- Email: [email protected]
Evidence for a defective protein synthesizing system in dormant spores of Bacillus cereus
Evidence
for a Defective
Protein
Spores
Received
October
Synthesizing
of Bacilios
System
in Dormant
cereus
11, 1967; accepted I>ecember
8, 1967
The rell-free ltmirlo xcid incorpor’:ltillg system from dormarlt spores of Bacillus cererrsis defective. After germinat.ion t,his activity rises to 50?;, of that of preparations from vegetat.ive cells. This increase is llot dependent upon either RN,4 or protein synthesis, and leads to an incorporating system essentially the same as that folmd from vegetative cells. Two defects ill protein synthesis were forlnd in dormant spores: (a) the absence, or near absence, of transfer enzyme activit,v, a11d (b) impaired bintling of mRN,4-aminoaeyl-sRNA by spore ribosomcs. The first defcrt is repaired bJ act,ivation of the spores and the second by germinatioll.
The suppression of protein synthesis during crypt,obiosis is a-e11 known. A fe\i minutes after the breaking of dormancy, :I rapid increase in protein synt’hesis is i&i:tted. The nature of the block in protein synthesis in some dormant systems is at the level of tmnscription (1 3), :md in others nt, the level of tr:mslation (4, 5). Investigations of these systems provide v:~lunble insights into t’he controls regulating the initiation of developmental changes. Hact,eri:Ll spores ;~re one of t)he most dormant, systems known. In lZacil/us cweus strzlin T, RXA synthesis commences npprosimntely 3 minutes after the initiution :md is followed sever4 of germination, minutes later by protein synthesis (1). Several lines of evidence, including the cl:rsscs of RX:1 p’csent in spores xrd the inhibition of ILKA, proteill, :md enzyme synt,hcsis by :tctinom>-tin 13, indicate that spores w-e devoid of st:thlc messenger RNA (mK.SA) :md that protein synthesis is dependent upon nctv t~ranscription (6). If the abscnco of protein synt.hesis in 1 Present address: Irlstitrtte of Applied !MicroI)iolog,v, Ulriversity of Tokyo, Tokyo, Japan. ? National Tnstitrlt es of Health Research Career Professor.
dormant and :tctivated spores is due only tu the :Lbsence of mRNA, t,hen one would prcdictj that. cell-free systems prepared from t,hcse org:misms should be devoid of basal amino acid incorporating tlctivit’y and should possess full capacity nhcn supplemcntcd n-it,h exogenous mRSA. To determine Ivhether blocks in the protein synthesizing system exist in addition to the apparent :~bscncc of mR?JA, cell-free :mlino ncid itlcorporxting syst,ems were prepared from sports of H. cer’eus str:rin T and comp:u-ed with the previously described system from vegetjativc cells of this same organism (7). The present p:tpcr deals wit,h the description of (a) the :Lctivntion of amino :Icid incorpornting activity during germination of spores of Hacillus ce~us str:tin T, (b) the char:Lctcriz:ttioll of the cell-free :unino ncid incorporating system from the germin:tted sports, and (c) the n&m-c of the defects in amino acid incorpornt,ing :IctivitJ. in dormwit spores.
Spores and vegetative wlls of II. ceu:rts straifi ‘I’ were employed ill Lhese studies. Spores were ~~11ivated in rwboys containing 10 liters of modi-
Uethod
.--~
-.__.
~-._--_-
f)ry grinding Sonic oscillation Nossal cell disintegrator Mini-mill Sand grinding .____ Mickle t.issue disintegrator”
Protein
-~~~
39.9 36 .2 13.2 64.5 32.2
~-_
20.5 22.5 8.4 26.4 10.8
-.-
Ribosomes"
1.85 5.-l 2.03 Not tested 0.76 Not t.est.ed 2.38 11.8 0.97 ___---~---^- 3.6 2.34
Yield of RNA(P)I'/;)
79 87 32 102 41 100
* RNA was determined by the orcinol method. h Phosphorous value was calculated by assuming 1 ml RNA contains 9% of phosphorolts. CRibosomes were isolated from the crude ext,racts by centrifugation at 105,oOOgfor 90 minutes. .\mount was calculated by assuming 1 mg ribosomes/ml in HZ0 has an absorbance at 260 rnp of 15. d Data from Fitz-James and Young (l-1).
Density-gradiellt. analysis was carried ollt by layering about a l-ml sample over the previously prepared linear sucrose density gradient from 5 to ZO”/c (a/a) containing standard hilffer. After rentIrifugation for 4 hours at. 25,000 rpm in a Bpinco model L SW-25 rotor, 25-drop samples (aborlt, 0.8-0.9 ml) were collected, atld the absorbance at, 2G0 mp was measured. -4 portion of each fraction was used for the assay of amino acid-incorporating :irtivil.y.
Crude extra& (S-30, see Ref. 7) were spuu at 42,040 rpm at. 4’ in a Spinco model E aualyticnl \lltracentrif\lge equipped with Schiieren optics. All rums were made ill a 12.mm standard rell fitted with a Kel-F centerpicre.
