Extracts of Vegetative and Sporulating Bacillus subtilis

Transcription

1 Proc. Nt. Acd. Sci. USA Vol. 71, No. 7, pp , July 1974 An Immunologicl Assy for the Sigm Subunit of RNA Polymerse in Extrcts of Vegettive nd Sporulting Bcillus subtilis (ntibody precipittion) ROBERT TJIAN AND RICHARD LOSICK The Biologicl Lbortories, Hrvrd University, Cmbridge, Msschusetts Communicted by John M. Buchnn, April 29, 1974 ABSTRACT The ctivity of the subunit of Bcillus subtilis RNA polymerse decreses mrkedly during the first hours of sporultion [T. G. Linn et l. (1973) Proc. Nt. Acd. Sci. USA 70, We he prepred ntibody ginst RNA polymerse holoenzyme to determine the fte of polypeptide during spore formtion. This ntiserum specificlly nd independently precipittes nd core polymerse from crude extrcts of B. subtilis s judged by both sodium dodecyl sulfte nd ure gel electrophoresis of the precipittes. We report tht crude extrcts of sporulting cells lcking ctivity contin s much polypeptide s extrcts of vegettive cells. However, polypeptide in extrcts from sporulting cells is pprently only wekly ssocited with RNA polymerse, s indicted by the filure of to co-purify efficiently with core enzyme during phse prtitioning. The loss of ctivity nd the wek binding of to core enzyme occurs normlly in mutnt blocked t n intermedite stge of sporultion (SpolI-4Z) nd in wild-type bcteri sporulting in 121B medium, Difco sporultion medium, or Sterlini-Mndelstm resuspension medium. In contrst, in two mutnts (SpoO-5NA nd SpoOb- 6Z) blocked t n erly stge of spore formtion remins ctive nd tightly ssocited with RNA polymerse during sttionry phse. The onset of sporultion by Bcillus subtilis is ssocited with chnge in the templte specificity of RNA polymerse (1). Enzyme isolted from sporulting bcteri is unble to trnscribe certin phge DNA templtes s ctively in vitro s RNA polymerse from vegettive B. subtilis. However, RNA polymerse in certin mutnts blocked t n erly stge of spore formtion retins, throughout sttionryphse, the templte recognition properties chrcteristic of enzyme from vegettive bcteri (2, 3). The chnge in templte specificity is pprently cused by mrked decrese in the ctivity of the subunit of RNA polymerse rther thn n ltertion of the core subunits,,b, f', nd (4, 5). However, it hs previously not been possible to determine whether sporulting bcteri ctully lck polypeptide or whether the polypeptide is present but its ctivity inhibited during spore formtion. To distinguish between these lterntives, we report n immunologicl ssy tht provides direct test for the presence of the polypeptide in extrcts of vegettive nd sporulting B. subtilis. METHODS Cells. Wild-type B. subtilis strin NCTC 3610 (ATCC Abbrevitions: SDS, sodium dodecyl sulfte; DSMI, Difco sporultion medium; SM\, Sterlini-Mndelstm medium ), Mrburg strin, ws used for ll experiments except where otherwise indicted. The sporogenous mutnts (6) were kindly provided by P. Scheffer. Medi nd Sporultion. Growth nd sporultion of B. subtilis 3610 ws in 121B medium (7) nd sporulting cells were hrvested 4.5 hr fter the end of logrithmic growth (T4.5) unless otherwise noted. For rdioctive lbeling of cells, the rdioctive precursor ws dded during erly logrithmic growth. Strin SMY nd the sporogenous mutnts were grown (8) in Difco sporultion medium (DSAI); cells were hrvested either t mid-logrithmic phse or llowed to enter sttionry phse by continued growth in DS'M or by resuspension of logrithmiclly growing cells in Sterlini-MNndelstm (SM) medium (9). In DSM nd SAM medi, sporulting or sttionry-phse cells were hrvested either 3 hr fter the end of logrithmic growth (T3) or 3 hr fter resuspending in SM medium (T3). After hrvesting, cells were wshed with buffer G (0.05 M Tris-HCl, ph 7.5, 0.01 MI MNlgCI2, 0.1 mmni ethylenediminetetrcette, 0.1 mmi dithiothreitol, 10% v/v glycerol) contining 1.0 AM KC1 (10) nd 5%lphenylmethylsulfonyl fluoride