Lactococcus lactis subsp. lactis C2

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1 JOURNAL OF BACTERIOLOGY, Oct. 1991, p /91/ $02.00/0 Copyright X) 1991, Americn Society for Microbiology Vol. 173, No. 19 A Membrne Protein Is Required for Bcteriophge c2 Infection of Lctococcus lctis subsp. lctis C2 R. VALYASEVI, W. E. SANDINE, AND B. L. GELLER* Deprtment of Microbiology nd The Center for Gene Reserch nd Biotechnology, Nsh 220, Oregon Stte University, Corvllis, Oregon Received 6 My 1991/Accepted 19 July 1991 Phge-resistnt mutnts, isolted from cultures of Lctococcus lctis subsp. lctis C2 infected with phge c2, did not form plques but bound phge normlly. The mutnts were sensitive to nother phge, skl, lthough the number of plques ws reduced -56% nd the plques were four times smller. Binding to phge skl ws reduced bout 10%. Another group of phge-resistnt mutnts, isolted from cultures infected with phge skl, bound normlly to both phges c2 nd skl but did not form plques with either phge. Crbohydrte nlyses by gs chromtogrphy of the ceul wlls showed no significnt differences in scchride compositions between the wild-type nd phge-resistnt cells. However, difference ws observed in the interctions of the phge with the cytoplsmic membrnes. Membrnes from the wild-type cells, but not mutnt cells, inctivted phge c2. Phge skl ws not inctivted by membrne from either strin. Tretment of wild-type membrnes with proteinse K eliminted the bility of the membrne to inctivte the phge, wheres tretment with mutnolysin hd no effect. On the bsis of this bility to inctivte the phge, membrne protein ws prtilly purified by gel filtrtion nd ion-exchnge chromtogrphy. Under nondenturing conditions, the phgeinctivting protein hs n pprent Mr of e350,000. The protein hs n pprent subunit size of 32 kd, which suggests tht it normlly exists s multimer with 10 to 12 subunits or in ssocition with other membrne components. It is proposed tht this protein is required for phge c2 infection. In lctococci, crbohydrtes of the exopolyscchride my be commonly used s phge receptors (8). Previous studies from our lbortory hve shown tht the binding determinnts of phges for Lctococcus lctis subsp. cremoris KH nd subsp. lctis C2 include the rhmnose of the extrcellulr wll polyscchrides (16, 17). An exception to this ws reported by Orm nd Reiter (12), who found tht membrnes from L. lctis subsp. lctis ML3 inctivted phge m13. The phge-inctivting mteril ppered to be protein, becuse trypsin digestion of the plsm membrne destroyed the phge-inctivting ctivity. In our previous study (16), tretment of the cell envelope (cytoplsmic membrne plus peptidoglycn nd exopolyscchrides) from L. lctis subsp. lctis C2 with either mutnolysin or sodium dodecyl sulfte (SDS) lone did not completely eliminte the dsorption of some phges (for exmple, c2). This suggested tht the dsorption of certin phges my require both membrne component nd scchride unit on the cell wll (peptidoglycn nd exopolyscchrides). In this pper, we report difference in the cytoplsmic membrnes of phge c2-sensitive nd -resistnt strins of L. lctis subsp. lctis C2. This difference correltes with the presence of protein nd ws used to purify nd identify protein with n pprent Mr of -350,000 nd subunit size of 32 kd. We propose tht this protein is required for phge c2 infection, pprently t step subsequent to dsorption. MATERIALS AND METHODS Bcteril strins, phges, nd medium. Bcteriophges c2 nd skl nd the bcteril host, L. lctis subsp. lctis C2, were grown nd mintined on M17 medium t 30 C (15). * Corresponding uthor Phge nd bcteril stocks were prepred s previously described (15) nd stored in 20% glycerol t -70 C. Isoltion nd solubiliztion of phge-inctivting protein. Cells of L. lctis subsp. lctis C2 from 9-liter culture were hrvested t n A6. of 1 to 1.2. Cytoplsmic membrnes were prepred by differentil centrifugtion of lysed, lysozyme-treted protoplsts, essentilly s previously described (13). Briefly, the cells were pelleted by centrifugtion (10,000 x g, 10 min, 4 C) nd wshed once in 150 ml of 0.1 M potssium