration) and greater than 400 p g / d of ampicillin.
In Vitro Translation Products Directed bypJM13 DNA-Supercoiled pJM13 was purified as described under "Experimental Procedures." The relative activities of the cell-free coupled transcription and translation systems isolated from E. coli and B. subtilis as measured by incorporation of r3H]lysine in response to supercoiled pJM13 DNA, phage $29 DNA, and T7 DNA are shown in Table I. The B. subtilis system is about 50% as active as the E. coli system on pJM13 which suggests that the Gram-positive staphylococcal DNA is transcriptionally and translationally active since the Gramnegative pMB9 DNA directs little or no protein synthesis in the B. subtilis system (data not shown). Digestion of supercoiled pJM13 with endonucleases Eco RI, HindIII, or Xba I reduces amino acid incorporation in the E. coli system to 88, 84, and 6570, respectively, of the value obtained with the supercoiled DNA, while digestion by Xba I lowers incorporation in the B. subtilis system further to 45% of the value obtained with supercoiled DNA.
The protein products of the pJM13 DNA-directed cell-free systems were analyzed by electrophoresis in a sodium dodecyl sulfate-polyacrylamide gel (Fig. 2). There are no detectable products in the absence of added DNA (11). A major protein synthesized in pJM13 DNA-directed reactions by both the E. coli (lane a ) and B. subtilis (lane c ) systems is a product of M , = 32,000. This protein is made very efficiently by both systems. In addition, significant amounts of this protein continue to be made by both the E. coli (lane 6) and B. subtilis (lane d ) systems when the salt wash fraction containing initiation factors is omitted from the assay. Although the amino acid incorporation in the absence of the initiation factor fraction in the experiment shown (Table I and Fig. 2) is 18% of the incorporation achieved in the presence of the initiation factor fraction, subsequent assays in the absence of salt wash have resulted in 28% of the incorporation observed in the complete system. We believe that this higher level is the more usual extent of factor-independent translation with pJM13 RNA. Based on the radioactivity in the 32-kilodalton band excised from a polyacrylamide gel relative to the remainder of labeled products in these later assays, 65% of the VHIlysine incorporated by the B. subtilis system in either the absence or presence of the initiation factor fraction is in the 32-kilodalton protein. These observations suggest that the 32-kilodalton protein is encoded in the Gram-positive portion of pJM13 since substantial initiation of protein synthesis in the absence of initiation factors is characteristic of messenger RNAs from +29 and other Gram-positive sources (3,12). No detectable 32-kilodalton protein is synthesized in reactions directed by Xba I-digested pJM13 in contrast to those directed  by Eco RI-or HindIII-digested pJMl3 (data not shown). The Xba I site has been mapped in the vicinity of the staphylococcal p-lactamase gene (17).
Peptide Mapping of the 32-kilodalton in Vitro Product-Although the extracellular p-lactamase of S. uureus cultures has M, = 28,823 (41), we recognized that the product formed from the same gene might be larger because of the presence of a leader amino acid sequence. There are 43 lysine residues and 4 arginine residues among the 257 amino acids of purified extracellular S. aureus p-lactamase (20). Lysine was therefore chosen to label the product formed in the in vitro synthesis to allow the detection of the maximum number of tryptic peptides if the 32-kilodalton protein were related to p-lactamase. The [:'H]lysine 32-kilodalton protein synthesized by the homologous B. subtilis transcription and translation system was eluted from a sodium dodecyl sulfate-polyacrylamide gel. Purified extracellular S. aureus p-lactamase from exponentially growing S. aureus cultures (30) was combined with the purified in vitro protein and digested with trypsin. The resulting peptides were chromatographed on the strongly acidic cation exchange resin Aminex A-5 (38). The column profile is shown in Fig. 3. The dotted line represents the radioactively labeled peptides of the in vitro 32-kilodalton protein. The solid line denotes peptides of S. aureus p-lactamase detected by ninhydrin assay (39). The large peak of ninhydrin reactivity at fractions 50-55 is due to free ammonia present in the sample applied to the column.
