Vol. 159, No. 2

Expression of the Gene Encoding Protein A in Staphylococcusaureus and Coagulase-Negative Staphylococci

MATHIAS UHLEN,"2 BENGT GUSS,2 BJORN NILSSON,' FRIEDRICH GOTZ,2t AND MARTIN LINDBERG2*Department of Biochemistry and Biotechnology, Royal Institute of Technology, S-100 44 Stockholm, and Department of

Microbiology, University of Uppsala, The Biomedical Center, S-751 23 Uppsala,2 Sweden

Received 24 February 1984/Accepted 11 May 1984

Two shuttle vectors containing the gene for protein A (spa) from Staphylococcus aureus have beenconstructed to study expression of the gene in various strains of S. aureus and in the coagulase-negative speciesStaphylococcus epidermidis, Staphylococcus capitis, and Staphylococcus xylosus. One plasmid, pSPA15,contains the complete structural gene for protein A, which binds to the cell wall in various Staphylococcusspecies. The other plasmid, pSPA16, codes for a truncated protein A lacking the C-terminal part called regionX. The latter is exclusively extracellular in all Staphylococcus species tested, which confirms the importance ofregion X for cell wall binding. The expression of the plasmid-coded protein A in various strains of S. aureus isstrongly correlated to the expression of the chromosomal spa gene. The coagulase-negative species expressingplasmid-encoded protein A produce 12 to 30% of the amount coded by the chromosomal spa gene in S. aureusstrains Cowan I and A676.

Protein A is a cell wall component in most strains ofStaphylococcus aureus (19). The protein is usually linkedcovalently to the peptidoglycan (40), but mutants and clinicalstrains have been isolated which secrete all of their protein Ainto the growth medium (19, 30). Clinical isolates withextracellular protein A are especially frequent among methi-cillin-resistant strains of S. aureus (20, 45). Protein A ischaracterized by its ability to bind selectively to the Fcregion of immunoglobulin G (IgG) (8), leaving the antigen-combining sites free.

Production of IgG-binding proteins of protein A type hasalso been reported for two other coagulase-positive staphy-lococcal species, Staphylococcus hyicus (31) and Staphylo-coccus intermedius (17). There are no reports on IgG-binding proteins of this type in coagulase-negativestaphylococci, but other serum-precipitating proteins havebeen reported being produced in this group of bacteria (19,34).

Protein A can be divided into two main domains, the IgG-binding region and the so-called region X, which anchors theprotein to the peptidoglycan of the cell wall (38). The aminoacid sequence of the IgG-binding region was published bySjodahl (39). The structural gene for protein A (spa) has beencloned in our laboratory (24) and independently by Dugglebyand Jones (6). We have sequenced the whole gene (11, 43),and the deduced amino acid sequence was compared withthe earlier published amino acid sequence of the IgG-bindingregion and a partial amino acid sequence of region X.

In the present paper, the gene for protein A has beencloned into the shuttle vector pHV33 (27) to study theexpression of the gene in various bacterial species. We havealso by in vitro techniques constructed a deleted spa genelacking the nucleotide sequence coding for region X toinvestigate the importance of this region for the attachmentof protein A to the cell wall.

* Corresponding author.t Present address: Institut fur Botanik und Mikrobiologie der

Technischen Universitat Munchen, D-8000 Munich 2, Federal Re-public of Germany.

713

MATERIALS AND METHODS

Bacterial strains and plasmids. The bacterial strains usedare listed in Table 1.Two shuttle vector plasmids, pSPA15 and pSPA16, con-

taining the spa gene were constructed to enable replicationin Escherichia coli, S. aureus, and coagulase-negative staph-ylococci. For these constructions the plasmid vectorspHV33 (27), pSPA8 (44), and pSPA10 (44) were used. Theplasmid pHV33 expresses ampicillin and tetracycline resist-ance in E. coli and chloramphenicol resistance in staphylo-cocci.DNA technology. Restriction endonucleases, T4 DNA li-

gase (New England Biolabs) and alkaline phosphatase (Sig-ma Chemical Co.) were used according to the recommenda-tions of the suppliers.