phosphte (ph 7.0)-0.2 mm phenylmethylsulfonyl fluoride (PMSF). The cells were gin pelleted by centrifugtion nd resuspended in 150 ml of 0.1 M potssium phosphte (ph 7.0)-10 mm MgSO4. Lysozyme (0.75 g) ws dded, nd the mixture ws incubted t 30 C for 30 min. Potssium sulfte nd PMSF were dded to finl concentrtions of 0.15 M nd 0.2 mm, respectively. After 5 min t 30 C, the mixture ws diluted to twice its volume with 0.1 M potssium phosphte (ph 7.0), nd RNse nd DNse were dded to finl concentrtions of 50,ug/ml ech. The mixture ws incubted 20 min t 30 C. A 0.1 M solution of K-EDTA (ph 7.2) ws dded to finl concentrtion of 15 mm. The mixture ws incubted 10 min t 30 C, nd then MgSO4 ws dded with gentle stirring so tht the concentrtion ws incresed to 20 mm. The mixture ws centrifuged t 48,000 x g for 30 min t 4 C. The pellet, contining the membrnes, cells, nd debris, ws resuspended in 75 ml of 50 mm potssium phosphte (ph 7.0)-10 mm MgSO4. The mixture ws centrifuged t 750 x g for 70 min t 4 C to remove the unbroken cells nd debris. The superntnt ws removed nd centrifuged t 48,000 x g for 30 min t 4 C. The pellet, contining the membrnes, ws resuspended in 10 mm [bis (2 - hydroxyethyl) imino - tris (hydroxymethyl) methne], ph 6.5. Membrnes (21 mg of protein) were solubilized on ice in buffer consisting of 10 mm bis(2-hydroxyethyl)iminotris(hydroxymethyl)methne (ph 6.5), 1% (wt/vol) Triton

2 6096 VALYASEVI ET AL. J. BACTERIOL. X-100 (Sigm Chemicl Co.), 10% (vol/vol) glycerol, nd 1 mm dithiothreitol (DTT) in finl volume of 1 ml. After 60 min, unextrcted mteril ws pelleted by centrifugtion t 4 C (120,000 x g for 60 min; Beckmn Airfuge Ultrcentrifuge). The superntnt ws collected nd stored t -70 C for lter use. The extrct usully contined bout 40% of input protein nd more thn 90% of phge-inctivting ctivity. Preprtion of cell wlls for gs chromtogrphy. Cells were hrvested t n A600 of 1.8 to 2 nd suspended in 10 mm KH2PO4 (ph 6.8) to finl concentrtion of 150 mg/ml (wet weight). Cell wlls (peptidoglycn nd exopolyscchrides) were isolted s previously described (16). Briefly, the resuspended cells were mechniclly disrupted by vigorous gittion with glss beds. The beds were removed by filtrtion through pper, nd the whole cells nd debris were removed by centrifugtion t 1,400 x g for 5 min t 4 C. Cell wlls were sedimented by centrifugtion t 15,000 x g for 10 min t 4 C. Resuspended cell wlls were treted with DNse nd RNse (40,ug/ml [ech]) nd PMSF (0.2 mm). The cell wll preprtion ws extrcted with hot (70 C) SDS (1 mg/ml) to remove the membrne. The cell wlls were wshed three times with wter, collected by centrifugtion, resuspended in wter, frozen in liquid nitrogen, nd stored t -70 C. Freeze-dried cell wlls (10 mg/ml) were mixed with ribose (20 jig/ml) s n internl stndrd in 2 N HCl, flushed with nitrogen gs, nd hydrolyzed t 100 C for 3 h. The hydrolystes were neutrlized with 15 N NH40H by using phenolphthlein s n indictor. Hydrolyzed cell wlls (1 mg/ml) were mixed with xylose (50,ug/ml) nd derivtized to lditol cettes s described previously (5). Gs chromtogrphy nd identifiction nd quntittion of lditol cettes. The lditol cette derivtives were seprted on 3% SP-2340 glss column (30.48 by 1.2 cm; Supelco Inc., Bellefort, P.) connected to model 5710A gs chromtogrph (Hewlett-Pckrd Co., Plo Alto, Clif.) equipped with flme ioniztion detector. Chromtogrphic conditions were djusted ccording to the specifictions of the mnufcturer (14). Pek res nd retention times were determined by using Hewlett-Pckrd modgl HW 3390A electronic integrtor. The derivtized cell wll polymer scchrides seprted by gs chromtogrphy were quntified s previously described (16). Isoltion of resistnt mutnts. L. lctis subsp. lctis C2 cells (108 CFU) were mixed with 1010 PFU of phge c2 or 108 PFU of phge skl in M17 top gr (0.4%) plus 15 mm CCl2. The phge-resistnt mutnt cells were collected by wshing cells off the top gr with M17 broth. Single colonies were isolted on M17 gr contining phge c2 (108 PFU/ml) or skl (107 PFU/ml) nd tested for their bilities to bind phge