Comparison of procedures for extracting transfer RNA from spores of Bacillus

,\Ii,'HIv1:S

OF tt,o~'t,t:2I,STr:Y

Comparison

\.s,,

tiIoI'IIYHI(!S

of Procedures

149, (iNi8

for

Spores

(1972)

Extracting

Transfer

RNA

From

of Bacillus’

Methods not involving physical disrttption for extracting tI:NA l’rom spores of using phenol, 8 M ttrea-mercaptoethanol, 8 M t~re:t-I hiogycolics acid, or dithiothreitol were compared. On the basis of these data, att improved estraction procedure is described which is applicable to spores, vegei ai ivc wlls a~td asporogrnotts mutanls OS Rm.ilhs scthiilis and Rmc~illos ~~~mqoleriim. Bacillus

Sjeveral methods c‘xist for cbxtract,irlg transfer RNA from bacterial cells in logarithmic growth. In the fen casw \vh(w tRn’As haw been clxt,ractcd from asporogcnous mukmts, thca same t,whniquw hnw bwn used LIS thaw for vegrtat,iw ~~~11s. Spores, ho\\-ww, prcwnt, a formidnbk (lxtraction problrlm. The sport’ has c~xtc&vc: coat structures which arc> difficult to pans t,ratcl. In addition, Bacillus mbtilis producw oxtracellular RKasw during sporulation. Th(b presence of large> amounts of activcb ZWases complicates cbxtraction prowdurw for both spores and asporogcwous mutants in the stationary phase of growth. Sawral proccdurw for cxt,ructing various component,s from spores huw bwn prcwntt>d in tlict literature. Among thaw arc physical prowdurcs involving ruptuw of uc% and dr? suspensions and prowdurw ctmploying trwtmcnt with 8 11 urw plus :I rr~ducing agcint. Thcl following prowdurcs for phgsical disrupt.ion haw bwn tricxd b>, ot,hcw : dr)r rupture using a dcntul amalgamator (1)) OI ground alumina (2), and I+-c%rupturci using :L tissue disintegrator (31, :L l’ott8w mill (4), grinding with alumin:~ (5), grinding \\-it11 glass beads (6, 7) or sonication (S). ’ Abbreviaiiotrs ttscd in this paper: Tris, TrisSDS, sodium(hydroxymethyl)aminometIlarle; dodecylsulfat,e; EDTA, ethylenediaminetet,raarctic acid; t RNA, transfer rihorrltrlric acid; atld ‘I’CA, trirhloronwtic acid.

WC’ prefcrwtl t 0 cotlccwtr:ltc~ cni IIOI~mechanical prowdurw .sinco thcly should provide 1~s opportunity for physically damaging the tRS,4 mol(~culrs and should bc wsier t#o apply to large> quantitiw of spores. On the basis of our comparisons, an improwd extr&ion proccdurc is described which is applicabl(l to sports, asporogcnous mut~ants in stationar\- plinsc and wgc+:it,ivc: cells.

.lIaleriaZs. The followitlg special chemicals ot enzymes mere ttscd: sttcrose (special enzyme grade, Mann Reseatch Laboratories), Itcnogra(itt76 (a solution of CiC,C ( tneglttmine diatrizoatr and 10’:; sodium diat,rizoate, 11:. l<. Squibb and Sons, Tnc.), lysozyme (3X cyrstallized, Sigma Chemical Co.), T3rij 58 (Pierce Chctnical Co.), IJ?Jase, illNaw free, Worthittqt on Hiochemical Corp.), (liesearch Organic/ diet hylp~roc:Lrl,ott:Lt c Inorganic Chemical Co.), dithiothreitol (Clclands Reagent., Calbiochem). P ronase I3 (Calbiochem) was self-digested before ~tsc by itlcltbatiott at 37” for 2 hr at a cotlcentrat ion of 20 rrtg;ml in 0.02 M ‘l’ris-Cl, pH 8.8. Gro7olh contlilions. :\I1 cells were growtt in I ryptotlr-yeast rstract mediltm (8). 1 liter pet 2.8 1 P’ernbach flask with cwttstan( shaking at. 37”. r,.Tryptophan, 40 ,~g per tttl, was added to cultures of tryptophan artxotrophs. Cldtrtres were st at%ed from a small incwuhtm of either heat -shocked spores or cells from :I freshly growl plate. Log phase cells were harvested at SO-50 Klott units (Klct t, Srtmmrrsotr w)loritnPtcr. red filteri aft ~1‘

