Proc. Nati Acad. Sci. USA Vol. 80, pp. 697-701, February 1983 Biochemistry Gene for staphylococcal protein A (cloning/DNA sequence determination/signal peptide/expression) SVEN LOFDAHL*, BENGT Guss, MATHIAS UHLINt, LENNART PHILIPSONt, AND MARTIN LINDBERG Department of Microbiology, University of Uppsala, The Biomedical Center, Box 581, S-751 23 Uppsala, Sweden Communicated by Jan G. Waldenstrom, October 26, 1982 ABSTRACT The gene for protein A from Staphylococcus au- reus was cloned into pBR322 in Escherichia coli An immunoassay was used to detect production of the protein. Protein A produced in E. coli was found in the periplasmic space and was purified and concentrated by IgG-Sepharose affinity chromatography. DNA sequence assay of the gene revealed a region with the general fea- tures of a prokaryotic signal peptide and a fifth structural region homologous to the four repetitive regions found earlier by amino acid sequence determination of the mature protein. Upstream from the structural gene there is a possible promoter region and a ribosomal binding sequence typical of gram-positive bacteria. The initiation codon is TTG. Protein A of Staphylococcus aureus is a cell wall component with a reported molecular weight of about 42,000 (1). The pro- tein has an extended shape (1), and sequence analysis has re- vealed two functionally distinct regions in the molecule (2, 3). The NH2-terminal part with a molecular weight of 27,000 con- sists of four continuous highly homologous IgG-binding units, each with a molecular weight of about 7,000. The COOH-ter- minal part with a molecular weight of about 15,000 (2) is a region covalently bound to the peptidoglycan with no IgG-binding ca- pacity. Because protein A interacts with the Fc fragment region of immunoglobulins from most mammalian species (4), the pro- tein has been used extensively for quantitative and qualitative immunological techniques (5). The biosynthesis of protein A occurs during the exponential growth phase of S. aureus (6). Most of the protein is bound to the cell wall, but in the stationary growth phase of the bacte- rium, some release probably occurs because of autolysis (7). There are also mutant strains of S. aureus that are unable to incorporate protein A into the cell wall; therefore, the protein is recovered in the growth medium. Extracellular production of protein A is common among methicillin-resistant strains (8). The present paper describes the cloning into Escherichia coli of the protein A gene (spa) from a staphylococcal strain with cell wall-associated protein A. The sequence of the cloned gene, which is expressed in E. coli, has been partially determined. The DNA sequence is compared with the available amino acid sequence of the protein (2, 3). An NH2-terminal signal peptide also has been identified that appears to function in E. coli. MATERIAL AND METHODS Bacterial Strains and Plasmids. E. coli K-12 strains AB259 (9), GM161 (10), and HB101 (11) were used as bacterial hosts under P1 containment conditions as approved by the Swedish Recombinant DNA Advisory Committee. The plasmid vectors were pBR322 (12), pBR328 (13), pTR262 (14), pHV14 (15), and pHV33 (16). S. aureus strain 8325-4 (011) mec4916 nov-142 str- 4916 (17) was used as the donor of spa. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertise- ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. DNA Preparations. Staphylococcal chromosomal DNA was prepared from cells grown to midlogarithmic phase, and pro- toplasts were prepared in 25% sucrose/50 mM Tris, pH 7.2, by lysostaphin treatment (15 pug/ml) at 370C for 30 min. The protoplasts were lysed by dilution of the sucrose to 10% and addition of Triton X-100 (0.1%) and EDTA (50 mM). DNA was treated with proteinase K (0.1 mg/ml) and NaDodSO4 (0.5%) for' 1 hr at 370C. After repeated phenol extractions, the DNA was precipitated with ethanol, dissolved in 10 mM Tris HCI buffer, pH 7.5/1 mM EDTA, and dialyzed against the same buffer. E. coli plasmid DNA was prepared as described by Tan- aka and Weisblum (18). For scoring large numbers of clones for plasmid DNA, an alkaline extraction method (19) was used. Experimental Procedures. Transformation of E. coli was car- ried out as described by Morrison (20). Restriction endonucleases, T4 DNA ligase (New England BioLabs, Boston), and alkaline phosphatase (Sigma) were used according to the supplier's recommendations. The 5' end of endonuclease-generated DNA fragments was labeled with [y-32P]ATP (New England Nuclear, 2,700 Ci/mmol; 1 Ci = 3.7 X 1010 becquerels) by using T4 