solution (6 mg/ml of 95% ethnol) to remove extrcellulr proteses nd then rpidly frozen. Cells Extrcts Were Prepred from bout 2 g of cells by disruption in Brun homogenizer nd high speed centrifugtion s previously described (5) except tht cells were initilly suspended in buffer I (0.01 AM Tris-HCl, ph 7.9, 1.0 mam ethylenediminetetrcette, 0.01 M AMgCl2, 0.3 mai dithiothreitol, nd 5% phenylethylsulfonyl fluoride solution). Hlf the superntnt fluid ws briefly sonicted to reduce viscosity nd then brought to 55% sturtion with solid mmonium sulfte (5). The resulting precipitte ws resuspended in 1 ml of buffer I contining 0.05 Al KCl nd 5% v/v glycerol, dilyzed ginst the sme buffer, nd is referred to s "mmonium sulfte enzyme." The remining hlf of the superntnt fluid ws prtitioned between phses of polyethylene glycol nd dextrn by modifiction (11) of the procedure of Bbinet (12). The finl mmonium sulfte precipitte of the phse-prtitioned enzyme ws resuspended in 1 ml of buffer I contining 0.05 AM KCl nd 5% v/v glycerol nd is referred to s "phse-prtitioned enzyme." Antiserum Preprtion nd Precipittion. The ntigen for the preprtion of ntiserum to holoenzyme ws mixture of highly purified (95%) nd core polymerse (5%) prepred s previously described (frction 6 protein from ref. 11). The ntigen ws first covlently coupled to Sephrose 2B tht

2 Proc. Nt. Acd. Sci. USA 71 (1974) hd been ctivted with cynogen bromide (13). Immuniztion of rbbits ginst the coupled ntigen, purifiction of gmm globulin from rbbit serum, precipittion of polymerse subunits, nd solubiliztion of the precipittes for gel electrophoresis were s previously described (5). One hundred microliters of the nti-holoenzyme ntiserum were sufficient to precipitte 5.0 1Ag of nd bout 10.0,gg of core polymerse. Sodium Dodecyl Sulfte (SDS) nd Ure Polycrylmide Gel Electrophoresis. High resolution SDS gels were 7.5% crylmide in Tris*glycine buffer (14). The ure (ph 8.7) gels were 5% crylmide; solutions were s described by Lemmli (15) except tht Tris ws 2.0 M, 6 M ure replced 0.1% SDS, nd no stcking gel ws used. RESULTS Sigm ctivity in extrcts of vegettive nd sporulting B. subtilis Ammonium sulfte enzyme ws prepred from vegettive nd sporulting B. subtilis s described in Methods nd ssyed for ctivity by mesuring the trnscription of phge <0e DNA nd poly(da-dt) in vitro. Since the trnscription of the phge templte is lrgely dependent on, wheres trnscription of the synthetic templte is not, the rtio of ctivity with 4e DNA templte to tht with poly(da-dt) serves s mesure of ctivity. The 4e to poly(da-dt) trnscription rtio for vegettive enzyme ws bout 5.0 (Fig. 1A), while the rtio for purified core polymerse lcking ws bout 1.0 (Fig. 1C). (Core enzyme hs significnt ctivity with Ye DNA s templte even though it contins only trce mounts of r.) In greement with previous reports, (1, 4), sporultion mmonium sulfte enzyme displyed very little ctivity nd hd 0e/poly(dA-dT) trnscription rtio (Fig. 1B) similr to tht of core polymerse (1.0). An immunologicl ssy for the polypeptide To ssy directly for the presence of the polypeptide in vegettive nd sporultion extrcts we prepred ntiserum ginst vegettive holoenzyme. This ntiserum independently precipittes either purified subunit (Fig. 2, Gel C), or core polymerse (not shown). The ntiserum ws first used to precipitte RNA polymerse from the mmonium sulfte enzyme from the experiment of Fig. 1A. The precipitte ws solubilized, nlyzed by SDS gel electrophoresis nd found to contin j3'f3 (150,000 dltons), (43,000 dltons), ntibody polypeptides nd, in ddition, polypeptide with the mobility of uthentic (55,000 dltons) (Fig. 2, Gel D). To confirm tht the 55,000- dlton polypeptide ws ctully o, this protein ws lso subjected to ure gel electrophoresis. The subunit migrtes more rpidly then (Fig. 2, Gels E nd F) during ure gel electrophoresis, even though migrtes fster then during SDS gel electrophoresis (Fig. 2, Gels A nd B). A slice contining puttive subunit ws cut from duplicte SDS gel nd subjected to electrophoresis on ure gel. Gel G (Fig. 2) shows tht this slice contined polypeptide with mobility of uthentic nd some contminting ntibody hevy chin. Thus, the ntibody directed ginst holoenzyme specificlly precipittes the polypeptide nd the subunits of core polymerse from extrcts of vegettive cells. Next, ntiserum ws used to precipitte RNA polymerse from the sporultion mmonium sulfte enzyme from the An Immunologicl Assy for Sigm Subunit 2873 experiment of Fig. 1B. SDS gel nlysis showed tht the precipitte contined #'/3, proteins of 100,000 nd 70,000 dltons,, ntibody polypeptides, nd pprently. From densitometer trcing of Gel A in Fig. 3 we conclude tht the stoichiometry of the core subunits ws fl'fl2. The 70,000-dlton protein hs previously been described (16) nd is new sporultion protein bound to RNA polymerse. It is not known whether the 100,000-dlton protein is lso ssocited with RNA polymerse. As further evidence tht the 55,000- dlton polypeptide in the SDS gel ws, this species ws found to migrte with uthentic during ure gel electrophoresis (Fig. 3, Gels B, C, nd D). Thus, the polypeptide is present in extrcts of sporulting cells nd is precipitted by ntiserum to holoenzyme. z U FIG. 1. x 'E A C lo 15 jjg PROTEIN IA 2.1 ACTIVITY WITH POLY(dA-dT) (14C CPM X 103) Trnscription of phge 4e DNA nd poly(da-dt) by mmonium sulfte enzyme nd phse-prtitioned enzyme from vegettive nd sporulting B. subtilis. The RNA polymerse ssy ws s previously described (1) except tht the specific ctivity of [14C]ATP ws incresed to 8,Ci/,umole. Assy mixtures contined either 6 jug of 4e 1)NA (-) or 10 Ag of poly(dadt) (A) s templte nd vrious mounts of either vegettive mmonium sulfte enzyme (A), sporultion mmonium sulfte enzyme (B), vegettive phse-extrcted enzyme (D), or sporultion phse-extrcted enzyme (E). The dt from A nd B were re-plotted in C s ctivity with te DNA versus ctivity with poly(da-dt) for the vegettive () nd the sporultion (A) enzymes. The dt in D nd E were similrly re-plotted in F. The ctivities ([1) for 160 ng, 320 ng, nd 640 ng of purified core polymerse (11) with He DNA nd poly(da-dt) were lso plotted in C nd F. D F

3 2874 Biochemistry: Tjin nd Losick Proc. Nt. Acd. Sci. USA 71 (1974) 2 SDS t i c PA B C D I. I HCH/ AIN _ UREA E F G _ H CHAIN 3 SDS [3 A- - 70,000 d *t H CHAIN A _ UREA,. T C D FIG. 2 (left). SDS nd ure polycrylmide gel electrophoresis ptterns of RNA polymerse subunits from vegettive cells. SDS gel electrophoresis: A, 2.6 pg of core polymerse plus 1.0 pg of purified ; B, 2.0 1g of purified ; C, ntibody precipitte from mixture of 2.0 ug of purified nd 200 Ml of ntiserum; D, ntibody precipitte from mixture of 600 pg of the mmonium sulfte enzyme from the experiment of Fig. 1A plus 100 Ml of ntiserum. Ure gel electrophoresis: E, 8 pug of holoenzyme; F, 3-mm slice contining cut from n unstined duplicte of SDS gel B; G, 3-mm slice cut from the region of n unstined duplicte of SDS gel D. H chin is the hevy chin of the ntibody. FIG. 3 (right). SDS nd ure polycrylmide gel electrophoresis ptterns of RNA polymerse subunits precipitted from sporultion mmonium sulfte enzyme by ntibody. SDS gel electrophoresis: A, one third of the solubilized ntibody precipitte from mixture contining 550 p&g of the mmonium sulfte enzyme from the experiment of Fig. 1B plus 100 ;4l of ntiserum. Ure gel electrophoresis: B, 3-mm slice contining cut from n unstined SDS gel of purified (sme s gel F of Fig. 2); C, 3-mm slice cut from the region of n unstined duplicte of SDS gel A; D, 3-mm slice cut from the region of n unstined SDS gel on which mixture of 2 pg of purified nd one third of the solubilized ntibody precipitte of gel A hd been subjected to electrophoresis. d = dltons. To compre quntittively the mount of in vegettive nd sporultion extrcts, mmonium sulfte enzyme ws