Protein was determined by t.he methods of Lowry et al. (13), and RNA was calcldat,ed by assuming 1 mg RNA~rnl H,O has an absorbance at 260 rn$ of 24
IIuiformly labeled r,-phet~ylalatlil~e-L”(: (X0 &/pmole) was obtained from Schwarg Bioresearch, Inc. The soIn-ces of the other chemicals have t)ectl dcscritrd
previously
0).
by several methods of spore disruption. .4s can be seen, the Mini-mill gave optimal results, comparable with t.hat report,ed by I’it,z-,l:tmes and Young (14). About 12 mg of ribosomes was obtained from 500 mg of spores by Mini-mill disrupt,ion. The ribosomes consisted of three major particles, 30, 50 and 70s. Similar ribosomnl distribution patterns were obtained by dry grinding or sand grinding, but t,hc yields were lower (50 and 3O’X, respectively). The Nossal cell disintegrator under the conditions empIoyed resulted in poor breakage; however, with Ionger treaments and a thicker suspension, maximal yields of RNA and protein can be obtained (S. Iiodenberg, personal communicat,ion). Decelopmerlt of antill acirl-incor?)oi-atirlg systei,~.4wiq wutgwwfi~. We have reported t,hat cell-free cxt,racts from previousI> dormant, spores lack amino a.cid incorporat.ing act,ivit.y (1). To further follow the development, of the prot,ein synt,hesizing q&em, ribosomes and supernat.ant fract.ions were prepared during various st,:tges of outgrowth, and the phenylalanine-‘“(2 incorporating activit,y was dct.ermined in t,he presence or absence of poly IT (Table TI). The first, column of Table 11 s11on~s the basal activity (phenylalanine incorporat,ion in the absence of poly TJ). Ten minutes after the init,iation of gcrminat.ior1, the system has about 60 5 of the act.ivit,\. of t,he vegetat,ive cells. The
‘I’ARLI~: I)EVELOPMENT
AC~UITY
OF
II
I’ROTEIN
IJURISG
t‘stmcts
SPIXTHESIXING
OUTGROWTHS Phenylalanine incorpzrated (~pmoles,fm~ ribosomal proteini -poly
spores I)orrnsnt I feat-activated spores (:erminatrd spores 5 mitt IO min 20 mitt 40 mitt Yegetativc
cells
9 9” 6.4”
u
+tloly
u
23
GG
38
4262 3550 5210 4438
88
9185
50
u Reaction mixture contained, in fimoles/ml; 45 Tris, pH 7.8; 10 magnesium acetate, JO NI14C1, :iO KCI, 0.2 GTP, 0.8 4TP, 4 PEP, 13.6 rg PEP kinase, 40 rrterc~aptoethatlol, 1 sperimiditte, 0.01 amino acid mixture, 0.02 L-phenylalanine-1X (10 pC/pmole), tiO0 pg R. cereus sRNA; and, where indicated, 100 pg poly I-. Total volume was 0.5 ml. A quantity of 55 rg of t,he supernatartt fractiotr (S-105.Sj and 23 gg of ribosomal (W-Rib-S) protein per 0.5 ml of the reaction mixture were rtaed. Samples were incitbated at 3G” for GO mill~ttes, and the hot trichloroaretir acid-ittsolttblc radioactivity was determined. ‘j These values are a limit of detect.iott, arttl, I twrefore, for all practical prtrposw, are zero.
in dormant and hea-activnted spores was less than 10 ~~moles per milligram ribosomal protein, and approximates the level of detection of radioact’ivit> in t,he zero-time control, and is, for all practical purposes, zero. This inactivit). might be due to (a) the nonsnturat~ing :mount of mRNA in t,he spore system, (b) defects in the prot,ein-synthesizing syst’em, or (c) the presence of inhibitor(s). To test t#hefirst possibility, phenylalanine incorporntion nxs examined in the presence of saturating amount of poly U. As shown in t,he second column, t,he addition of poly I!’ to the system from germinat#ed spores or \:eget,ative cells increases phenylalanine incorporation approximately IOO-fold, but, the system from dormant and heat +&v:tt,cd spores responds very poorly. These findings indicate th)at the dormant spores, in :tddit8ion to t,he apparent lack of mRSA, have a defect’ive protein svnt h&zing systc~m. There is ;i dramatic ‘irlincorporation
crease ill amiuo acid incorporating activity. during the transit’ion stage from dormant spores to germinated spores. Five minutes after t’hr init,iation of germination, the q-stem responds to the added pol;\r IT. Is this change a11 activatjion or a rle HOW synt’hesis of some component of the incorpornt ing system? To test, this, spores were germinated for 10 minutes in the presence of 100 p,g chlor:Lmpheuicol/ml and 25 pg actinomycin II/ml to inhibit prot.ein and RN4 synthesis. Cell-free extra&s wercs prcpnrcd and amino acid incorporating &ivity ~-as measured. Table III sl~ows th(L results. The addition of the nnt,ibiotics did not, inhibit the appearance of the incorporat ing activity-, clearly she\\-ing that the appearawe of t,he activity after germination is not due to the synthesis of some component of the system. When spores QW~ germinated in the presence of chloramphenicol, higher incorporating :Ictivit,ies ww observed. It is ~~11 known (15) that, wider these conditions, I
the amino acid incorporating sgst,em in sI )orw, it is first necessaq to determine whether the characteristics are identical to those: from vcget:ltive cells. Table IV
118
s-30-s s-30-s
(CM)
s-30-s
(CM + Act. I)1
338
X8
‘I Extracts (superrtatant fraction was obtained by certtrifngat.ion at) 30,000 g for 30 minutes aittl then dialyzed overnight against standard buffer) were prepared from the spores germituted for 10 minrttcs itt the nbsenw of antibiotics (S-30-8); it1 t,he presence of chlor:tmpheilicol, lOO&ml,S-30-F (CM) ; and chloramphenicol, 100 pg/tnl; and HCtinomycirr I), 25 rg/ml, S-30-S (CR1 + Acti I)), respectively. For inctlbat ion condit,ions see Table IT. The zero-timp valt~e was sttbtrapted frl)m all samples.