Identification of 32-kilodalton Protein As p-Lactamase-The peptides formed by tryptic digestion of the 32-kilodalton protein formed in the in vitro system correspond very closely to those formed from a similar digest of purified S. aureus plactamase (Fig. 3). Every ['Hllysine peptide of the in vitro product corresponds to a ninhydrin peptide of p-lactamase produced in vivo except for the three in the region where the ninhydrin peptides are obscured by ammonia. The peptides detected by ninhydrin that do not have corresponding [:'HI lysine peptides may result from any of the four unlabeled arginine peptides or from the unlabeled COOH-terminal peptide. The similarity of the tryptic peptide maps of the in vitro 32-kilodalton protein and S. aureus p-lactamase indicates that the major product synthesized by the B. subtilis coupled system is authentic S. aureus p-lactamase. The abundance of lysine residues in p-lactamase does not account for its appearance as the major in vitro product since incorporation of ['%] methionine also results in /3-lactamase as the major product (data not shown). concentration of protein in the cell-free protein synthesis reaction. Mercaptoethanol, present at 10 mM in cell-free reactions, accounts for some yellow to red color production in the absence of pJMl3 DNA corresponding to 26.5 milliunits (22). Reaction-mixtures that contained pJM13 DNA resulted in the production of 11.8 milliunits of /3-lactamase activity over this background. Similar levels of activity were consistently observed. The production of 3.2 milliunits of activity over background in assays directed by pJM13 DNA in the absence of the salt wash fraction suggests that this activity is a result of in uitro /3-lactamase synthesis. That is, the ratio of  RNA in reactions a-fwas 1.7, 1.2, 0.57, 0.84, 0.45, and 0.85 nmol, respectively. Transcripts were analyzed by electrophoresis and autoradiography using a 1.75% acrylamide and 0.5% agarose slab gel. 3 X lo5 cpm of acid-insoluble radioactivity were applied to each lane. protein than estimated on the basis of enzyme activity.
Transcription of Supercoiled pJMI3 DNA in Vitro-Having demonstrated that the major protein synthesized in the B.
subtilis cell-free system by pJMl3 DNA-directed transcription and translation system is S. aureus /3-lactamase, we chose to map the position for this gene on the plasmid molecule. The transcription products formed by both purified E. coli RNA polymerase (27) and B. subtilis RNA polymerase (9) directed by supercoiled pJM13 DNA at an enzyme/DNA ratio = 5 are shown in Fig. 4. Increasing the ionic strength from 2 mM (lanes c and e) to 160 mM (lanes d and f ) not only stimulated synthesis (1.5-fold for B. subtilis RNA polymerase and 1.9-fold for E. coli RNA polymerase), but also enhanced the production of RNAs of discrete size classes as previously observed for transcription of $29 DNA (9). As shown in lane d of Fig. 4, B. subtilis RNA polymerase produces products containing about 750, 1250, and a population ranging in sue from roughly 3700 to 5900 bases. E. coli RNA polymerase (lane f, Fig. 4) also produces products containing 1250 and those ranging from 3700 to 5900 bases. E. coli polymerase produces an additional product of about 8000 bases (Fig. 4, lane f). The RNA marker of 1150 bases (Fig. 4, lane a) appears to co-migrate with a pJM13 transcript (Fig. 4, lanes d and f ) that has been determined to be 1250 bases in length from a number of gels (such as that shown in  -f and h, Fig. 5, and Fig. 4). Therefore, it is a run-off RNAinitiated 2000 base pairs from one of the Eco RI ends of the staphylococcal DNA. The 3700-base RNA (Fig. 5, lanes c-f and h ) results from initiation at this same promoter and transcription across the Eco RI restriction site to a partially efficient terminator in the pMB9 portion of the plasmid. The 3700-base transcript can be placed as shown on Fig. 6, since HindIII-digested substrate is observed to leave its transcription unaltered (Fig. 5, lanes d and e). If this RNA were initiated at a promoter 2000 base pairs from the other end of the staphylococcal DNA, transcription of HindIII-digested pJM13 would result in an RNA of about 2330 bases. The 3700-base RNA was also found to hybridize to all three Eco RI fragments of pJMl3 (data not shown), which lends further support to its position indicated in the map (Fig. 6).