tI:NA

(3

FROM WOREP

1-6 hr ixtcubat~ion. The cells reached the beginning of stationary phase in 6-8 hr. Asporogenous mutants were harvest,ed at 24 hr and spores were harvested at 21 or 72 hr. Pt~$calion of spores. Before extraction, spores were purified by treat.ment with lysozyme and SDS (9) followed by centrifugation through GO:; Renografin (details are given in Results Section on Met hod 15) or separation in polyethylene glycol (10). Bac/ct,ia2 slrains. ‘L’he following strains were obt,ained from the collection in this laboratory and were described by Hoch and Spizizen (11) : B. s~blilis 168 trpZ-, a st,rain which forms normal sporll8, an early blorked spores ; 11. sublilis asporog?nous mutant), and B. s~btilis Rog22 (spot) an asporogenous mutant with a Iatcr block. Racillzrs nzcgaleriut)L KM was obtained from Philip C. Fit.z-James. I
Method d, rlirecf $lelzol. Transfer RKA can b(l cxt,racted from vcgetat,ivc cells directly b!- a phenol trcat’mcnt (12, 13). This procedure has been very successfully applied to large quantit,ics of cells of Escherichia co/l (14) and yeast (I;). WC applied this phenol procedure a.nd sc~cral variations to spores of B. subtibis. Each variation began with 100 mg lyophilizod sporw which had prrviously been purifkd a~ described in Slatcrials and Methods. Thai basic procedure involved the suspcwion of lyophilizcd spores in 0.4 31 n’aC1, 10 rnir MgCIZ , 1 rn>I EDTA, 2 rnlr Ka2S203 and ‘LOmnl Tris, pH 7.5 and agitation for 30 min with one-third volume of watwsut,uratcd phwol. The phenol-aqucow suspension was thrn hft ovrrnight, atI 4’. The nuckic acid fraction was rccovcred from t#hc suspension by cxtruct’ing thcl scparatcd aqueous phascb with CHCl,: isowith amylalcoliol (24: 1) and prccipitatind ethanol. The tR?;;2 was then scpnratecl by chromatography on Sqhadcx G-100 01 DF::\E-ccllulosc~.

The rclativct efficicncics of several variations on this phenol extraction procedure n-crc made by comparing the lysine acceptor activity of t,he extracted tRNAs. The variations &d and their rclativc efficicncics wcrc as follows: overnight phenol with no addition (1.00) + phenethylalcohol (0.19), + Rrij .X (O.Sl), prctrcat,ed with 8 11urea and 10 ‘%, 2-mcrcaptoethnnol (0.22) and pretrcnted b3 fr~~czP-t,hrt~~-ing (1.19). The results &OW th:bt the direct phc>nol mt%hod can be used to cbxtract’ tRNA from spores. However, t$hc \-iclld ~-as vcrv IOK, O.l-l.,j A&units p(sr g \vet weight, and considerable ribonuclrasc :lctivit,v was dctcctrd during the phenol cxxtrnction of sporrs and asporogenous mutants, although no RNaw activit’y was detectable in an overnight phenol cxt8ract’ion of log phase cells. A time study dont on t,hc extract,ion of t.RKA and DlVA from sports by t(hc phenol yroccdurc ehowd that, t,he maximum amount, of tRSA was cxtractcd during 4-5 hr and DNA during 5-G hr. lifter 5 hr, howwr, RKasc activity was still dttcctable cvcn in the prwcncc of phenol. On t,he basis of t,his, direct, extraction with phenol was unsuitable for sports or asporogcnous mut,ants in *tationary phaw. A freeze-thaw, direct phenol m&hod for extracting tKXA has also been applied to conidia of Aspergillus orgzae and was found tmobe unsuccessful (16). Procedures