nd form plques. The cell wlls of selected mutnts were prepred nd tested for losses in phge binding s previously described (16). The bility of cell wlls to bind phge is expressed s the percentge of phge inctivted by the sme weight of cell wlls from the wild-type strin C2. Phge inctivtion ssy nd units of phge inctivtion. Membrnes were mixed nd shken with phge c2 (5 x 105 PFU/ml) in 10 mm bis(2-hydroxyethyl)imino-tris(hydroxymethyl)methne (ph 6.5) t 25 C for 20 min. The mixtures were centrifuged (12,000 x g, 4 min), nd titers of phge in the superntnt were ssyed s previously described (15). The mount of phge inctivted ws clculted by dividing the difference of the totl phge dded minus the phge in the superntnt by the totl phge dded nd then multiplying by 100. Our phge inctivtion ssy contined phge c2 (5 x 105 PFU/ml) in 10 mm [bis(2-hydroxyethyl)imino-tris(hydroxymethyl) methne] (ph 6.8) nd n pproprite mount of smple to decrese the titer of the phge by 27 to 77% when shken t 25 C for 20 min. The mount of phge inctivted under these conditions ws liner function of the mount of smple dded. One unit of phge-inctivting ctivity ws defined s the mount of smple required to inctivte 52% of the phge dded, which is the midpoint of the liner portion of the phge inctivtion curve. Tretments with mutnolysin nd proteinse K. Menbrnes in solution of 50 mm K2PO4 (ph 7.0) nd 10 mm Mg2SO4 t concentrtions which inctivted 90% of phge (5 x 105 PFU/ml) were treted with mutnolysin (20 U/ml) t 25 C for 16 h or proteinse K (0.1 mg/ml) t 4 C for 16 h. The proteinse K-treted smples were further incubted with PMSF (0.2 mm) t 4 C for 30 min. The cell envelopes (membrne plus peptidoglycn nd exopolyscchrides) were treted with mutnolysin s described previously (16). Gel filtrtion chromtogrphy. The solubilized membrnes were pplied to Sephcryl S300 (Phrmci LKB Biotechnology) column (1.6 by 60 cm) in buffer consisting of 10 mm bis(2-hydroxyethyl)imino-tris(hydroxymethyl)methne (ph 6.5), 0.1% Triton X-100, 10% glycerol, nd 1 mm DTT. The sme buffer ws used for elution t flow rte of 20 ml/h. Frctions (1.5 ml ech) were collected nd ssyed for phge-inctivting ctivity nd totl protein (3; ovlbumin stndrd). Frctions with the highest phge-inctivting ctivity were combined. Ion-exchnge chromtogrphy. A DEAE-cellulose (DE52; Whtmn Biosystems Ltd., Kent, Englnd) column (0.8 by 6 cm) ws equilibrted with n equilibrtion buffer consisting of 10 mm bis (2 - hydroxyethyl) imino - tris (hydroxymethyl) methne (ph 6.5), 0.1% Triton X-100, 10% glycerol, nd 1 mm DTT. The combined frctions from the gel filtrtion column which contined phge-inctivting ctivity were pplied to the column. The column ws developed with three-step grdient t 10 ml/h. Ech step ws 1 bed volume of equilibrtion buffer plus 0.15, 0.25, nd 0.50 M NCl. The phge-inctivting ctivity eluted in the 0.25 NCl step. Frctions (1 ml ech) were collected, nd liquots were ssyed for phge-inctivting ctivity nd totl protein. SDS-polycrylmide gel electrophoresis. SDS-polycrylmide gel electrophoresis ws performed by the procedure of Lemmli (9). The gels were silver stined s previously described (2). Phospholipid ddition to colunm frctions. Attempts to increse the phge-inctivting ctivity of the DEAE-purified frctions were done s follows: cetone- nd etherextrcted Escherichi coli phospholipids (50 jxg) were lyophilized nd then dissolved by dding 200,ul of the combined pek ctivity frctions from the DEAE column. The mixture ws ssyed for phge-inctivting ctivity s described bove. Mterils. Mutnolysin, lysozyme (EC ), bovine serum lbumin, glycerldehyde-3-phosphte dehydrogense (EC ), crbonic nhydrse (EC ), soyben trypsin inhibitor, -lctlbumin, thyroglobulin, poferritin,,-mylse (EC ), nd ovlbumin were purchsed from Sigm Chemicl Compny. Proteinse K (EC ) ws from Boehringer Mnnheim Biochemicls. Totl E. coli phospholipids were purchsed from Avnti Polr Lipids, further purified by cetone nd diethyl ether extrction in the presence of 2 mm 2-mercptoethnol s previously described (7), nd stored in chloroform under N2 t -70'C.