A significant amount of RNA polymerase reads through the termination site for 3700-base RNA to produce an RNA species some 8000 bases in length. The exact size of this The acid-insoluble radioactivity loaded was 3.7 X 10" cpm in all lanes except g, h, and i which contained 2.9 X lo", 0.48 X l b , and 0.17 X 10" cpm, respectively. Transcripts were analyzed by electrophoresis and autoradiography using a 2.0% acrylamide and 0.7% agarose slab gel with +29 transcripts serving as the markers indicated. transcript has not been determined. However, it is reduced to a length of 5300 bases when Sma I-digested pJM13 is transcribed (Fig. 5, lane fi. Additional digestion with Eco RI results in a further shortening of this transcript to 2000 bases (Fig. 5, lane g).

Cell-free Transcription and Translation
A clue to the position of the 1250-base RNA within the staphylococcal DNA is provided by transcription of Xba Idigested pJM13 which results in shortening of the 1250-base RNA to 750 bases (Fig. 5, lane c). Thus, the 1250-base transcript is initiated at a promoter 750 base pairs from the Xba I site. The location of this promoter relative to the Xba I site was established as described in the following section.
Transcription of Exonuclease 111-treated pJM13 DNA-Consecutive digestion of DNA molecules with accessible 3' termini with exoIII and S1 nuclease for increasing time periods results in progressive and synchronous shortening of these molecules from both ends. Transcription of exoIII-treated DNAs has been used as a method for defining the position of major promoters by correlating the shortening or disappear-ance of transcripts with the size of exoIII-treated substrates (10,35,42,43). We decided to determine the position of the promoter and the direction of transcription for the 1250-base RNA and also to substantiate the location of the promoter for the 3700-and the approximately 8000-base RNAs through this approach.
If the 1250-base transcript is initiated at a promoter near the HindIII site and runs toward the Xba I site as shown in Fig. 6, then a short ex0111 and S1 nuclease treatment of HindIII-digested pJM13 should result in a loss of the 1250base RNA since its promoter would be removed. If transcription proceeds in the opposite direction, this treatment should have no effect on the 1250-base RNA. The HindIII and truncated HindIII digests of pJMl3 DNA were analyzed on an agarose gel to quantitate the extent of shortening (Fig. 7a,   lanes d and e). Since the HindIII site is located 840 base pairs from the Xba I site, removal of 220 base pairs (rate from Fig.  76) from HindIII-digested DNA would be sufficient to remove the promoter if transcription proceeds in the direction indicated in Fig. 6. The HindIII and truncated HindIII digests were transcribed by E. coli RNA polymerase. Whereas the HindIII-digested DNA directs the synthesis of the 1250-base transcript (Fig. 7c, lanejl, the exoIII-and S1 nuclease-treated substrate does not (Fig. 7c, lane i). The promoter for the 1250 RNA is therefore mapped as shown in Fig. 6.
T o c o n f i the position of the other major staphylococcal promoter, Eco RI-digested pJMl3 DNA was treated with ex0111 for various time periods followed by S1 nuclease digestion. The digests were analyzed on an agarose gel (Fig. 7a) and found to be shortened at a constant rate of 220 nucleotides/min/end (Fig. 76). Transcription of these progressively shortened DNAs by E. coli RNA polymerase results in shortening of the 2000-base RNA at a rate consistent with the template size (Fig. 7c, lanes 6, c, d, and g). The shortest template (Fig. 7c, lane g) should result in a transcript of 240 \ €co R I €io RI FIG. 6. In vitro transcription map of pJM13 DNA. Locations of the major promoter and termination sites utilized by E. coli RNA polymerase were determined using restricted and exoIII shortened-DNA as described in the text. That the exact location of the termination site for the read-through RNA has not been determined is represented by the dashed line. Both promoters are also utilized by B. subtilis RNA polymerase as indicated in Fig. 4, but the long readthrough RNA apparently terminates earlier nearer the Sma I site.