polynucleotide kinase (Boeh- ringer Mannheim). The DNA sequence was determined as de- scribed by Maxam and Gilbert (21). Cell extracts for assay and purification of protein A from E. coli were made by several methods, including sonication in a buffer containing protease inhibitors (22), disintegration in a pressure cell (X-press, Biotec, Stockholm, Sweden) in polymix buffer (23), or lysozyme treatment in 50 mM Tris'HCI, pH 8.5/ 50 mM EDTA. The cell debris was spun down, and the super- natant was saved for analysis. An osmotic shock procedure was used to release proteins from the periplasmic space (24). Alkaline phosphatase was assayed as described by Heppel et aL (25). Phenylalanine-tRNA synthetase was tested as described by Wagner et aL (26). For detection and quantification of protein A, the enzyme- linked immunosorbent assay (ELISA) was used (27). Human IgG (Kabi, Stockholm, Sweden) was used to coat wells in a microtiter dish (Titertek, Amstelstad, The Netherlands). Serial 1:2 dilutions of the cell extracts were added to allow binding of protein A to the IgG. Free IgG-binding sites were then ti- trated by adding alkaline phosphatase-linked protein A. Pure protein A was used in parallel for quantitative determinations. The method allows detection of nanogram quantities of protein A. IgG-Sepharose 4B (Pharmacia, Uppsala, Sweden) affinity Abbreviations: kb, kilobase(s); ELISA, enzyme-linked immunosorbent assay. * Present address: Dept. of Bacteriology, National Bacteriological Lab- oratory, S-105 21 Stockholm, Sweden. t Present address: Dept. of Biochemistry, Royal Institute of Technol- ogy, S-100 44 Stockholm, Sweden. * Present address: European Molecular Biology Laboratory, Heidel- berg, Federal Republic of Germany. 697
Transcript
Proc. Nati Acad. Sci. USAVol. 80, pp. 697-701, February 1983Biochemistry
Gene for staphylococcal protein A(cloning/DNA sequence determination/signal peptide/expression)
SVEN LOFDAHL*, BENGT Guss, MATHIAS UHLINt, LENNART PHILIPSONt, AND MARTIN LINDBERG
Department of Microbiology, University of Uppsala, The Biomedical Center, Box 581, S-751 23 Uppsala, Sweden
Communicated by Jan G. Waldenstrom, October 26, 1982
ABSTRACT The gene for protein A from Staphylococcus au-reus was cloned into pBR322 in Escherichia coli An immunoassaywas used to detect production of the protein. Protein A producedin E. coli was found in the periplasmic space and was purified andconcentrated by IgG-Sepharose affinity chromatography. DNAsequence assay of the gene revealed a region with the general fea-tures of a prokaryotic signal peptide and a fifth structural regionhomologous to the four repetitive regions found earlier by aminoacid sequence determination of the mature protein. Upstreamfrom the structural gene there is a possible promoter region anda ribosomal binding sequence typical of gram-positive bacteria.The initiation codon is TTG.
Protein A of Staphylococcus aureus is a cell wall componentwith a reported molecular weight of about 42,000 (1). The pro-tein has an extended shape (1), and sequence analysis has re-vealed two functionally distinct regions in the molecule (2, 3).The NH2-terminal part with a molecular weight of 27,000 con-sists of four continuous highly homologous IgG-binding units,each with a molecular weight of about 7,000. The COOH-ter-minal part with a molecular weight ofabout 15,000 (2) is a regioncovalently bound to the peptidoglycan with no IgG-binding ca-pacity. Because protein A interacts with the Fc fragment regionofimmunoglobulins from most mammalian species (4), the pro-tein has been used extensively for quantitative and qualitativeimmunological techniques (5).The biosynthesis of protein A occurs during the exponential
growth phase of S. aureus (6). Most of the protein is bound tothe cell wall, but in the stationary growth phase of the bacte-rium, some release probably occurs because of autolysis (7).There are also mutant strains of S. aureus that are unable toincorporate protein A into the cell wall; therefore, the proteinis recovered in the growth medium. Extracellular productionof protein A is common among methicillin-resistant strains (8).The present paper describes the cloning into Escherichia coli
ofthe protein A gene (spa) from a staphylococcal strain with cellwall-associated protein A. The sequence of the cloned gene,which is expressed in E. coli, has been partially determined.The DNA sequence is compared with the available amino acidsequence of the protein (2, 3). An NH2-terminal signal peptidealso has been identified that appears to function in E. coli.