3 VOL. 173, 1991 TABLE 1. Anlysis of phge binding to cell wll, sensitivity to infection, nd phge inctivtion by membrne % Cell wll % Sensitivity % Membrne binding tphg inctivtion Strin of phgeb to phgec of phged c2 skl c2 skl c2 skl C RMC2/ e 0 0 RMC2/ e 0 0 RMC2/ e 0 0 RMC2/ e 0 0 RMSK1/ RMSK1/ C2, wild-type strin C2; RMC2 strins, phge c2-resistnt mutnts; RMSK1 strins, phge skl-resistnt mutnts. b Percentge of totl PFU in the superntnt of mixture of phge nd cell wlls fter 20-min incubtion nd centrifugtion. c Number of plques divided by the number of plques formed by the sme mount of smple on wild-type strin C2 nd then multiplied by 100. d Percentge of totl PFU fter incubtion with membrnes. e Plque dimeters reduced by 50%. RESULTS Isoltion nd properties of phge-resistnt mutnts. Spontneously occurring phge-resistnt mutnts of L. lctis subsp. lctis C2 were selected by infecting mid-exponentil-phse culture with phge c2 t multiplicity of infection of 100. The frequency of the selected phenotype ws 106. Fifty rndomly picked mutnts were tested for their sensitivities to phge by plque ssy, nd ll were found to be totlly insensitive to phge c2 (no plques were formed). Four of these mutnts were studied further, nd the results re listed in Tble 1 (RMC2/4, -2/8, -2/9, nd -2/13). These phge c2-resistnt mutnts were chllenged with nother phge, skl, which lso infects this strin. There ws 54 to 58% reduction in the sensitivities to phge skl (Tble 1) nd fourfold reduction in the plque sizes. None of the 50 rndomly picked mutnts hd detectble defects in binding of whole cells to phge c2. To corroborte this, cell wlls (we use this term to men the peptidoglycn nd exopolyscchrides) were isolted from 4 of the 50 mutnts nd tested for phge binding. All were found to bind phge c2 to the sme extent s the phge-sensitive prentl strin, lthough binding to phge skl ws reduced by pproximtely 10% (Tble 1). This suggests tht the loss of sensitivity to these phges ws not the result of loss of the cell surfce phge receptors. To nlyze further the cellulr requirements for phge infection, spontneously occurring phge-resistnt mutnts of the sme host were selected by infecting mid-exponentil-phse culture with phge skl t multiplicity of infection of 1. The frequency of occurrence of the phge skl-resistnt mutnt ws Of the 30 mutnts tht were rndomly picked, ll but three bound the sme mount of phges c2 nd skl s the phge-sensitive prentl strin. Further nlysis of the three binding-defective mutnts will be presented elsewhere (17). All of the mutnts tht hd no decrese in the mount of phge which could bind were tested for sensitivity to phges skl nd c2 by plque ssy nd were found to be totlly insensitive to both phges. Two representtive nlyses re listed in Tble 1 (RMSK1/1 nd RMSK1/2). Cell wlls were prepred from 3 of the 27 mutnts which ppered to bind the phges normlly nd tested for binding to phge skl nd c2. In ll three mutnts tested, there ws no reduction in binding to either phge. PHAGE c2-inactivating PROTEIN 6097 TABLE 2. Crbohydrtes present in cid-hydrolyzed cell wll preprtions of wild type nd phge-resistnt mutnts of L. lctis subsp. lctis C2 determined by gs chromtogrphic nlysis of lditol cette derivtives Strin (n) Amt (pug/mg of cell wll) of crbohydrteb (men ± SD) Rh Gl Glc GlcNAc C2 (5) ± 6.2 RMC2/4 (3) ± ± ± 29 RMC2/8 (3) 255 ± ± ± 8.8 RMC2/9 (3) 241 ± ± ± RMC2/13 (3) 255 ± ± ± 7.1 RMSK1/1 (3) ± ± ± 14 RMSK1/2 (3) ± ± C2, wild-type strin C2; RMC2 strins, phge c2-resistnt mutnts; RMSK1 strins, phge skl-resistnt mutnts. b Rh, rhmnose; Gl, glctose; Glu, glucose; GlcNAc, N-cetylglucosmine. These results demonstrte tht phge-binding mutnts of this strin re much less frequent thn those in n L. lctis subsp. cremoris strin tht we hve chrcterized previously (16). It lso shows tht the loss of the phge receptor is not common mechnism of phge resistnce for this host nd these two phges. Chemicl nlyses of the cell wlls from the mutnts by gs chromtogrphy showed tht there were no significnt chnges in the cell wll crbohydrte components (Tble 2). This confirmed tht the insensitivity to phge skl nd c2 infections is not relted to ny chnges