Plasmid DNA from E. coli was prepared by the alkalineextraction method (1) and from staphylococci by centrifuga-tion of cleared lysates in CsCl gradients containing ethidiumbromide (22, 41). Electrophoresis of endonuclease-digestedplasmid DNA and elution and purification offragments wereperformed as described earlier (43).Growth media and transformations. E. coli strains were

propagated in Luria broth (25) with 0.1% glucose. Transfor-mation of E. coli HB101 was done by the method ofMorrison (28). Transformants were selected on Luria brothplates containing ampicillin (50 ,ug/ml) or tetracycline (10 jig/ml). The Staphylococcus species were grown in TryptoneSoya Broth, (Oxoid Ltd., London, England), supplementedwith thiamine (1 mg/liter), nicotinic acid (1.5 mg/liter), andcalcium pantothenate (1.5 mg/liter). Tryptone Soya Agarwas used as solid medium.

Preparation and transformation of protoplasts of S. aui-e-us, Staphylococcus epidermidis and Staphylococcus xylosuiswere done as previously described (10, 21). The cell wall-regenerating medium was DM3, a Casamino Acids-yeastextract-bovine serum albumin medium containing 0.5 Msodium succinate and 8 g of agar per liter according to Changand Cohen (5). For selection of chloramphenicol-resistanttransformants, CY broth (33) containing 0.5 M sodiumsuccinate, 0.020 M MgCl2, 0.8 g of bovine serum albumin,

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714 UHLEN ET AL.

TABLE 1. Bacterial strains

Species Strain Characteristics and Referencederivation

S. aureus Cowan I (NCTC High-level producer of 78530) cell wall-bound

protein AA676 Produces only extra- 23

cellular protein A;Methicillin resistant

SA113 Restriction-deficient 12mutant of strain 8325

8325-4 Donor of the cloned 24spa gene

U320 Protein A-deficient 13mutant derived fromstrain SA113lysogenized withphage 83A

Wood 46 Protein A-negative 15reference strain

S. epidermidis 247 Contains two cryptic 35plasmids

S. xylosus KL117 Xylose-positive and 37novobiocin-resistantwhich are speciescharacters

S. capitis LK499 Resistant to 14lysostaphin

E. coli HB101 Host for plasmid 4constructions

and 4 g of agar per liter was used as a soft agar overlay.Chloramphenicol was added to a final concentration of 10p.g/ml in the whole agar medium. Phenotypic expression wasallowed for 3 h before the addition of soft agar. The plateswere usually incubated for 3 days at 37°C. Plasmid DNAfrom transformants was isolated and checked by restrictionanalysis.

Since Staphylococcus capitis is resistant to lysostaphin,L-form cells were made by plating 109 CFUs on DM3 agarplates containing 25 p.g of methicillin per ml. Small L-formcolonies appeared after 4 to 5 days at 37°C at a frequency ofca. 10-7. These primary L-form colonies were subculturedonce on the same agar medium with 100 ,ug of methicillin perml and then three times on medium with 400 ,ug of methicillinper ml. From these last plates, L-form cells were resus-pended in the hypertonic buffer used for preparation ofprotoplasts and then used in transformation experiments.To decrease the restriction of incoming DNA when S.

aureus was transformed with plasmid DNA from E. coli, arestriction-deficient mutant SA113 (12) was used. To furthereliminate restriction, the protoplasts were heated at 56°C for30 s in volumes of 0.2 to 0.4 ml immediately before theaddition of DNA (42).

Qualitative and quantitative measurements of protein A. Aqualitative screening test for production of protein A wasmade by plating cells on brain heart infusion agar platescontaining 0.5 to 1% dog serum. A precipitation halo ap-peared around protein A-producing colonies after incubationfor 24 h at 37°C and 24 h at 4°C. To detect weak production, aheavy streak of cells was needed.

Cell wall-associated protein A was measured quantitative-ly by testing the binding of 125I-labeled human IgG (Kabi,Stockholm, Sweden) to the cells (15) or by using the enzyme-linked immunosorbent assay (ELISA) as previously de-scribed (24) after complete lysis of the cells by lysostaphintreatment.