involoing

8 31 urea

and

a reduc-

reagent. Gould and Hit,chins reported tdlat bact,erial spores could be sensit,ized to lysozymc by treat,ment, with reagent&s which rupt’ure disulfide bonds (17), presumably dw to solubilization of a coat protein fraction (1X). The twntmmt, of spores with reducing rcagent,s follo\vcJd by lysozymc breakage ~-as subscqucnt~ly adapted by various laborat,ories and has been uwd for c>xt,racting tR.NA from Badlz6s me@erium (19, 20). Nc.vcrt,hclcss, this mtthod of opcwing spores has bccln criticized :IS n form of nonphysiological germination (21) sinw :I phase darkening of sporw, :L loss in optSicnl dcnsit> and a loss in vinbl(l hc&rwi~tant tipores was obswvcd afkr scwsitizcd spores I\-erc k-cat cd Ivitli lysozyme (17, 22). Such a loss in r(:fractility has hwn obscrvcd h\, Ipitz-.Jamw in!/

64

\‘OLI)

ANT)

(8) after lysozymr treat.mcnt of spores sensitized with a reducing reagent, however, he showed that this uxs due to membrane rupture followed by leaching and lysis of the spore core. When the proper conditions were used to stabilize protoplast integrity, FitzJames obtained intact protoplasts which retained spore-like characteristics in electron The st,abilization of spore microscopy. spheroplast#s has also been reported by Sakakibara and Ikeda (30) adter sensitization with thioglycolic acid and urea followed by treatment with lysozyme. The authors reported that these spore spheroplasts retained characterist’ics specific to intact dormant spores. Method B, urea-.G?aercaptoethanol. We had originally used a urea-2-mercaptoethanol and lysoayme procedure to extract tRNAs from B. subtilis spores (23). However, the method was long and the yields varied between 1 and 2.5 A260 units per g wet weight of spores. The dithiothreitol procedure which will be described later was found to be much more effective in rendering the spores susceptible to lgsis by lysozyme (see Fig. 1) and yield of tRNA. Method C, urea-thioglycolic acid. A method using 8 M urea, 10 70 thioglycolic acid and 0.01 M EDTA has been described by Sakakibara and Ikeda (30). The original procedure was developed for making protoplast,s, and we adapted it for tRNA isolation. No RNase activity was detectable in the supernatant fractions which contained the tRNA, but the yield of tRiSA was highly variable. A yield of 2.2 A2e0 units after DEAE-cellulose chromat’ography per g wet weight of spores was obtained on a small quantity of spores. However, it was not, possible to obtain this yield when large amounts of spores were used. In fact, sometimes we recovered no tRNA at all. This was probably due to our difficulty in stabilizing sphcroplasts from mature spores of our strain (B. s&ibis 168) under our condit,ions. We were not able to stabilize the spore sphcroplasts by using either 20 % sucrose or 0.73 M lactose although protoplasts from all stages of veget,ative cells arc easily stabilized in 20% sucrose. This instability of spore sphcroplasts has also been notcad by ot,hers (8). Therefore, MY concluded that, :I sphrro-

RIINATOGAWA

0

10

time [min)

20

30

FIQ. 1. Susceptibility of spores treated with urea-2-mercaptoethanol or dithiothreitol to lysis by lysozyme. Treated B. megaterium spores were incubated at 37“ with 100 pg/ml lysozyme in 0.25 M sucrose, 20 mu Tris pH 7.5,20 mM CaClz . Initial optical density of each sample was approximately 1. Two ages of spores and two different pretreatment methods were used. Young spores (24 hr) were used for samples A and C and mature spores (72 hr) for samples B and D. The urea-2-mercaptoethanol pretreatment (Method B) was used for samples A and B, and the dithiothreitol pretreatment (Method D) for samples C and D. The fall in optical density with t,ime is due to spore lysis since t,his buffered sucrose medium is not suitable for stabilizing spore protoplasts for longer than 5 min. This does not represent germination, since Fitz-James has employed electron microscopy to show that t.he loss in optical density is accompanied by a rupturing of the plasma membrane of the spore (10).