in the cell wll crbohydrte components, which contin the receptors for phge skl nd c2 in this strin (16, 17). The mechnism of phge resistnce in these cses pprently involves step subsequent to the binding of the phge to the exopolyscchride receptor. It is lso importnt to note tht the lditol cette derivtives of neither glycerol nor ribitol were detected, lthough internl stndrds of the two were redily detected nd resolved from the other monoscchrides. This suggests tht this strin lcks teichoic cids. Membrne inctivtion of phge. Plsm membrnes were prepred from prentl nd mutnt cells. When phge c2 ws mixed with membrnes from the phge-sensitive, prentl strin, 94% of the PFU were inctivted nd no longer hd the bility to infect sensitive cells (Tble 1). The inctivtion ws specific for phge c2, s phges skl, kh, nd were not inctivted by the prentl membrnes. Moreover, membrnes from the phge-resistnt mutnts (selected from infections with phge c2) did not inctivte phge c2 (Tble 1), skl, kh, or (dt not shown). These results suggested tht membrne component of the prentl strin, but not the mutnt strins, irreversibly intercts with phge c2, resulting in phge ihctivtion. To test whether the phge-inctivting membrne component ws proteinceous, prentl membrnes were treted with proteinse K. 'This destroyed the phge-inctivting ctivity of the membrnes (Tble 3). Incubtion of the sme wll membrnes with mutnolysin, which hydrolyzes cell glycosidic bonds, did not inctivte the membrne. For control, to show tht the mutnolysin tretment would hve effectively hydrolyzed ny contminting cell wll components in the membrne preprtion, the cell envelope (wll plus membrne) from the prentl strin ws similrly treted with mutnolysin. This destroyed the polyscchride, which is the phge receptor (16), s indicted by 76%

4 6098 VALYASEVI ET AL. TABLE 3. Inctivtion of phge c2 by membrnes nd cell envelope treted with proteinse K nd mutnolysin Tretment % of totl PFU inctivted by: Treted membrnes Treted envelopes None (untreted) Proteinse K 4 NAb Mutnolysin Envelope preprtions contin cell wll plus cytoplsmic membrne. b NA, not pplicble. reduction in phge binding to the mutnolysin-treted cell envelopes compred with tht of the untreted cell envelopes. These results suggest tht membrne protein inctivtes the phge. Identifiction nd purifiction of phge-inctivting membrne protein. The purifictjon of the phge-inctivting protein is summrized in Tble 4. The mount of membrne mteril necessry to inctivte 50% of the 5 x 105 phge per ml in 20 min ws defined s 1 U of ctivity. Membrnes prepred from n exponentil-phse culture of L. lctis subsp. lctis C2 hd specific ctivity of 205 U/mg of protein. To purify the phge-inctivting protein, membrnes were solubilized in solution consisting of 1% Triton X-100, 10% glycerol, 10 mm bis(2-hydroxyethyl)imino-tris (hydroxymethyl)methne (ph 6.5), nd 1 mm DTT. The insoluble proteins were removed by centrifugtion (120,000 x g, 1 h). More thn 90% of phge-inctivting ctivity ws retined in the superntnt. Becuse 48% of the totl protein ws not solubilized under these conditions, the specific ctivity fter solubiliztion incresed bout twofold, to 384 U/mg of protein. The solubilized mteril ws pssed through Sephcryl S300 gel filtrtion column. Frctions were collected nd ssyed for phge-inctivting ctivity nd totl protein (Fig. 1). One brod pek of ctivity eluted from the column, centered t position corresponding to n Mr of 350,000. The frctions with pek ctivity were pooled for further purifiction. The pooled frctions contined >90% of the eluted ctivity, which ccounted for bout 24% of the totl ctivity pplied to the column. The specific ctivity of the pooled frctions ws 701 U/mg, n increse of bout twofold from the pplied mteril. The pooled frctions from the gel filtrtion column were pplied to DEAE-cellulose column. After being wshed with 2 volumes of equilibrtion buffer (see Mterils nd Methods), phge-inctivting inctivity ws eluted with three-step grdient of 0.15, 0.25, nd 0.50 M NCl in equilibrtion buffer. Most (>90%) of the phge-inctivting ctivity eluted in single pek in the 0.25 M slt step (Fig. 2). TABLE 4. Purifiction of