The restriction sites shown are those used to establish the map. Out of 13,350 f 300 total base pairs, the restriction sites are mapped clockwise around the plasmid as follows: Eco RI at 0 or 13,350 base pairs, HindIII at 330 base pairs, Sma I at 3140 base pairs, Eco RI at 5450 base pairs, Eco RI at 6250 base pairs, Xba I at 8750 base pairs, and HindIII at 9590 base pairs. solid circles represent the Eco RI A or staphylococcal DNA fragment bases, slightly shorter than the $29 Ala transcript, which is probably unresolved at the bottom of the gel. Thus the 2000base RNA is initiated at a promoter 2000 base pairs from the Eco RI site and results from RNA polymerase running off the end of Eco RI-digested DNA (Fig. 6). Translation Products Directed by exoIII-treated pJM13 DNAs-Having mapped the two major transcripts directed by the staphylococcal portion of pJM13 DNA, we wanted to know which one encoded the p-lactamase protein.  for reactions b, c, d, g, i, and;, respectively. Equal amounts of acid-insoluble radioactivity (800,000 cpm) were loaded on the gel for all reactions except f (450,000 cpm) and g (307,000 cpm). (Fig. 8, lanes a-h, j , 1, and m), but no P-lactamase is made when the promoter for 1250-base RNA is removed as in the case of the exoIII-treated HindIII digest (Fig. 8, lane k). The small amount of p-lactamase which is apparently produced (Fig. 8, lane k) most likely results from 1250-base RNA transcribed from a fraction of DNA which is nuclease resistant (10). Furthermore, when only the 1250-base RNA and a 240base RNA from the promoter for the 2000-base RNA are present (Fig. 7, lane g), p-lactamase is synthesized (Fig. 8,   lane g). Thus, the 1250-base RNA is the mRNA encoding Pscription and translation assays directed by the same trun-lactamase. cated DNAs used in Fig. 7 is shown in Fig. 8. P-Lactamase is Several lower molecular weight products are also made produced in all reactions in which 1250-base RNA is present when only the 1250-base RNA is present. The most major of  a-rn was 541,000, 175,340, 477,000, 264,860, 244,500,  225,370, 291,000, 539,600, 344,500, 421,200, 312,430, 120,700, and 320,230 cpm, respectively. Marker proteins are indicated with radioactive ink and included bovine serum albumin, purified extracellular S. aureus p-lactamase, chymotrypsin, and cytochrome c. these products (Fig. 8, lane g) have approximate M , = 17,600 f 400, 14,300 f 400, and 11,400 f 400. Reduced amounts of products (but in a comparable ratio to p-lactamase) with similar molecular weights are formed in the presence of the staphylococcal DNA only (Fig. 8, lane 0. Since the 1250-base RNA has only enough coding capacity for a protein of about M, = 12,000 in addition to p-lactamase, the major products larger than M, = 11,400 f 400 are probably related to plactamase. Furthermore, these products are not present when the 1250-base RNA is not made (Fig. 8, lane k). The 11,400dalton protein may be encoded in the 3' end of the 1250-base RNA.
It is likely that the product of M, = 11,400 (Fig. 8, lanes f   and g) is the upper band of a doublet which has been resolved in other experiments (data not shown). The lower band of the doublet of M, = 10,500 f 400 is present in lanes c, d, e, and h   (Fig. 8) but it is no longer synthesized in lanes f and g as most strongly indicated by the reduced intensity of this region relative to p-lactamase. It is this smaller protein of the doublet that is still made in the absence of the 1250-base RNA (fig. 8,   lane k). The protein of M , = 10,500 may therefore be encoded in the RNA initiated at a promoter 2,000 base pairs from the end of the staphylococcal DNA. The genes for resistance to cadmium, lead, and bismuth have been mapped in this region (16). Further work must be done to c o n f i the location of the genes for the 11,400-dalton and 10,500-dalton proteins on the staphylococcal DNA.