Binding of 125I-labeled IgG was measured with bacteriagrown to the early stationary growth phase in Tryptone SoyaBroth. After measuring absorbance at 540 nm in a Linsonspectrophotometer, 1 ml of cell suspension was centrifugedin an Eppendorf tube, washed in phosphate-buffered saline(PBS)-0.1% Tween 20, and incubated with 3 x 104 cpm of125I-labeled IgG in 200 pLI of PBS-0.1% Tween 20. Afterincubation for 1 h at room temperature, the bacteria werecentrifuged and then washed three times in PBS-0.1%Tween 20; the radioactivity associated with the pellet wasmeasured. IgG was iodinated by the chloramine-T method(26). Extracellular protein A was measured in cell-freegrowth medium at the early stationary growth phase by theELISA test.

Production of protein A from E. coli clones was measuredby the ELISA test after growth in Luria broth medium to thestationary growth phase and release of the protein from theperiplasmic space by osmotic shock as previously described(24).

Affinity chromatography and sodium dodecyl sulfate-poly-acrylamide gel electrophoresis (SDS-PAGE). IgG-Sepharose4B (Pharmacia Fine Chemicals, Uppsala, Sweden) affinitychromatography was used to purify and concentrate proteinA from cell extracts and growth media as previously de-scribed (24). The protein was eluted from the column with0.35 M NaCl in 0.1 M glycine-HCl buffer, pH 3.0. The elutedfraction was dialyzed against distilled water, lyophilized,and then analyzed by SDS-PAGE by the method of Laemmli(18) in a 13% or 10 to 15% gradient gel. The gels were stainedwith Coomassie brilliant blue, destained, and photographed.Marker proteins were obtained from Bio-Rad Laboratories,Richmond, Calif.

RESULTSConstruction of shuttle plasmids containing the spa gene. To

study the expression of the cloned spa gene (Fig. 1) in S.aureus and coagulase-negative staphylococci and to investi-gate the importance of region X for the binding of protein Ato the cell wall, two shuttle plasmids were constructed. Theplasmid pHV33 (27), derived from the E. coli plasmidpBR322 and the S. aureus plasmid pC194, was used for thecloning. Replication of pHV33 occurs in both bacterialspecies expressing ampicillin and tetracycline resistance inE. coli and chloramphenicol resistance in the Staphylococ-cus species.

In a previous report (24), we described the construction ofa plasmid called pSPA3, which contained the spa genecloned into pBR328. The 2.1-kilobase EcoRV fragment

A.

Sau3ATaql BcIl Sau3A Hindlil

L 1, ,0

B. sE D A B c

Sau3A "') coCRV

1, .k/1. kb

x pSPA15

C. pSPA16FIG. 1. (A) Restriction map of the DNA sequence encoding

protein A from S. aureus strain 8325-4 (43). The heavy line repre-sents the structural gene. (B) Schematic drawing of the gene with itsvarious regions. S is a signal sequence, A to D are IgG-bindingregions, E is a region homologous to A to D, and X is the C-terminalpart of protein A with cell wall-binding activity. (C) The geneencoding the truncated protein A lacking region X.

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EXPRESSION OF spa IN STAPHYLOCOCCI AND E. COLI 715

pHV33HindIII EcoRV

11

pSPA101. DOe1st with

IXf) NT2. >sJ,1tA

t roatmnt

1. Dinest[;CORI

12. Puriff r.arr

ERI.HindI I I

*- EcoRV"" BamHI

S E A'

pSPA8t with 1. Dw

716 UHLEN ET AL.

TABLE 2. Expression of the spa gene in various Staphylococcusspecies"

cpm bound RelativeSpecies Strain per 8 x 109 binding

CFU (%)

S. aureus Cowan I 6,770 100SA113 1,140 17SA113(pSPA15) 2,310 34U320 250 4U320(pSPA15) 5,790 86A676 310 5A676(pSPA15) 4,910 73Wood 46 220 3Wood 46(pSPA15) 280 4

S. epidermidis 247 220 3247(pSPA15) 1,580 23

S. xylosus KL117 200 3KL117(pSPA15) 1,490 22

S. capitis LK499 280 4LK499(pSPA15) 1,840 27

a The measurements were performed on cells at early stationary growthphase by determination of the IgG-binding capacity (equals cell wall-boundprotein A) of strains with or without pSPA15, using 25I-labeled human IgG.