the phge-inctivting protein isolted from L. lctis subsp. lctis C2 Totl Totl Spt Yield' Purifi- Purifiction step protein ctivity (Umg) Sp ct (%) ction (mg) (U (fold) Crude membrne 21 4, Soluble membrne 7.5 2, Sephcryl S , DE-52b <1 <1 Expressed in percent b ctivity. Averge of two vlues. A M._ 0._ 0 U) 0 L Frction Number B egeg 5 g g ~_ std * ~~~~~~-.ff K -45 K I _K~ FRACTION NUNBER FIG. 1. (A) Sephcryl S300 chromtogrphy of the Triton X-100- solubilized membrne preprtion. Smples (7.5 mg ech) of Triton X-100-soluble membrne components were pplied to column of Sephcryl S300 (1.6 by 60 cm) equilibrted in buffer consisting of 10 mm bis(2-hydroxyethyl)imino-tris(hydroxymethyl)methne (ph 6.5), 0.1% Triton X-100, 10% glycerol, nd 1 mm DDT. Frctions (1.5 ml ech) were collected nd nlyzed for phge-inctivting ctivity (0) nd totl protein (+). Moleculr weight stndrds eluted in the positions indicted t the top of pnel A: thyroglobulin (670,000 [670K]), poferritin (442K), P-mylse (200K), nd bovine serum lbumin (66K). (B) SDS-polycrylmide gel electrophoretic nlysis of frctions contining phge-inctivting ctivity from the Sephcryl S300 column. The gel ws silver stined. The open rrowhed indictes the position of the 32-kD protein which coelutes with the phge-inctivting ctivity. The frction numbers re shown below ech lne. The lne on the right contined moleculr weight stndrds: bovine serum lbumin (66K), ovlbumin (45K), glycerldehyde-3-phosphte dehydrogense (36K), crbonic nhydrse (29K), soyben trypsin inhibitor (20.1K), nd - lctlbumin (14.3K). Usully this ws the only detectble ctivity, lthough occsionlly smll mount of ctivity eluted in the 0.15 M step. Unfortuntely, bout 96% of the ctivity ws lost during this step, reducing the specific ctivity to 52 U/mg, or bout 75% less thn the crude membrnes. A number of ttempts were mde to reconstitute the ctivity to the pooled frctions from the DEAE column nd to discover the reson for the loss of ctivity. (i) An equl volume from ll frctions ws mixed nd ssyed for ctivity. Another mixture contined just the pek protein-contining frctions from ech step, including the initil wsh. c 0 C.) 0 cu L- 0) 4- C._ 0 L-

5 VOL. 173, 1991 A %..o z 0.1 B LL 20- `1= z =310. > 1= C, o./ FRACTION NUMBER Lne : _ K 14.3 K.- oo o z 400jW 2o0 I FIG. 2. (A) DEAE-cellulose chromtogrphy of the phge-inctivting protein from the Sephcryl S300 column. The combined, pek ctivity frctions 32 to 36 (inclusive) from the Sephcryl column (1.5 mg of protein) were pplied to DEAE-cellulose column (0.8 by 6 cm) nd eluted with three-step NCl grdient. Frctions were collected nd nlyzed for phge-inctivting ctivity (0), totl protein (D), nd conductivity (*). (B) SDS-polycrylmide gel electrophoresis nd silver stining. Lnes 1 to 4 contin 15-,ul smples of frctions 15 to 18, respectively, from the DEAE column shown in pnel A. Lnes 6 to 8 contined pooled frctions from the Triton X-100 extrction, Sephcryl S300, nd DEAE columns, respectively. The mounts of protein loded in lnes 6 to 8 were 4, 1.5, nd 1 p.g, respectively. Lne 5 contins the sme moleculr weight stndrds used in Fig. 1. The open rrowhed indictes the 32-kD protein bnd which coelutes with the phgeinctivting ctivity. The totl ctivity of the mixtures did not chnge. (ii) E. coli phospholipids were dded to the pek frctions but hd no effect on the ctivity. (iii) Solubilized membrnes from the phge-resistnt strin were mixed with the purified mteril, but this did not increse the ctivity. (iv) A smple of Triton X-100-solubilized phge-inctivting mteril ws dsorbed to smll mount of equilibrted DEAE-ceilulose in tube. The undsorbed mteril hd less thn 2% of the ctivity. When 0.25 M NCl ws dded, 76% of the ctivity ws desorbed from the resin nd recovered in the superntnt fter the resin ws pelleted by centrifugtion. Incresing the NCl concentrtion from 0.25 to 0.5 M did not increse the ctivity in the undsorbed frction. (v) The crude membrnes, Triton X-100-extrcted mteril, nd pooled frctions from the gel filtrtion column were stored t 4 C for 1 week without loss of ctivity. Electrophoretic nlysis. Frctions from the gel filtrtion PHAGE c2-inactivating PROTEIN 6099 nd DEAE