DISCUSSION
S. aureus p-lactamase is the major protein synthesized in a cell-free B. subtilis transcription and translation system directed by pJM13 DNA. A similar amount of p-lactamase is produced by the analogous E. coli system. The peptide map of the in vitro product correlates extremely well with that of purified extracellular S. aureus PC1 p-lactamase. The in vitro p-lactamase synthesized by both B. subtilis and E. coli systems, however, is larger than the extracellular enzyme on SDS-polyacrylamide gels by an apparent M, = 3100. Since p-lactamase is a secreted enzyme, the additional molecular weight of the in vitro product suggests the presence of a signal or leader peptide of 20 to 25 amino acids. Although the signal peptides of p-lactamases from Bacillus licheniformis 749/C, Bacillus cereus 569/H, and E. coli pBR322 (R-TEM) have been studied (44), this is the f i t reported characterization of the S. aureus signal peptide. Our determination of the nucleotide sequence of this region (see accompanying report) provides the cognate primary sequence of this leader peptide.
The capability of B. subtilis ribosomes to efficiently and accurately synthesize a precursor form of S. aureus p-lactamase allowed a determination of the enzymatic activity of this form. Although significant p-lactamase activity was detected over background, this activity was about 17-fold less than predicted from the amount of labeled protein synthesized in vitro. There are two possible interpretations of this observation. First, the precursor form of the enzyme may not be active because intracellular p-lactamase activity is unnecessary since penicillin and related substrates probably do not cross the cell's inner membrane. The observed activity could result from a low level of processing during the transcription and translation reaction. Second, the unprocessed form of the enzyme may be only about 6% as active as the processed form. We have not distinguished between these two alternatives. There is no evidence of inhibition of p-lactamase activity in the in vitro assay since addition of purified p-lactamase to a control cell-free assay yields the activity expected for purified enzyme alone. Although it has been reported that a cell-bound form of B. licheniformis p-lactamase from which the extracellular enzyme is cleaved has enzymatic activity (45) and that the signal sequence need not inhibit activity, this is apparently not the case with the precursor p-lactamase from S. aureus.
in addition to the efficient and productive recognition of the S. aureus p-lactamase gene by B. subtilis ribosomes, this gene has another feature commonly observed for mRNAs of Gram-positive origin (3,11,12). A significant amount of plactamase is synthesized in the absence of the initiation factor fraction. Since 28% of the P-lactamase synthesized in the presence of the salt wash fraction by the B. subtilis system is produced in the absence of the salt wash fraction, p-lactamase synthesis is stimulated only 3.6-fold by the salt wash addition. Furthermore, assays in the absence of the salt wash fraction contained about 30% of the p-lactamase activity produced in the presence of the salt wash, indicating that the products of both assays have similar properties. This 3.6-fold stimulation is in marked contrast to the 50-60-fold stimulation of translation by salt wash addition of T7 and other Gram-negative mRNAs. An inverse correlation has been observed between the dependence upon initiation factors (salt wash components) and the degree of mRNA-16 S rRNA complementarity for stimulation of ribosome binding to initiator regions (14).
Whether the feature of S. aureus p-lactamase mRNA that facilitates protein synthesis in the absence of initiation factors is related in any way to a stronger RNA-RNA complementarity should be revealed upon analysis of the primary sequence ofthe ribosome binding site region (see accompanying report). In order to position the S. aureus p-lactamase gene on pJM13, a map of the major RNA transcripts produced in vitro was constructed. P-Lactamase is encoded on a 1,250-base RNA initiated at one of two major plasmid promoters. The promoter is mapped 90 f 20 base pairs from the HindIII site in the staphylococcal DNA and is utilized by both E. coli and B. subtilis RNA polymerases. This 1250-base RNA has about 370 bases more coding capacity than required for p-lactamase (including 40 bases for ribosome binding). We have preliminary evidence to suggest that a protein of about M , = 11,400 is also encoded in this RNA.