the same level as Cowan I. It is noteworthy that strain A676,which exclusively produces extracellular protein A in pres-ence of plasmid pSPA15, also contains cell wall-boundprotein A at a high level as well as the normal level ofextracellular protein A. The coagulase-negative species withthe plasmid pSPA15 produce 20 to 30% of the amount ofprotein A in Cowan I associated with the cell wall. The lowamounts of extracellular protein A from strains with mainlycell wall-bound protein A is probably the result of autolysisof cells (29).The expression of the deleted spa gene of pSPA16 in

various strains follows the same pattern as for the completegene of pSPA15 (Table 3). All of the pSPA16-coded proteinA was extracellular in all species and strains, which confirmsthe importance of region X for the association of protein A tothe cell wall. Strain A676 carrying plasmid pSPA16 producedfive times as much protein A as the plasmid-free parentalstrain.

Analysis of the chromosomally and plasmid-encoded spaproducts by SDS-PAGE. Figures 5 and 6 show polyacryl-

amide gels of protein A, produced by E. coli HB101 andvarious staphylococcal species, purified by IgG affinitychromatography. The chromosotnally encoded protein Afrom S. aureus strains Cowan I and 8325-4, released bylysostaphin treatment, were heterogenous in size, in contrastto the extracellular protein A from strain A676 (Fig. 5, lanes1 to 3). Protein A, encoded by plasmid pSPA15 and releasedby lysostaphin, was also heterogenous in size (Fig. 5, lanes5, 6, 8, 9, and 10), although S. aureus U320, S. epidermidisand S. xylosus give one major band corresponding to amolecular weight of ca. 53,000 which fits well with themolecular weight predicted from the DNA sequence (43). Incontrast, only a little degradation is observed in the proteinpurified from the medium of strains carrying plasmidpSPA16 encoding the truncated protein lacking region X,with a predicted molecular weight of 30,963 (Fig. 6, lanes 2to 5). Note that strain A676 produces a homogenous extra-cellular protein A (Fig. 5, lane 1 and Fig. 6, lanes 5 and 6) anda heterogenous product when the plasmid-encoded, cellwall-bound protein is released by lysostaphin (Fig. 5, lane 8).These results indicate that degradation of cell wall-boundprotein A may occur by the action of intracellular proteasesreleased during lysostaphin treatment.The protein A isolated from E. coli is extensively degraded

(Fig. 5, lane 4 and Fig. 6, lane 1). However, bands corre-sponding to the full-sized proteins can be seen in both lanes.The band from the protein encoded by the deleted spa geneofpSPA16 was strong (Fig. 6, lane 1), in contrast to the weakband from the protein encoded by the complete spa gene inpSPA15 (Fig. 5, late 4), indicating that region X is moresensitive to proteolytic degradation in E. coli than the IgG-binding portion of the protein. However, it cannot beexcluded that the heterogeneity in size is due to earlytermination at the level of transcription or translation.

DISCUSSIONProtein A of S. aureus is probably the most studied cell

surface protein in gram-positive bacteria. The reasons forthe interests in this protein are many, but the specific bindingof protein A to the Fc portion of immunoglobulins (16) hasattracted the most attention. This property has made proteinA useful for qualitative and quantitative immunologicaltechniques (9).

In previous papers, the cloning and the determination ofthe nucleotide sequence of the spa gene from S. aureus weredescribed (11, 24, 43). In this report, we have described the

TABLE 3. Cell wall-bound and extracellular protein A production (mg/liter)"Plasmid free pSPA15 pSPA16

Species Strain Cell wall Extracellular Cell wall Extracellular Cell wall Extracellularbound bound bound

S. aureus Cowan I 100 12 ncb nc nc ncSA113 15 0 30 3 15 30U320 0 0 50 6 0 100A676 0 100 50 100 0 500Wood 46 0 0 0 0 0 0

S. epidermidis 247 0 0 30 3 0 12S. xylosus KL117 0 0 30 3 0 25S. capitis LK499 nd' 0 nd 3 nd 25

a Production of cell wall-bound (released by lysostaphin treatment) and extracellular (present in growth medium) protein A by various staphylococcal species,with or without plasmids pSPA15 and pSPA16, at early stationary growth phase measured by the ELISA test. All values refer to 8 x 109 CFU/ml. Zero values cor-respond to