columns were nlyzed by SDS-polycrylmide gel electrophoresis nd silver stining (Fig. 1B nd 2B, respectively). In ech gel, only one bnd (rrowhed) correlted with the ctivity pek. This protein hd n pprent size of bout 32 kd. It ws the only mjor silver-stined bnd present fter the DEAE chromtogrphy, lthough severl minor bnds could be seen. This suggests tht the 32-kD protein is required for phge c2 infection. Considering the size of the ctivity which eluted from the gel filtrtion column under nondenturing conditions, the 32-kD phgeinctivting protein is pprently ssocited with other components or itself in multimeric complex. DISCUSSION All of the phge-resistnt mutnts isolted from n infection of L. lctis subsp. lctis C2 with phge c2 dsorbed phge normlly, even though ll were incpble of forming plques (Tble 1). The muttion in phge c2-resistnt cells lso ffected the sensitivity to nother phge, skl. Although the phge c2-resistnt cells formed plques when chllenged with phge skl, the number of plques ws -56% less nd the plques were fourfold smller. The phge c2-resistnt cells were reduced -10% in binding phge skl. A similr result ws lso observed with the phge-resistnt mutnts isolted from infection with phge skl. Almost ll the mutnt cells nlyzed, with the exception of three, bound phges c2 nd skl normlly, but none formed plques when chllenged with either phge (Tble 1). The three mutnts with loss of dsorption to phge hve chnges in their exopolyscchride composition (17). The chemicl nlyses of the cell wlls from six of the phge-resistnt mutnts by gs chromtogrphy showed tht there were no significnt chnges in the cell wll crbohydrte components compred with the wild type (Tble 2). This suggests tht phge resistnce ws not due to muttions of the phge receptor on the cell wll but rther to chnge in cellulr component required for phge infection t step subsequent to dsorption. Our results demonstrte tht membrnes from phgesensitive cells inctivte phge c2 (Tble 1). Proteinse K tretment of the membrnes completely eliminted the bility of the membrnes to inctivte the phge (Tble 2), strongly suggesting tht protein is involved in the inctivtion of phge. Although these dt lone might suggest nonspecific effect of the membrnes to inctivte the phge, the fct tht membrnes from phge-resistnt strins did not inctivte the phge mkes this very unlikely. At present, there is no evidence tht crbohydrte moiety ssocited with the membrne is involved in the inctivtion of phge, becuse mutnolysin did not reduce the phge-inctivting ctivity of the membrnes. However, mutnolysin hs restricted hydrolytic specificity, nd we cnnot rule out the possibility tht smll, hydrolytic frgments of the exopolyscchride, glycoproteins, or membrne-bound lipoteichoic cids re involved in the phge-inctivting ctivity. We speculte tht the dsorption of phge c2 to the cell surfce my involve t lest two steps, in process similr to tht of mny well-chrcterized grm-negtive bcteriophges (10). An initil, reversible binding to the cell wll polyscchride my be followed by n irreversible step, s the phge intercts with the proteinceous membrne component. In E. coli, the second, irreversible step tht leds to phge inctivtion is sometimes ssocited with the relese of the phge DNA from the cpsid (4, 6, 10). Incubtion of the membrnes from L. lctis subsp. lctis

6 6100 VALYASEVI ET AL. C2 did not inctivte phge skl, yet membrnes from strins tht were completely resistnt to phge skl were lso incpble of inctivting phge c2. This suggests tht phge c2 nd skl require the sme membrne protein for infection but tht phge skl does not interct irreversibly with this protein in vitro, wheres phge c2 does. Consistent with this ide re the results tht phge c2-resistnt cells were prtilly resistnt to phge skl nd phge skl-resistnt cells were completely resistnt to phge c2. However, the mechnism of interction between the phge-inctivting ctivity nd both phges needs further study. The phge-inctivting protein ws purified from crude membrnes by Triton X-100 extrction, gel filtrtion, nd ion-exchnge chromtogrphy. The moleculr mss of the phge-inctivting protein under nondenturing conditions ws estimted