plast-procedure was not useful for spores of Raciblus subtilis 168. Method D, dithiothreitol. Recently, Fita.James has presented a thorough study of the susceptibility of spores of three species of Bacillus to coat removal by treatment with dithiothreitol and SDS at alkaline pH (8). This procedure resulted in a semi or completely costless spore, depending on the species. The remaining cortex and germ cell wall were then digested with lysozyme. FitzJames characterized t#hesc various spore struct,ures with phase and electron micros. . copy, mgrosm smears, some biochemical properties and viability t’csts. We compared t.he efficiency of the urea-mercaptoet,hanol procedure (described above as Method B) with the dithiothreitol procedure of E’itzJames with respect t,o coat removal and lysozyme scnsit,ivity by t,hc following experiment. Purified sport’s of I?. rnegaterium T
were treated with 8 11urea-10 % 2-mercaptoethanol pH 3.0, 1 hr, room temperature or in 100 rnM dithiothreitol and 0.5% SDS in 0.1 M NaCl, pH 11.0, room temperature, 18 hr. After incubation, the spores were washed four times in 0.15 M Tu’aCl and suspended in 0.25 M sucrose, 20 ml1 Tris, pH 7.5 and 20 mM CaClz containing 100 pg lysoeyme per ml at 37”. As noted before, spore spheroplasts were not stabilized against lysis under these conditions. Rupture of the spores was measured by observing the loss in optical density at 650 nm. Spores of two ages were used, 24 and 72 hr from the time of inoculation. The former stage represents young, newly released spores and endospores and the latter represents older spores. The results are shown in Fig. 1. The dithiothreitol procedure is clearly superior for promoting lysis of spores. In addition, the yield of tRNA from B. subtiEis 168 is 14-16 AW units after Sephadex G-100 chromatography per g wet weight of spores which is considerably higher than the yield obtained with the urea-mercaptoethanol procedure. Since the yield of tRNA was the best with the dithiothreitol procedure, this was chosen as the basis of the improved extraction procedure which will be described in Improved Extraction Procedure. RIBONUCLEME ACTIVITY INHIBITORS

AKD ITS

Ribonuclease activity is detectable in supernatants of sporulating B. subtilis cultures in the early phase of sporulation (24). The characteristics of B. subtilis ribonucleases have been summarized by Nishimura (‘25). He discussed the presence of three ribonuclcases in B. subtilis skain H; two extracellular and one intracellular. The intracellular enzyme had no base specificity, had a pH optimum of 5.8 and was inhibited by 1.7 rntiI EDTA. The cxtracellular nucleases were specific for the secondary phosphate esters of purine ribonucleoside 3’-phosphates of RNA, had a pH optimum at 7.5, and were insensitive to inhibition by 0.01 M EDTA, polyvinylsulfate, and most metal ions except Hg2+. In order to dctermioe the optimal conditions for protecting RP\‘A from RNase attack

during extraction, we characterized the RXase act,ivity in clxtracts of B. subtilis mit,h respect t.o pH optimum and effect of inhibitors. pH Optima. The act,ivity of RKA in the culture fluid and exkacts of B. subtilis log phase cells, &ationary phase cells, and sports showed that sporulating cells and spores possessed intracellular Rr\‘ase activity \vith an acid pH opt,imum and extracellular RNase activit,y with an alkaline pH optimum. None of these activities was present in appreciable amounts in log phase cells. E$ect of inhibitors. Alt#hough our data are in agreement \vith t.hat of Xishimura (‘25) on the pH optima of the intra- and extracellular RNase activities, and on the inhibition of intracellular RPITase by EDTA, we fomld that our extracellular RNase was inhibikd by EDTA. Under our condit’ions with our strain, the RKase activity of all three fractions was inhibited by 0.01 M EDTA, 0.1% diethylpyrocarbonate, and 1.5 mg/ml bcntonitc. IMPROVED EXTRACTION PROCEDURE