MATERIAL AND METHODSBacterial Strains and Plasmids. E. coli K-12 strains AB259
(9), GM161 (10), and HB101 (11) were used as bacterial hostsunder P1 containment conditions as approved by the SwedishRecombinant DNA Advisory Committee. The plasmid vectorswere pBR322 (12), pBR328 (13), pTR262 (14), pHV14 (15), andpHV33 (16). S. aureus strain 8325-4 (011) mec4916 nov-142 str-4916 (17) was used as the donor of spa.
The publication costs ofthis article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertise-ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
DNA Preparations. Staphylococcal chromosomal DNA wasprepared from cells grown to midlogarithmic phase, and pro-toplasts were prepared in 25% sucrose/50 mM Tris, pH 7.2,by lysostaphin treatment (15 pug/ml) at 370C for 30 min. Theprotoplasts were lysed by dilution of the sucrose to 10% andaddition of Triton X-100 (0.1%) and EDTA (50 mM). DNA wastreated with proteinase K (0.1 mg/ml) and NaDodSO4 (0.5%)for' 1 hr at 370C. After repeated phenol extractions, the DNAwas precipitated with ethanol, dissolved in 10 mM Tris HCIbuffer, pH 7.5/1 mM EDTA, and dialyzed against the samebuffer. E. coli plasmid DNA was prepared as described by Tan-aka and Weisblum (18). For scoring large numbers ofclones forplasmid DNA, an alkaline extraction method (19) was used.
Experimental Procedures. Transformation ofE. coli was car-ried out as described by Morrison (20).
Restriction endonucleases, T4 DNA ligase (New EnglandBioLabs, Boston), and alkaline phosphatase (Sigma) were usedaccording to the supplier's recommendations. The 5' end ofendonuclease-generated DNA fragments was labeled with[y-32P]ATP (New England Nuclear, 2,700 Ci/mmol; 1 Ci = 3.7X 1010 becquerels) by using T4 polynucleotide kinase (Boeh-ringer Mannheim). The DNA sequence was determined as de-scribed by Maxam and Gilbert (21).
Cell extracts for assay and purification of protein A from E.coli were made by several methods, including sonication in abuffer containing protease inhibitors (22), disintegration in apressure cell (X-press, Biotec, Stockholm, Sweden) in polymixbuffer (23), or lysozyme treatment in 50 mM Tris'HCI, pH 8.5/50 mM EDTA. The cell debris was spun down, and the super-natant was saved for analysis.An osmotic shock procedure was used to release proteins
from the periplasmic space (24).Alkaline phosphatase was assayed as described by Heppel et
aL (25). Phenylalanine-tRNA synthetase was tested as describedby Wagner et aL (26).
For detection and quantification of protein A, the enzyme-linked immunosorbent assay (ELISA) was used (27). HumanIgG (Kabi, Stockholm, Sweden) was used to coat wells in amicrotiter dish (Titertek, Amstelstad, The Netherlands). Serial1:2 dilutions of the cell extracts were added to allow bindingof protein A to the IgG. Free IgG-binding sites were then ti-trated by adding alkaline phosphatase-linked protein A. Pureprotein A was used in parallel for quantitative determinations.The method allows detection ofnanogram quantities of proteinA.
Abbreviations: kb, kilobase(s); ELISA, enzyme-linked immunosorbentassay.* Present address: Dept. of Bacteriology, National Bacteriological Lab-oratory, S-105 21 Stockholm, Sweden.
t Present address: Dept. of Biochemistry, Royal Institute of Technol-ogy, S-100 44 Stockholm, Sweden.
* Present address: European Molecular Biology Laboratory, Heidel-berg, Federal Republic of Germany.