EXPRESSION OF spa IN STAPHYLOCOCCI AND E. COLI 717

Ml 234 5 6 78 910M

92_66

45 -

31-

22

14FIG. 5. SDS-PAGE of protein A from bacterial strains, with or

without the plasmid pSPA15, purified by lgG-Sepharose 4B affinitychromatography. Cell wall-bound protein A from Staphylococcusspecies was released by lysostaphin treatment, and periplasmicprotein A from E. coli was obtained by the osmotic shock proce-dure. The purified proteins were separated orl a 10-15% gradient gel.Lanes: (1) S. aureus A676 (culture medium); (2) S. aureus CoWan I(lysostaphin released); (3) S. aureus 8325-4 (lysostaphin released);(4) E. coli HB101(pSPA15) (from periplasm); (5) S. aureus SA113(pSPA15) (lysostaphin released); (6) S. aureus U320(pSPA15) (lyso-staphin released); (7) S. aureus A676(pSPA15) (culture medium); (8)S. aureus A676(pSPA15) (lysostaphin released); (9) S. epidermidis247(pSPA15) (lysostaphin released); and (10) S. xylosusKL117(pSPA15) (lysostaphin released). Lanes M, marker proteinswith the sizes shown as kilodaltons.

construction of shuttle vectors, which were used to transferthe spa gene between E. coli, S. aureus and various coagu-lase-negative staphylococci, to study the expression andregulation of the gene as well as the localization of the geneproduct.The availability of a system for transferring the spa gene

between various strains and mutants of S. aureus as well asother Staphylococcus species greatly facilitates studies onthe biological and immunological effects of protein A. Thesystem has already been used ih a study of protein A as apotential virulence factor in mastitis (P. Jonsson and M.Lindberg, unpublished data). To facilitate the production ofprotein A itself and for the production of fused proteinsbased on the spa gene (44), it is desirable to find apatho-genic, gram-positive bacteria as alternatives to S. aureus.There is a considerable variation in the production of

protein A in various strains of S. aureus (19). The expressionof the spa gene in plasmids pSPA15 and pSPA16 is correlat-ed to the expression of the chromosomal spa genes in thevarious S. aureus strains, with the exception of strain A676with the deleted spa gene in plasmid pSPA16, which has aconsiderably increased production compared with the plas-mid-free parental strain (Table 3). On the other hand, strainA676 with the complete spa gene in plasmid pSPA15 onlyproduces twice as much protein A compared with theplasmid-free strain, with approximately half the amount ofprotein A bound to the cell wall (Table 3). In strain SA113,the expression levels with either of the plasmids wereincreased 100% compared with the plasmid-free strain, but

still the expression was considerably repressed comparedwith strains Cowan I and A676 (Tables 2 and 3). The mutantstrain U320 was derived from a derepressed mutant ofSA113 (13); consequently, the expression levels of theplasmid-coded genes are comparable to Cowan I and A676(Tables 2 and 3). In strain Wood 46, the plasmid-coded spagenes were completely repressed as was the chromosomalspa gene.

Several authors have suggested a common regulation ofthe synthesis of various exoproteins, including protein A,mainly based on the isolation of pleiotropic exoprotein-negative mutants (3, 7). Strain 8325-4, which was the donorof the spa gene for cloning in E. coli (24), produces minuteamounts of protein A (32), much less than the related strainSA113, which was used in this investigation (Tables 2 and 3).In an earlier study on the production of protein A in 8325-4(32), it was shown that derepressed production of protein Awas obtained by growing streptomycin- or novobiocin-resis-tant mutants of this strain in the presence of the antibiotics.Furthermore, derepressed production of protein A wascorrelated with inhibition of synthesis of alpha- and beta-hemolysin. It was suggested that the structural spa gene wasregulated by a repressor protein, the synthesis of which wasinhibited by streptomycin or novobiocin. This repressorhypothesis is supported by the finding that mutants of SA113with high-level production of protein A can be isolated, asdescribed for the isolation of the protein A-deficient mutantU320 (13). According to the repressor hypothesis, our re-sults suggest that strains Cowan I and A676 are derepressed,strain SA113 is partially derepressed, and strain Wood 46 isstrongly repressed (Tables 2 and 3). In fact, we recentlyfound that Wood 46 produces minute amounts of extracellu-lar, low-molecular-weight protein A, which was detected byconcentration of a 3-liter volume of growth medium on anIgG-Sepharose 4B column. Derepressed mutants of thisstrain have also been isolated which produce extracellularprotein A at high levels but simultaneously have lost theproduction of alpha-hemolysin and extracellular proteases(M. Lindberg, unpublished results). These data are congru-