The following method has been developed on the basis of the preceding data to give a gentle but efficient’ rxtraction of tRNA witshout, RKase activit’y. Purijcation and extraction of tRNA .from spores. After cent.rifugation from the culture media, sport preparat,ions were treated wit.h lysozyme and SDS as described by Lazzarini (9). The spore preparation free of vegetative cells was then made 30% (v/v) in Renografin, (taking cont’cnts of bottle as lOO%), layered over 60% Renografin and centrifuged at X,OOOg, 30 min 5”.Spores of normal density p&t,. The pellet was then washed once with water. Purified spores were suspended in 50 rnbf Cleland’s Reagent (dithiolhreitol), 0.5 % SDS, pH 11 (‘10 ml/S g wet weight) and left for 18 hr at room temperature. Spores were then washed by crntrifugation four times wit’h 0.15 M Y&l and OIUY with 50 rnfir Tris, pH 8.0, 10 rn>I MgC12 , 1 rnhI EDTA, 2 ml1 2-mercaptort,hanol and 0.05 % diethylpyrocarbonak (extraction solution). The addit,ion of diethylpgrocarbonat,e to the ctxkact.ion solution drscribed did not inhibit. the

activity of lysozymc although it was a powerful inhibitor of RNasc. Diethylpyrocarbonate has also been reported to have an inhibitory effect on the germination of intact, spores (26). The washed spores were suspended in extraction solut8ion (100 ml/5 g wt weight), adjust’ed to pH 8.0, and incubat.ed for 30 min at 37” wit,h 1 mg/ml Iysozyme. Self-digested pronaw, 1 mg/ml was added and t,hc incubation continued for 3 min. Sodium dodecyl sulfak, 0.2 R. (\vt/v) was then added and the suspension ;;tirrcd for 30 min at room tcmpwat8uro. The tRKA was recovered and purified from thcl rxt’ruct,ion solution as dcwribcd for cells in logurithmic growth. Ihtraction of tRSA from log and stationary phase cells. After centrifugat8ion from thtb culture media, log and stationary phase cells wre washed by ccntrifugat’ion \\-ith cold 10 rnh[ Tris pH 8.0, 0.15 JI 9&l, 1 rnlr EDTA, and 20 o/o(w-t/v) sucrose (Tris-sucrow). Ttw pellets were disperwd in prcheatcd Trissucrose at 37” to malw a .5c/( suspension (wet weight/vol). Lysozymc, 100 pg/ml, was added and the cells incubated at, 37” for 15 min. The prot,oplnsts wrr ccntrifugcd at 13,OOOy,10 min, and suspcxndcd in extraction tiolution (dcscribcd above). Sodium dodclcyl sulfate>, 0.2 % (wt#/v) was thcxn added, and thca tRNA was recovcwd and purifkd as dwcribed brlolv. Recovery and puvijication of t.IiNA. hhalf volumt! water sat,urated pht~lol \vas added to t,hr suspension of spews or protoplasts in extraction solution and SDS and this suspension uxs st,irrcd 30 min, room t11v ccnt8rifugation, Afkr temperature. aqueous phase was wit#hdrawn and sawd. The phenol was \vashcld with a small volumcl of cbxtraction solution, and this aqueous phasr, after ccnt’rifugation, was combined with the first. Th(> combined aqueous phases \vwc: t,hen extractcbd onw with an equal volume of CHCl, : isoamylalcohol (24 : 1, v/v) and prrcipit&cd &h c%hanol. The collcctrd prccipitatr was dissolved in 10 rnlr Tris pH 7.5, 10 rnlr :\lgCl, , 1 ml1 EDTA, 2 mlr 2-mcrcaptor,t,llanol (TMEM), extrackd again with phenol and CHCls: isoamylalcohol as bcforc and prc>cipit8atrd wit,h c,thanol. Thcb