697
698 Biochemistry: L6fdahl et al.
chromatography was used to purify and concentrate protein Afrom cell extracts (28). The protein was eluted from the columnby using 0.35 M NaCl/0. 1 M glycine HCI buffer, pH 3.0. Theacid-insoluble residue of the eluted fraction was analyzed byNaDodSO4/polyacrylamide gel electrophoresis. The gel wasstained with amido black (0.1% in 45% methanol/10% aceticacid).
RESULTSCloning of the Gene for Protein A. A gene bank of S. aureus
strain 8325-4(441) mec-4916, nov-142, str-4916 was con-structed. Chromosomal DNA was partially digested with MboI and sedimented through a 10-30% sucrose gradient. The sizeofthe DNA in the different fractions was determined by agarosegel electrophoresis, and fractions containing 8- to 10-kilobase(kb) DNA fragments were mixed with the vector pBR322 pre-digested with BamHI and treated with alkaline phosphatase.The mixture was ligated with T4 DNA ligase and transformedinto E. coli strain 259 with selection for ampicillin resistance.Recombinants were recognized by their sensitivity to tetracy-cline. Five hundred tetracycline-sensitive clones were pooledand grown in groups of 25. The cell extracts from these poolswere tested for protein A by the ELISA technique (27). Oneof the pools was positive for protein A. It was subdivided intofive pools with five clones in each. Finally, in a last series oftests, a single protein A-producing clone, pSPA1, was identi-fied.
Restriction Map and Subeloning of pSPA1. The restrictionmap ofpSPAL was made by single, double, or triple digests withthe enzymes indicated in Figs. 1 and 2. By adding up the sizesof the various restriction fragments, a total size for the pSPA1plasmid of 12 kb was obtained, indicating that the cloned frag-ment is around 7.6 kb.
In order to determine the minimum size of the cloned DNAthat is necessary to express a functional protein A, various re-striction fragments were subcloned into E. coli HB101. AHindIII/Pst I fragment spanning from map position 0 to 2.1 kb(Fig. 2) was cloned into pTR262 forming pSPA2. The insertedfragment includes 346 bases of pBR322 sequences, but the re-
0/12
90 PSPA\1A
Bam HI
FIG. 1. Circular restriction map of pSPA1, with the size in kbstarting at the EcoRI site within the vector pBR322 at 12 o'clock. Thepositions of the EcoRI, EcoRV, HindIl, and Pst I site are indicated aswell as the single BamHI site. The junctions between the vector andthe inserted DNA are indicated with thick arrows.
AS E D A B C X
, ,_i- I
EcoRI Hind III
EcoRVB || |I
0
Taq I BclRsal Hind III
PstlRsal EcoRV
2 kb
FIG. 2. (A) Schematic drawing of the gene coding for protein Awith its different regions; the heavy line represents pBR322 DNA. (B)Detailed restriction map of the correspondingDNA sequence, with thesize in kb starting at the same EcoRJ site as in Fig. 1. The junctionbetween the vectorpBR322 and clonedDNA is indicatedwith an arrow.The sites for Taq I (two) andRsa I (one) within the vector sequence havenot been included.
maining 1.8 kb of staphylococcal DNA is enough to code for afunctional protein A. An EcoRV fragment of 2.15 kb (Fig. 2)spanning the same region with 200 additional bases of staphy-lococcal DNA was cloned into pBR328 forming pSPA3. Thissubcloning was carried out to obtain inserts of spa in both ori-entations, thereby establishing whether spa can be transcribedfrom its own promoter or iftranscription is due to a read-throughfrom the tet promoter. However, out ofeight subelones, all hadthe inserted fragment in the same orientation as in the clonepSPAl. The failure to obtain clones with reversed orientationmay be caused by an unstable inverted repeat of 190 bases gen-erated under these conditions. Instead, the EcoRV fragmentwas cloned into two plasmids, pHV14 and pHV33. The differ-ence between these plasmids is that the tet promoter, whichis located upstream from the EcoRV site in the tet gene in bothplasmids, is not functional in pHV14 (15). Protein A-positiveclones were obtained with both vectors, suggesting that the spapromoter can be recognized by E. coli RNA polymerase.DNA Sequence Determination. The results of the subclon-
ing directed us to start the DNA sequence assay from theHindIII site at map position 1.4 kb and to go counterclockwise(Fig. 1). By comparing the known protein sequence of proteinA (2, 3) with the obtained DNA sequence, we could locate theposition of the HindIII site in the gene. As shown in Figs. 2 and3, this site is within region A of protein A. Further analysis re-vealed restriction sites for the enzymes Taq I, Rsa I, and BclI at map positions 0.70, 0.87, and 0.98 kb, respectively (Fig.2). These three sites were used for sequence determinations inone or both directions, giving nucleotide sequences in mostcases from both strands.