to be 350,000. The size is in the sme rnge s tht of the phge-inctivting protein of strin ML3, reported previously (11). The brod elution profile my suggest some heterogeneity of structure. This could result from the presence of ttched lipids or crbohydrte or loss of some of the components of such lrge membrne complex during purifiction. Although the specific ctivity decresed s result of the ion-exchnge step, the purity of one protein ws improved fter ech step. This protein hs n pprent size of 32 kd, on the bsis of its migrtion on SDS-polycrylmide electrophoretic gels. The mss yield of the 32-kD protein ws estimted from densitometer scns of silverstined gels to be 45%. The elution pttern of the 32-kD protein is the only pprent protein which coincides with the phge-inctivting ctivity. Ech of the other contminting proteins re from side frctions of incompletely resolved peks which did not coelute with the phge-inctivting ctivity. Attempts to rectivte the protein hve thus fr been unsuccessful. This is not uncommon for membrne proteins with biologicl ctivity, including other lctococcl membrne proteins in generl (1) nd phge-inctivting proteins specificlly (11). Although we hve tken some precution to void inctivtion, such s the use of glycerol s n osmolyte nd the ddition of phospholipids during detergent solubiliztion (1), further ttempts to mintin ctivity fter solubiliztion re currently in progress. ACKNOWLEDGMENTS This reserch is supported by grnt from the Western Diry Foods Reserch Center nd the Ntionl Diry Reserch nd Promotion Bord. We thnk Todd Klenhmmer for supplying phges nd bcteril host strins. REFERENCES 1. Ambudkr, S. V., nd P. C. Mloney Bcteril nion exchnge: use of osmolytes during solubiliztion nd reconsti- J. BACTERIOL. tution of phosphte-linked ntiport from Streptococcus lctis. J. Biol. Chem. 261: Ansorge, W Fst visuliztion of protein bnds by impregntion in potssium permngnte nd silver nitrte, p In D. Stthkos (ed.), Electrophoresis '82. Wlter de Gruyter & Co., Berlin. 3. Brdford, M. M A rpid nd sensitive method for the quntittion of microgrm quntities of protein utilizing the principle of protein-dye binding. Anl. Biochem. 72: Furukw, H., T. Kuroiw, nd S. Mizushim DNA injection during bcteriophge infection of Escherichi coli. J. Bcteriol. 154: Hrris, P. J., A. B. Blkeney, R. J. Henry, nd B. A. Stone Gs chromtogrphy determintion of the monoscchride composition of plnt cell preprtions. J. Assoc. Off. Anl. Chem. 71: Hyshi, M., A. Aoym, D. L. Richrdson, Jr., nd M. N. Hyshi Biology of the bcteriophge (X174, p In R. Clendr (ed.), The bcteriophges, vol. 2. Plenum Press, New York. 7. Kgw, Y., nd E. Rcker Prtil resolution of the enzymes ctlyzing oxidtive phosphoryltion. XXV. Reconstitution of vesicles ctlyzing 32P-denosine triphosphte exchnge. J. Biol. Chem. 246: Keogh, B. P., nd G. Pettingill Adsorption of bcteriophge eb7 on Streptococcus cremoris EB7. Appl. Environ. Microbiol. 45: Lemmli, U. K Clevge of structurl proteins during the ssembly of the hed of bcteriophge T4. Nture (London) 227: Lindberg, A. A Bcteriophge receptors. Annu. Rev. Microbiol. 27: Orm, J. D Isoltion nd properties of phge receptor substnce from the plsm membrne of Streptococcus lctis ML3. J. Virol. 13: Orm, J. D., nd B. Reiter The dsorption of phge to group N streptococci. The specificity of dsorption nd the loction of phge receptor substnces in cell-wll nd plsmmembrne frctions. J. Gen. Virol. 3: Otto, R., R. G. Lgeveen, H. Veldkmp, nd W. N. Konings Lctte efflux-induced electricl potentil in membrne vesicles of Streptococcus cremoris. J. Bcteriol. 149: Supelco Inc GLC/HPLC bulletin 774B. Supelco Inc. Bellefort, P. 15. Terzghi, B. E., nd W. E. Sndine Improved medi for lctic streptococci nd their bcteriophges. Appl. Microbiol. 29: Vlysevi, R., W. E. Sndine, nd B. L. Geller The bcteriophge kh receptor of Lctococcus lctis subsp. cremoris KH is the rhmnose of the extrcellulr wll polyscchride. Appl. Environ. Microbiol. 56: Vlysevi, R., W. E. Sndine, nd B. L. Gelier. The bcteriophge skl receptor is the rhmnose nd glucose of the exopolyscchride of Lctococcus lctis subsp. lctis C2. Submitted for publiction.