M 1 2 3 4 5 6 M

9266

45

31

221 4

FIG. 6. SDS-PAGE of protein A from bacterial strains, with orwithout the plasmid pSPA16, purified by IgG-Sepharose 4B affinitychromatography. The proteins were purified from the culture medi-um of various Staphylococcus species and from an osmotic shocklysate of E. coli and separated on a homogenous 13% gel. Lanes: (1)E. coli HB101(pSPA16) (from periplasm); (2) S. aureus U320(pSPA16); (3) S. epidermidis 247(pSPA16); (4) S. xylosusKL117(pSPA16); (5) S. aureus A676(pSPA16); and (6) S. aureusA676. Lanes M, marker proteins with the sizes shown as kilodal-tons.

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718 UHLEN ET AL.

ent with the model for coregulation of extracellular proteinsin S. aureus suggested by Bjorklind and Arvidson (3).

Protein A coded by the chromosomal spa gene in S.aureus strains Cowan I and 8325-4 and released bylysostaphin from the cell walls was heterogenous, but theslowest migrating band in each, strain was of similar sizecorresponding to molecular weights of ca. 52,000 and 53,000,respectively (Fig. 5, lanes 2 and 3). Protein A encoded byplasmid pSPA15 containing the complete spa gene expressedin various staphylococcal hosts was of similar size and morehomogenous (Fig. 5, lanes 5, 6, 8, 9, and 10). Earlierdeterminations of the molecular weight of cell wall-boundprotein A from strain Cowan I have given contradictoryresults, depending on the methods used. Bjork et al.. (2)reported a molecular weight of 42,000 when determined bygel filtration on Sepharose 6B in 6 M guanidine hydrochlo-ride and 56,000 when determined by disc electrophoresis inpolyacrylamide gels containing 0.1% SDS. These data wereconfirmed by Movitz et al. (30). The values obtained by gelfiltration were considered more reliable. However, from theknowledge of the DNA sequence of the cloned spa genefrom strain 8325-4 (43), it is possible to predict a molecularweight of 52,752 of the mature protein. The gene productencoded by pSPA16, which is homogenous, has a tnolecularweight as determinied by gel electrophoresis (Fig. 6) which isclose to the predicted value of 30,963.The present investigation has finally confirmed the impor-

tance of region X for the association of protein A to the cellwall. The spa gene of strain A676, which produces exclu-sively extracellular protein A, was recently cloned in ourlaboratory. The gene will be further characterized to investi-gate the interaction of protein A and the cell wall. Analternative approach will be to introduce in vitro mutationsor deletions in region X of the gene encoding cell wall-boundprotein A and to study the capacity of cell wall binding of thealtered proteins.

ACKNOWLEDGMENTS

This investigation was supported by grants from the SwedishMedical Research Council (to M.L., project no. 16X-03778), theSwedish National Board for Technical Development (to M.U. andB.N.) and Pharmacia Fine Chemicals, Uppsala, Sweden (to M.L.).We thank L. Philipson for valuable discussions and K.-E. Johans-

son for expert help and advice in the SDS-PAGE. We thank H.-O.Pettersson for skillful technical assistance and C. Pellettieri forskillful secretarial help.

ADDENDUM IN PROOFWe recently found an error in our earlier DNA sequence

of the protein A gene (43). After nucleotide 1617, close to the3' end of the structural gene, a G should be inserted, whichshifts the reading frame anid adds 15 amino acid residues tothe protein. Thus, the total length of the mature proteinshould be 488 amino acids, giving a predicted Mr of 53,697.The correction does not influence the general discussion ofthe present paper, but gives a stretch of hydrophobic aminoacids which might suggest that the C-terminal part of regionX is associated with the cell membrane.

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