Fig. 3 shows the DNA sequence ofspa from the HindIII siteat map position 1.4 kb and upstream. The amino acid sequencededuced from the DNA sequence also is presented, togetherwith the amino acids that are changed from the protein se-quence reported by Sj6dahl (2, 3).The DNA sequence (Fig. 4) reveals an NH2-terminal region
called E similar to the repetitive protein regions D-A-B-C re-ported by Sjodahl (2, 3). This region of 50 amino acids has 42amino acids that are identical to those in protein region D.
Region E is preceded by a leader sequence with the char-acteristics ofa signal peptide containing a basic region, followedby a hydrophobic stretch of amino acids (29). The initiation co-don for translation is probably TTG, an initiation codon so faronly identified in gram-positive bacteria (30). Six nucleotidesupstream from the TTG codon, a Shine-Dalgarno sequence(31) is found that has many features in common with other gram-positive ribosome binding sites (30). Further upstream two pos-sible promoters were found (Fig. 3).
By calculating the number ofbases necessary to code for thewhole protein, it seems that pSPA3 but not pSPA2 contains thecomplete gene.
FIG. 3. The base sequence-for the 5' end of theprotein A gene. Twopossible promoters (-35 and -10) and a possible Shine-Dalgarno se-
quence (=) are indicated. The amino acid sequence as deduced fromthe DNA sequence is also-shown. The three amino acids (residues 99,101, and 120) that are changed compared to the amino acid sequenceof Sj6dahl (2,-3) are indicated as well as the 8 residues of 50 in regionE that differ from the corresponding amino acids of region D. The ini-tial residues of regions S. E, D, and A are indicated by arrows.
The Gene Product of spa in E. coli. Cell extracts of the E.coli clone pSPAl were prepared in order to study the gene prod-uct. IgG-Sepharose affinity chromatography was used to purifyand concentrate protein A from cell extracts. The protein was
analyzed by NaDodSO4/polyacrylamide gel electrophoresis.ProteinA produced inE. coli (pSPA1)migrated close to the pureprotein A from S. aureus (Pharmacia, Uppsala, Sweden) (Fig.5). The extracts from cells carrying pBR322 had no correspond-ing protein.
Transport of Protein A into the Periplasmic Space. Becausespa contains a leader sequence with the characteristics of a sig-nal peptide and protein A is found outside the cell membranein S. aureus, we investigated the localization ofthe synthesizedprotein A also in E. coli. Cells carrying pSPA1 were treated intwo ways. One portion was disintegrated in the X-press to re-
lease the contents of the whole cell; Another portion was sub-jected to osmotic shock, which releases proteins in the peri-plasmic space but not intracellular material (24). The two ex-
tracts were assayed for activity of alkaline phosphatase as an
example ofa periplasmic enzyme, phenylalanine-tRNA synthe-tase as an intracellular enzyme, and protein A. Table 1 showsthat protein A was found in the periplasmic space, thus indi-cating that the signal peptide from S. aureus is functional alsoin E. coli.
DISCUSSIONProtein A from S. aureus is a macromolecule of considerablegeneral interest. It has been suspected to play a role in the vir-ulence of S. aureus (33-35) and is an important reagent for thecharacterization of products by immunoprecipitation. Recently
Proc. Natd Acad. Sci. USA 80 (1983k, 699
G G/A T/C C
ER CW AAAATAACAA
region D TC= ~~Gt * GA
reqion E _- GCAn is CT
A_--A__ T
A
A
e~g C
CT
_ A_ A_ A
_M lo GC
A
TC
_m AG
_. T
O* ~A
Tm T
* C- CAA
jT
G
A
CX TC
* 4 TA
* G
M.' A
A
- T
A
T
* * fir G
* A
A
FIG. 4. Autoradiograph of a nucleotide sequence gel showing thejunction between regions- D and E. The sequence- determination wascarried out according to Maxam and Gilbert (21) on a DNA fragmentlabeled at the Bcl I site at the position corresponding to 0.98kb in Fig.2. The partially chemically degraded products were resolved in an 8%polyacrylamide sequence assay gel (21).
protein A has been tested in the treatment of cancer and im-munocomplex disorders (36).