ethanol prccipit’atc> K~S tlissolwd in TA115,\1 and incubated for 30 min 37” \I-it11 10 pg/ml Dn’asc (RNasc frw) and rc~prwipitatc~tl \\.it11 clthanol. This ethanol prccipitat,n was dissolwd in 1 11 K&l, 10 m\r Tris, pH 7.5, 1 ml1 EDTA, 2 m;\r 2-mc~ralptoctll:lnol, and 1 % mcxthanol and applied to a Soph:~dcx G-100 column (2.5 X 75 cm). Thr column was c>quilibratcd and elutchd wibh tht> sarn(’ buffer at, room tempcbraturc, and the cont&s of tubes cont,aining tRNA n-w‘ combiwd nrd prccipitatc>d wit,h ethanol. Deacylafion oj endogenous amino acids. Thcl c%hanol precipitate> \\-a~ dissolved in 13 11Tris (pH S.0 at 37”) ant1 incubated at 37” for 30 min. Thcb t1iN.k \vcr(a t,hnn prccipit’atod I\-ith &anol and the prccipit,ate was dissolwd in TMEJI and stored at, - 15”. Yielrl. Thch yields of t Id, howwrr, assays on unfractionatrd material arc’ subjwt to wror on tho high side brcauw of cont,aminat,ing material. Thcl charactcbrization of somcl physical and biological propcrtirs of tRYAs exkacted from logarithmicall\growing cells, asporogcw~ous mutants in stationary growth phase, and sports lvill br prcscntc>d clscwhtw (30). On the basis of hypcrchromicity, absorption sprctra, or orcinol and diphrnq~laminc color reactions, all tRXA samples gave similar results n-hich indicated t,llc: samplw wrc grclat,c,r than ST,“Z pure:. .IS an indicat~ion of RS:w tlamagc of the

3. POWISLL, J. F. AND STRANGE, 1:. Ti;., Biochem. tlLKL4 samples, we twtcd t’he amino acid J. 54, 205 (1953 j acceptor activity before and after heating. 4. LRVINSON, H. s. .ixD SEV.\G, &I. G., ;iTch. Nishimura and Novelli (31) studied this Biochem. Biophys. 50, 507 (1951). propertq of Escherichia coli tRN\‘A which 5. BISHOP, H. L., hhGIT.\, L. K. .\SD &I, 1;. fI., had been treated w&h R. subtilis ext8raccllular J. HarferioZ. 99, 771 (19(i9). RSase and demonstrated a difference bc6. SPIEGELM\S, G., ~)IcICINSo3, !2:., IDRIm, J., t,wwn trcakd and untrratcd samples. ThrrcSTIX~BKllG, W., I:~DI~;xIIIGRG, 8. .\ND fore I\‘(’ assayed tRKA samples extracted H \LVORSON, 1%. o., in “Spores IT!" (I,. I,. from B. suOtilis ~11s in logarithmic grolvth Campbell and II. 0. Halvorson, I~&.), p. 235, American Society for Microbiology, pliaw, asporogcnous mutant’s in stationaq Ann Brbor, MI, (1969). phase, and spores for amino acid acceptor 7. Gomu, (;. W. .AXD HITCHISS, A. I)., J. Germ. activity for phc, arg, ilcu and val brforr and ViClObiOT. 33, 413 (1963). after heating at S.5” for A min in 0.2 nI SnCl, 8. FITZ-J.\~~I,:S, I’. C., .J. Baclwiol. 105, 1119 10 ml1 Tris pH 7.2 at a cone&ration of 2 (1971). mg tRSA/ml. All samplrs showed grcatclr 9. LIZZ~IKI, R. A., Proc. Sal. ~lcatl. Sci. C.S.B. than 90 ‘Z rrtc>ntion of amino acid acwptor 56, 185 (1954). activity for phc>, arg and ileu although all 10. S.uxs, L. E. \ND ALDWTON, (+., J. Bncferiol. samples showed a slight loss of act’ivit8y in 82, 331 (1961). vulinr-t,RNA. Since fragmrr& of tRX\‘As 11. HOCH, J. .mD SPIZIZEN, J., in “Bpores IV,” have brcn shown to renaturc after heating, (1,. L. Campbell and H. 0. Halvorson, Eds.)? p. 112, ilmerican Society for Microbiology, this proccdurr is not an absolute mrasure of Ann i2rbor, MI (1969). lack of RNasc damagch. Howvrr, it doc>s L. H. AND M&ORQUOU.\LI~:, 1). J., giw an indicat,ion that’ no gross damage leas 12. BRVB.\I;M, Biochim. Biophys. .Acfa 76, 18 (1963). occurred. CONCLUSION

The improved extraction procedure prescnkd in t,his paper gives an efficient reproducible met8hod for the extraction of tR?;A from vegetative cells and spores of Bacillus subtilis and B. megateCm. Four other methods for cxtract,ing tRNA from spores wrc investigated and found to bc less efficicwt

13. ZUB.\Y, G., J. Mol. Bid. 4, 347 (1962). 14. HANCHER, C. W., PHARI~CS,14;.F., NO~XLLI, 0. T).\vID .\ND KELMERS, ii. I).. Iliofechnol.