prepred from vegettive cells rdioctively lbeled with 8H nd sporulting cells lbeled with "S. The extrcts were first mixed nd then precipitted with ntiserum. The precipitte ws solubilized nd subjected to electrophoresis on n SDS gel. After stining, slices contining either 13' nd or were TABLE 1. cut from the SDS gel nd the rdioctivity in ech slice ws mesured. Tble 1 (Post mixing) presents the rtio of rdioctivity in the nd,s' subunits from sporulting (585) nd vegettive ('H) cells. The '"S/3H rtio for the j#'# subunits compred to the rtio for the subunit is mesure of the reltive mount of in vegettive nd sporultion extrcts. Since S/81H for 1'13 ws 0.56 nd the rtio for ws 0.58, we conclude tht there re pproximtely equl mounts of o, reltive to core polymerse, in both vegettive nd sporultion extrcts. By dividing the 35S/'H rtio for by the *S/'H rtio for it'd we clculte normlized rtio of 1.06 for the mount of in the sporultion extrct reltive to the vegettive extrct. Therefore, despite the low level of ctivity, sporultion extrcts contin s much polypeptide s vegettive extrcts. Co-purifiction of f with core polymerse during phse prtitioning Although the - polypeptide is present in sporultion extrcts' it ws not known whether is ctully bound to polymerse. As test of the binding of to core enzyme in sporultion extrcts, we determined whether, co-purifies with RNA polymerse during prtitioning of crude extrcts between phses of polyethyelene glycol nd dextrn. This procedure efficiently purifies holoenzyme from vegettive extrcts (11), lthough the 70,000-dlton protein ssocited with RNA polymerse in sporulting cells does not remin bound to core enzyme during this purifiction procedure (17). RNA polymerse purified by phse prtitioning from vegettive extrcts hd fe/poly(da-dt) trnscription rtio of bout 5.0 (Fig. 1D), while the trnscription rtio for sporultion enzyme ws bout 1.0, rtio chrcteristic of polymerse lcking (Fig. 1E). Although enzyme from the sporultion extrct lcked ctivity fter phse prtitioning, this enzyme responded normlly to dded purified in vitro. (The ddition of 0.5 ig of purified to 10 Mug of phse-extrcted sporultion enzyme incresed the Oe/poly(dA-dT) trnscription rtio from 1.0 to 5.3.) An ntibody precipitte of the prtilly purified vegettive enzyme contined 13', 13,, nd in ddition to ntibody polypeptides (Fig. 4, Gel A). In contrst, n ntibody precipitte from the phse-prtitioned sporultion enzyme lcked, lthough it contined the core subunits Reltive mounts of e in extrcts of vegettive nd sporulting B. subtili8 ft',3 Subunits Subunit Normlized 'H cpm 'S cpm 36S/3H 3H cpm 3S cpm 35S/3H rtio Post mixing Ammonium sulfte enzyme 3,390 1, ,710 1, Phse-prtitioned enzyme 4,183 2, , Prior mixing Phse-prtitioned enzyme 2,856 2, , Post mixing experiment. Ammonium sulfte enzyme nd phse-prtitioned enzyme were prepred from sporulting cells (2 g) lbeled with 2.2 mci of [1'S]methionine nd vegettive cells (2 g) lbeled with 4.8 mci of [3H]methionine. The vegettive nd sporultion mmonium sulfte enzymes were mixed nd RNA polymerse precipitted from 10% portion of the mixture with 150 Ml of ntiserum. RNA polymerse ws similrly precipitted from 10% portion of mixture of the vegettive nd sporultion phse-extrcted enzymes with 150 Mul of nti-serum. Antibody precipittes were solubilized nd subjected to electrophoresis on SDS gels. After stining, 1-mm slices contining o nd, 'fl were cut from the gels nd the rdioctivity from 35S nd 3H ws mesured. The normlized rtio ws clculted from the 35S/'H rtios for nd #'f3. Prior mixing experiment. Sporulting cells (0.3 g) lbeled with 2.5 mci of ['1S] methionine nd hrvested 10 hr fter the end of logrithmic growth were mixed with vegettive cells (2.5 g) lbeled with 1.2 mci of [3Hlmethionine. Phse-prtitioned enzyme ws then prepred from the mixture. RNA polymerse ws isolted by ntibody precipittion nd nlyzed by SDS gel electrophoresis s described bove.