Although p-lactamase is produced in simiiar amounts by both E. coli and B. subtilis cell-free systems, it is a higher percentage of the total protein synthesized in the B. subtilis system (65%) than in the E. coli system (39%). That is, except for a product of about M , = 14,000 (Fig. 2, lane c), the B. subtilis system yields lower amounts of most of the additional proteins synthesized by the E. coli system. The pattern of major translation products from the E . coli system directed by supercoiled (Fig. 2, lane a) Eco RI-or HindIII-cleaved (Fig. 8, lanes h and j ) or truncated Eco RI-cleaved pJM13 (Fig. 8, lanes d and e) is quite similar. pMB9 DNA is reported to cause the synthesis in minicells of polypeptides of 34,000, 18,OOO, and approximately 14,000 daltons (46). Although two of the major additional products synthesized by the E. coli cell-free system are about M , = 17,600 and 14,300, we do not believe these arise from pMB9 coding sequences for the following reasons. These proteins are synthesized when some 1,760 base pairs are removed from each end of Eco RI-digested pMB9 (Fig. 8, lane g ) leaving about 1,930 base pairs of pMB9 DNA intact. Although this would contain ample coding capacity for these proteins if a promoter were present, it would not explain the disappearance of these products upon exonucleolytic removal of 220 base pairs from HindIII-cleaved plasmid (Fig. 8, lane k ) . Furthermore, small amounts of products with similar molecular weights are synthesized in the presence of the purified staphylococcal DNA fragment (Fig. 8, lane 1). This analysis leads to the conclusion that many of the products including those of M, = 17,600 and 14,300 synthesized by the E. coli system are related to ,8-lactamase since they are made in the presence of 1,250-base RNA which encodes plactamase and perhaps a protein of 12,000 daltons or less. The reason for the greater amino acid incorporation of the E . coli system relative to the B. subtilis system is not well understood. One contributing factor may be the synthesis of slightly more 1,250-base RNA by E. coli RNA polymerase than by B. subtilis RNA polymerase (Fig. 4).
The stronger of the two major promoters which has been mapped is located 2,000 base pairs from the end of the staphylococcal DNA. The absence of a termination site in staphylococcal DNA for this RNA is consistent with the finding that the cadmium resistance locus spans this Eco RI site in pI258 from which this fragment was derived (17). Either the complete transcript encodes a protein larger than M, = 73,000 (approximate coding capacity of a 2,000-base RNA) and is interrupted by the Eco RI site, or two or more genes for resistance to cadmium and possibly even to bismuth and lead which are mapped in this region (17) may be organized on an operon which contains an Eco RI site. The only major in vitro protein product which may be encoded in this RNA transcript is M , = 10,500 since the other in vitro products in a supercoiled pJM13 DNA-directed assay are also present when all but 240 bases of the 2,000-base RNA are missing (Fig. 8, lane g). The identity of this protein is unknown.
A promoter for an operon containing the arsenate, arsenite, and antimony resistance genes is thought to be within 900 base pairs of the other end of the staphylococcal DNA fragment and transcribed in a clockwise direction (17). Since arsenate resistance is inducible, it is not surprising that a major transcript does not arise from this region. It is tempting to speculate that the minor 750-base transcript produced by B. subtilis RNA polymerase (Fig. 4, lane d) results from initiation at a promoter for these genes. E. coli RNA polymerase might not form this transcript due to interference from transcription of the opposite strand. Such transcription would occur during synthesis of the greater than 8,000-base readthrough RNA and possibly during utilization of a promoter near the Eco RI site (47).
Since pJM13 confers in vivo tetracycline resistance, there must be some transcription of pMB9 DNA in the direction opposing the synthesis of the 3700-base and its read-through RNA. The promoter responsible for this transcription is thought to be located at the HindIII site in pMB9 (48) and is salt sensitive (49). It is perhaps not unexpected that this promoter is much weaker than the staphylococcal promoters since the proteins encoded in pMB9 that are involved in tetracycline resistance act either intracellularly or at the membrane (46) and would not be expressed at the levels required for p-lactamase which is often dispersed extracellularly.
The S. aureus p-lactamase gene has been mapped on a plasmid which directs its efficient synthesis in both B. subtilis and E. coli cell-free transcription and translation systems. It is a good candidate for characterization of a ribosome binding site region since it has features typical of mRNAs from Grampositive bacteria and this information will result in the elucidation of the signal peptide sequence of a Gram-positive protein.