15. Hf$ry’ ‘19 1o55(19Gg). 1,; , I:. W., Biochew. 16.

17. 18.

. ACKNOWLEDGMENTS

We t,hank (+. David Novelli and John Spizizen for helpful discussions. We thank Philip C. Fit,zJames for sending us Bacillus megafeGum KRI. This investigation was supported by PHS Research Grant No. GM 17421 from the Xational Institllte of General Medical Sciences and b,v a Brown Hazel1 Grant from the lIesearch Corporat,inn.

19. 20. 21. 22.

23. 24. 2. T,.\WRICNCE, N. L. .\ND H.\~~vo~cso~~, H. ORIS., Rartcriol. 68, 331 (1954).

Hiophp.

ILIes.

(‘otnmm. 10, 186 (1963). T.\N.\Ic.z, K., h,~OTOH.\SHI, A., bk~.\, K.-I. .WD Y.\N.UXTA, T., J. Gen. Appl. Miw~biol. 12, 277 (1966). (~XLD, C. W. .\su HITCHINS, A. I>.! J. Gcn. Microhioi. 33, 413 (1963). SOMERVILLE:, 11. J., T)EL.\FII:LD, F. P. .\ND RITTISXHI:IE, S. C., J. Jkwferiol. 101, 551 (19iO). CH.WISON, P., I)uP~un-, 1,:. J. .\ND KoI~NI%I:I<(:, A., J. Biol. Chem., 243, 5101 (1968). ~)I,:UTSCHICR, M. P., CH.\hmOK, P. .\SD KORSBI.:RG, A., J. Biol. (‘hem., 243, 5117 (1968). IDMSS, .J. M. .ANU HIILVORSON. H. O., .lwh. Biochem. Bioph~ys. 133, 442 (1969). (:OULI), (i. W. .lsu KII\.G, W. I,., in “Spores I\‘” (1,. I,. Campbell and H. 0. Halvorson, ISds.), p. 276, American Society for Alimxbiology, Ann Arbor, MI (1969). Vow, I<. S., J. Uacferiol. 102, ill (1970). (irow? 11”. M.\sDI..LsT.\Y, J., in “Microbial. 1:. 377, 19th Symposium of the Socie1.y 01 General hZicrobiology, Cambridge, Unvrrsity Press. Cambridge, E~ql:rnd (1969).

68

T’OLD

AND

S., in “Procedures in Nucleic 25. NISHIMURA, Acid Research” (G. L. Cantoni and D. R. Davies, Eds). p. 564, Harper and Row, NY (1966). G. W., in “The Action, Use and 26. GOULD, Natural Occurrence of Microbial Inhibitors in Foods”, p. 17, 4th Int. Symp. on Food Microbiology, Goteborg, Sweden (1964). J. E., JR., NAGS, E. H. AND 27. DONNELLAN, LEVINSON, H. S., in “Spores III” (L. L. Campbell and H. 0. Halvorson, Eds.), p.

MINATOGAWA 152, American Ann Arbor, MI

Society for SIicrobiology, (1965). P. C., Cm. J. Mzhobiol. 1, 525

28. FITZ-JAMES, (1955). 29. WOESE, c. 11. AND I;OItnO, J. l!., .I. k&riol. 30, 811 (1969). S., in “Spores 30. VOLD, B. S. AND MINATOWWA, V” (L. L. Campbell and H. 0. Halvorson, Eds.) American Society for Microbiology, Ann Arbor, MI, in press (1971). S. AND NOVELLI, G. D., hoc. 31. NISHIYUKA, Nat. Ad. Sci. U.8.n. 63, 178 (1965).