4 Proc. Nt. A cd. Sci. USA 71 (1974) 4 A, A B C /3,,,0 1-M _ * W- p _ " A B C D E F G FIG. 4 (left). SDS polycrylmide gel electrophoresis ptterns of RNA polymerse subunits precipitted by ntibody from phse-prtitioned vegettive nd sporultion enzymes. A, solubilized ntibody precipitte from mixture contining 260 jxg of phse-prtitioned vegettive enzyme from the experiment of Fig. id plus 100 Mul of ntiserum; B, the solubilized ntibody precipitte from mixture contining 270 ug of phse-prtitioned sporultion enzyme from the experiment of Fig. 1E plus 100 Ml of ntiserum; C, the solubilized ntibody precipitte from mixture contining 230 lg of the first polyethylene glycol superntnt frction from the phse-extrcted sportiltion enzyme from the experiment of Fig. le plus 100,uI of ntiserum. FIG. 5 (right). SDS polycrylmide gel electrophoresis ptterns of phse-prtitioned enzyme from sporogenous mutnts. Antibody precipittes were prepred by incubting 100 ul of ntiserum with 300,ug of phse-extrcted enzyme from: A, vegettive SMY grown in DSM; B, sporulting SMY grown in DSM; C, sporulting SMY, in SM resuspension medium; D, sttionry-phse SpoO-5NA grown in DSM; E, sttionryphse SpoO-5NA in SM resuspension medium; F, sttionryphse SpoOb-6Z grown in DSM; G, sttionry-phse SpoII-4Z grown in DSM. The enzymes were from the experiment of Tble 2. in stoichiometry of #'#2 (Fig. 4, Gel B). However, some polypeptide could be detected in the first polyethylene glycol superntnt of this purifiction procedure for both the sporultion (Fig. 4, Gel C) nd the vegettive (not shown) extrcts, but we hve not quntitted the mount of in these two cses. To compre quntittively the mount of r tht co-purifies with core enzyme during prtil purifiction of RNA polymerse from vegettive nd sporulting cells, extrcts prepred from vegettive cells lbeled with 3H nd sporulting TABLE 2. Trnscription rtios for RNA polymerse from sporogenous mutnts SMY Veg DSM SMY T3 DSM SMY T3 SM SpoO-5N T3 DSM SpoO-5NA T3 SM SpoOb-6Z T3 DSM SpoII4Z T3 DSM Ammonium Phsesulfte prtitioned Strin Time Medium enzyme enzyme Vrious mounts of either mmonium sulfte enzyme or phse-extrcted enzyme prepred from ech of the indicted strins of bcteri were ssyed with 6 jug of Oe DNA or 10 jsg of poly(da-dt) s templte. The ctivities were plotted ginst protein concentrtion s illustrted in Fig. 1 nd the e/poly(dadt) trnscription rtios were computed from the liner portions of the ctivity curves. Veg vegettive. An Immunologicl Assy for Sigm Subunit 2875 cells lbeled with 4S were phse prtitioned. The phseprtitioned enzymes were then mixed nd RNA polymerse ws precipitted by the ntiserum to holoenzyme. SDS gel nlysis of the precipitte reveled tht 4S/3H for the P subunits ws 0.60, while the rdioctivity rtio for ws 0.11, giving normlized rtio of 0.18 for the mounts of in the sporultion extrct reltive to the vegettive extrct (Tble 1, Post mixing). Thus, in the sporultion extrct does not co-purify efficiently with core enzyme during phse prtitioning. Since from sporultion extrcts ppers to co-purify poorly with core polymerse during phse prtitioning, one possibility ws tht the polypeptide is ltered nd cnnot bind to core enzyme. Another possibility ws tht remins unltered but is unble to bind to the core polymerse in sporulting cells. To investigte these lterntives, we mixed sporulting cells lbeled with 35S with n 8-fold excess of vegettive cells lbeled with 3H nd prtilly purified RNA polymerse from n extrct of the mixture by phse pttitioning. RNA polymerse ws precipitted from the phseprtitioned enzyme by ntiserum to holoenzyme nd nlyzed by SDS gel electrophoresis. We found tht under conditions of excess vegettive cells from