Considerable work on the biosynthesis and chemistry ofpro-tein A has been carried out during the last 15 years, but littleis known of its genetics. This report is the first in a series de-
700 Biochemistry: Lofdahl et al.
A B C D E
120-
85-66-
48.5-
24 -
18.5- v
FIG. 5. NaDodSO4/polyacrylamide gel electrophoresis in 13%gelsof IgG-Sepharose-purified cell extracts. Lanes: B and C, extracts of E.coli cells carrying the respective plasmids pBR322 and pSPA1; D, com-mercially available protein A from S. aureus (Pharmacia, Uppsala,Sweden); A and E, adenovirus 2 proteins used as size markers (shownas Mr x 10-3).
scribing the isolation of the gene for protein A and its charac-teristics.The-protein A gene spa was cloned into pBR322 in E. coli
by using an immunoassay to detect production of the protein.To locate the gene, several subclones were constructed, andfinally a partial DNA sequence was determined. Because theFe-binding part of the protein consists of four repetitive sub-units (D, A, B, and C), it was not obvious where to locate thefirst DNA sequence, which was determined from the HindIIIsite at map position 1.4 kb (Fig. 2). However, Sjodahl (2, 3) hasdemonstrated that there are some minor differences betweenprotein regions D, A, B, and C because single amino acids atthe same position in the four subunits can vary. Furthermore,three extra amino acids are inserted in the NH2-terminal partof region D. Taking this into account, it was possible to locatethe HindIII site within the sequence coding for region A.
Within regions D and A, a high degree.of conformity is dem-onstrated between the published amino acid sequence and thesequence deduced from the DNA data (Fig. 3). Only 3 out of95 amino acids are different, and all changes can be explainedby single-point mutations. The divergence is probably due to
Table 1. Enzyme activities and protein A content of E. coli cellscontaining pSPA1*
* Cells were grown in a phosphate-limiting medium (32) in order toderepress the synthesis of alkaline phosphatase. One liter of cell cul-ture was divided into two portions. One portion was osmoticallyshocked to release periplasmic proteins (24); the other portion wasdisintegrated in an X-press to release the whole cell content. All fig-ures in the table are calculated per 500 ml of overnight cell culture("7.5 x 108 colony forming units per ml).
t One unit of activity represents a change in A410 per min of 1.0.t One unit of activity isdefinedasthe formation of 1 pmol/min of Phe-tRNA.
§ Because the determination was made in serial 1:2 dilutions, theamounts are presented as being. within the range of two dilutionsteps.
strain variation. The amino acid sequence was made on strainCowan I, whereas the DNA sequence was obtained from strain8325, where the genetics is better known. The DNA sequencereveals a fifth region (Fig. 3), homologous to the repetitive re-gions D, A, B, and C described earlier. This region, named E,is nearest to the NH2-terminus, and, of its 50 amino acids, 42are identical to those of region D. It has been reported thatprotein A has four IgG~binding sites, one in each of the regionsD, A, B, and C (2, 3). The four Fc-binding sites contain thesequence Phe-Tyr-Glu, which is thought to be necessary forbinding. The corresponding sequence in 'E is Phe-Tyr-Gln.Moreover, E lacks eight residues compared to regions A, B, andC and lacks 11 compared to D at the NH2-terminus. Whetherprotein A from strain 8325 has five- Fc-binding sites or whetherthe fifth region cannot bind IgG because of these differencesremains to be tested.
As protein A is found outside the cell membrane in S. aureus,a signal peptide should precede the mature protein. This was
confirmed from the deduced amino acid sequence, where a re-
gion with the general features of a prokaryotic signal peptidewas identified upstream from region E. The NH2-terminal re-
gion of 11 amino acids is basic, containing one tyrosine, one
arginine, and four lysine residues. This is followed by a hydro-phobic stretch of23 amino acids, including several flexible res-
idues, such as five glycines and one proline. In many prokaryot-ic signal sequences, an alanine residue is located at the cleavagesite; in accordance, downstream from the hydrophobic region,
Table 2. Initiation sites recognized by B. subtilis or S. aureus ribosomes or by bothSource Sequence
S. aureusprotein A .. .UAC AGGGGGUAUUAAUUG...
S. aureus1-lactamase .. .AUAUCGGAGGGUUUAUUUUG...
B. licheniformis3-lactamase ... CAAACGGAGGAGACGAUUUUUGAUG.
The sequence of protein A is compared to sequences obtained from a variety of sources as referred toby McLaughlin et al. (30). Sequences are aligned at the G-rich stretch (large capitals) complementary tothe C-C-U-C-C sequence at the 3' end of B. subtilis 16S rRNA. The initiation codon and nucleotides inthe Shine-Dalgarno sequence that match the 165 rRNA are underlined.
Proc. Natl. Acad. Sci. USA 80 (1983)
Proc. Nad. Acad. Sci. USA 80 (1983) 701
several alanines can be identified that could be cleavage sitesfor a protease. Lindmark et al (37) reported an NH2-terminalsequence Ala-Gln-Asp-Glx-Ala-Lys for extracellular protein A.This may be compared with residues 37-42 in' the deduced- se-
quence, which are Ala-Gln-His-Asp-Glu-Ala. The similarity isobvious provided that the His residue was not detected byamino acid sequence analysis. The cleavage site would then belocated at the' alanine residue number 36, which gives a signalpeptide of 36 amino acids. This is considerably larger than thereported signal peptides from E. coli (29) but fits well with signalpeptides identified in gram-positive bacteria (38, 39). However,the protein A signal peptide seems to be recognized by the E.coli membrane because the protein is found in the periplasmicspace (Table 1).The initiation codon appears to be UUG, so far only identified
in gram-positive bacteria (Table 2). UUG has been reported toinitiate the synthesis of staphylococcal &lactamase with me-
thionine in Bacillus subtilis (30). The criteria for a strong Shine-Dalgarno sequence typical ofgenes from gram-positive bacteria(30) are also fulfilled in the sequence. Six bases upstream fromthe initiation codon, a G-rich stretch of bases is found with 8of 11 bases complementary to the 3' terminus ofB. subtilis 16SrRNA. For comparison, different gram-positive initiation sitesare listed in Table 2. Three of five have been predicted to use
UUGas the initiation codon. A fourth, ,3-lactamase from B. lich-eniformis has the possibility to use UUG, which precedes theAUG codon, giving a more suitable distance to the Shine-Dal-garno sequence. It is not known ifall these proteins are initiatedwith a methionine.
There are two possible overlapping promoters, both showinga high degree of similarity to an "ideal" E. coli promoter (40).The plasmids pSPAL, pSPA2, and pSPA3 all have functional E.coli promoters derived either from the tetracycline gene ofpBR322 and pBR328 or the A-repressor gene ofpTR262. There-fore, it was not clear if the staphylococcal spa promoter is usedin E. coli. However, when the spa gene was inserted into theEcoRV site ofpHV14 or pHV33, both expressed protein A. Thedifference between pHV14 and pHV33 is that the tet promoteris not functional in pHV14. These results indicate that the staph-ylococcal promoter can be recognized by E. coli RNA poly-merase.The protein A synthesized in E. coli possesses the property
to bind to IgG. Therefore, it was possible to purify the proteinand to characterize it by gel electrophoresis. Fig. 5 shows thatits size is approximately the same as that of pure protein A iso-lated from S. aureus, but limited proteolysis may have oc-
curred.We are grateful to Dr. J. Sjoquist and Dr. H. Wigzell for stimulating
discussions and to Dr. H. Wigzell for introducing us to the ELISA tech-nique for assay of protein A. We thank H. 0. Pettersson for excellenttechnical assistance and C. Sjoholm for typing the manuscript. Thiswork was supported by grants from the Swedish Medical ResearchCouncil.
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