sporulting bcteri copurified with core polymerse s efficiently s from vegettive cells (Tble 1, Prior mixing). Apprently, from the sporulting cells is unltered nd is not intrinsiclly reduced in its bility to bind to core polymerse. The subunit of RNA polymerse in sporogenous mutnts RNA polymerse in certin mutnts blocked t Stge 0 of sporultion fil to lose u ctivity during sttionry phse (2, 3). We hve checked whether the subunit from two such mutnts hrvested t sttionry phse co-purifies with RNA polymerse during phse prtitioning. First, enzyme ws prepred by both mmonium sulfte frctiontion nd phse prtitioning. The 0e/poly(dA-dT) trnscription rtio for enzyme from wild-type bcteri (SMY) sporulting in either Difco sporultion medium (DSM) or Sterlini-Mndelstm (SM) resuspension medium ws bout 0.6, rtio similr to tht of core polymerse (Tble 2). In contrst, enzyme from sttionry-phse cells of Stge 0 mutnt (SpoO-5NA) hrvested either from DSM or SM resuspension medium retined trnscription rtio similr to tht for RNA polymerse in vegettive extrcts (4.0). Likewise, RNA polymerse from nother Stge 0 mutnt (SpoOb-6Z) exhibited high oe/- poly(da-dt) trnscription rtio. However, enzyme from mutnt blocked t Stge II of sporultion (Spoll4Z) hd undergone the chnge in templte specificity normlly. Precipittion by ntiserum to holoenzyme reveled tht RNA polymerse purified by phse prtitioning from sttionryphse cells of the wild type nd Stge II mutnt contined only low mounts of, while phse-extrcted enzyme from the erly blocked mutnts pprently contined norml mounts of the subunit (Fig. 5). DISCUSSION Extrcts prepred from sporulting B. subtilis contin polypeptide even though RNA polymerse in these extrcts displys only very low levels of ctivity. Moreover, the mount of subunit in extrcts from sporulting cells is s gret, reltive to the subunits of core RNA polymerse, s

5 2876 Biochemistry: Tjin nd Losick the mount of in extrcts from vegettive bcteri. However, polypeptide in crude extrcts from sporulting cells is pprently not tightly ssocited with RNA polymerse, s indicted by the filure of - to efficiently co-purify with core polymerse during phse prtitioning. (It is possible, however, tht is wekly bound to polymerse nd would not be removed from the core enzyme during other purifiction procedures.) Thus, the lck of ctivity in sporultion extrcts my be cused by the filure of to bind tightly to core polymerse. The low level of of ctivity in sporultion extrcts nd the filure of to bind tightly to core polymerse could be. cused by n ltertion of o, n ltertion of core polymerse, or new component of sporulting cells tht inhibits the binding of to core enzyme. It is unlikely tht f is ltered, since under the conditions of the "Prior mixing" experiment of Tble 1, from the sporulting bcteri co-purified s efficiently with RNA polymerse s from the vegettive bcteri. It is lso unlikely tht n ltertion of core polymerse interferes with the function of o, since the core subunits f3', il, nd isolted erly during sporultion re pprently indistinguishble from those of vegettive enzyme nd since sporultion core polymerse is stimulted to the sme extent in vitro s vegettive core, polymerse by vrious mounts of purified r (T. Linn, unpublished dt, nd ref. 5). We therefore suggest tht sporulting bcteri contin n inhibitor of ctivity nd tht this inhibitor cts by interfering with the binding of the subunit to RNA polymerse. Proof of this hypothesis will require the isoltion of such n inhibitor from sporulting bcteri. It is interesting to note tht Stevens (18) hs recently isolted frction from Escherichi coli infected with phge T4 tht inhibits ctivity in vitro. Brevet nd Sonenshein (2) hve shown tht the loss of ctivity chrcteristic of RNA polymerse from wild-type cells undergoing sporultion does not occur in certin mutnts blocked t Stge 0, but occurs normlly in mutnts tht proceed to Stge II nd beyond. Our finding tht phse-prtitioned enzyme from sttionry-phse cells of two Stge 0 mutnts contins polypeptide, while RNA polymerse from mutnt blocked t Stge II contined little or no is consistent with the pttern of ctivity in extrcts from these mutnts. The finding tht certin Stge 0 mutnts retin ctivity demonstrtes tht the ltertion of RNA polymerse is ssocited with events occurring erly during sporultion nd is not merely consequence of sttionry-phse growth. It should be noted, however, tht the stge of morphologicl block need not correspond temporlly to the loss of ctivity, since morphologicl events re not necessrily directly relted to the time of trnscription of different clsses of sporultion genes. For instnce, certin stge 0 mutnts tht prtilly lose ctivity (2, 10, 19) could be blocked t n event other thn ltertion of RNA polymerse tht is required for lter stges of sporultion. Similrly, the retention of ctivity by Stge II mutnt (Spol 46-7, ref. 19) could indicte tht loss of ctivity is not necessry to chieve Stge II of spore formtion but my be required for subsequent stges of sporultion. A preliminry report by Struss (20) suggested tht B. eubtilis resuspended in SM sporultion medium does not undergo the loss of ctivity chrcteristic of bcteri Proc. Nt. Acd. Sci. USA 71 (1974) sporulting either in DSM or 121B medium. In our hnds, however, RNA polymerse from bcteri sporulting in SM medi lcks ctivity nd RNA polymerse prtilly purified by phse prtitioning lcks polypeptide, lthough phse-prtitioned enzyme from Stge 0 mutnt resuspended in SM medium displys high levels of ctivity nd contins polypeptide (Tble 2 nd Fig. 5). It ws previously suggested tht the filure of phge 4e to grow on sporulting bcteri could be ccounted for by the loss of ctivity, since the decrese in phge burst size during spore formtion closely prllels the loss of the bility of RNA polymerse to trnscribe phge ye DNA s mesured in vitro (1). Moreover, sporultion-defective mutnt tht prtilly retins ctivity supports the continued growth of He during lte sttionry phse (3). This proposl hs now received strong support from the finding (J. Segll, R. Tjin, J. Pero, nd R. Losick, in preprtion) tht the rte of Oe trnscription in vivo fter infection of wild-type sporulting cells or fter infection of sttionry-phse cells of Stge 0 mutnt closely reflects the templte specificity of RNA polymerse from these bcteri. Note Added in Proof. Recent experiments indicte tht chlormphenicol tretment of sporulting cells rpidly relieves the inhibition of ctivity s mesured in vitro nd tht drugtreted sporulting bcteri support trnscription by Be more ctively in vivo thn untreted sporulting bcteri. These findings could indicte tht the proposed inhibitor of is physiologiclly unstble. We thnk A. M. Pppenheimer, Jr. nd J. Murphy for ssistnce in the preprtion of ntiserum. This work ws supported by grnt from the Ntionl Institutes of Helth (GM ). 1. Losick, R. & Sonenshein, A. L. (1969) Nture 224, Brevet, J. & Sonenshein, A. L. (1972) J. Bcteriol. 112, Sonenshein, A. L. & Losick, R. (1970) Nture 227, Brevet, J. (1974) Mol. Gen. Genet. 128, Linn, T. G., Greenlef, A. L., Shorenstein, R. G. & Losick, R. (1973) Proc. Nt. Acd. Sdi. USA 70, 1865r Ionesco, H., Michel, J., Cmi, B. & Scheffer, P. (1970) J. Appl. Bcteriol. 33, Sonenshein, A. L.' & Roscoe, D. (1969) Virology 39, Scheffer, P., Millet, J. & Aubert, J. (1965) Proc. Nt. Acd. Sci. 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