TABLE
I \.
:&minct illCorI)or:LtioI1. Amino acid incorpcnw tion :rt, :31” continues more than 1 hour in both systems. Table V compnrcs other charxturistjics oi’ polo. Cstimulnt,ed phenykkmine itl-
I:syl.”
1 Complete minus NII,Cl minus GTP rninlls ATP, PEP atltl pyruvnte Itimtse rninlls merraptoet hnnol minrls spermiditre
4.3 2.6 2.1 0.57
10.X
4.5 2.t;
x,.5 7.8
2 Complete minris spernatant minrls rilwsomes
3 .3 0 0.26
10.8 0 Il.28
3 Complete minus sILNA minus poly U minus amino acitl mixt we phw ribonuclease plus deoxyrihonnclease~
3.0 0.8 0.04 3.1
8.i 0.1 0.1 7.x
0 2.5
0 7.6
6.9 X.6
0
li For inclbtion conditions see Table II. Total volume was 0.25 ml. The germinnt ed sport system contained 44 rg srlpernatarit protrill (S-105-S) and 20 ~g ribosomal protein (W-RibS). The veget at ivcl s!-stem contained 80 pg prcilrculuted s~ipcrl~atnril protein (Inc-9-105-S) and 48 rg rit)osom:il proleilr (Inc.W-ILibS). h J)ata from (7). c In both systems 10 pg rihol,rtclease or 40 pg deoxyribolillrle;se/ml rcac t ion mixtrlre were 11sed.
requirements of poly Udependent I-‘hen?-l:tlaniIle-‘4C incorporntion into prot,ein of germinnt~ed cell-free extracts and extracts of vegetative cells. The amino acid incorporat,ion is dependent upon ATP, the suJwrt~:tt;u~t, fraction, rihosomcs, sllNA,
summarizes
and
poly
t,hc
Ij.
The
systcrn
is
sensitive
t,o
ItKase but insensitive t,o DN:wc. Omission of SH ,Cl, GTI’, or qermidine led to :L loss of :lbout~ 30 50 ‘:; of the incorpor:Lt,ing :ICtivity. These those obscrwd CCllS.
ch:w:lct.cristics in extracts
are
similar
from vegct~ativc
t,o
TIME (MINI FIG. 1. l‘imr cotwse of ami11o acid incorpora1ion of cst rcwts from gcrmiri:tted spores (---j nntl vpgctitt ive cells (~ ). For incllhation cotkdit ions see ‘l’:tl)le II. Tot al vol~~mr, 0.25 ml. l’oI,- I-, 25 pg, was :tddcd. (;errniriated (5 mitlrltes) spore sgstcm contained 48 pg S-105-S protein :clrd 20 ~g ItIc-W-RilbS proteill.
Optimal bIg3’ cotIreIItratioil (mu) Optimal rewtioll tenpernture (“C) ITalf-sntrrrated cont. of sRNA (rg/ml) Half-satrlruted conic. ol poly li (hg/ml)
11
!I
3i
3li
ItiX
13ti
“4
32
Conditiotls are the same as in Table II. Total vollmrc, 0.25 ml. Germinated spore system cow tail& 44 pg sllpernnt ant fractiotr (P-105-S) and 19 pg ribosomal (W-Rib-S) proteins, alltl the vegetative cell system contained 48 pg prrillcubated srlpernntant frwtion (Inc.S-105-S) alIt 21 pg rit)osornal ITn>-W-Ribs) protrill.
I)P:F15CTT\~E t’t:OTETN
I )orma11t spores
+ + + +
+
f + + + +
+ +
+ + + + +
+ + + +
SYh’THE$TS
10700 32 2192 I(iifi 3)
7313 10530 6177 28 12 110
--c +
+
+
932 3172
+ +
131-H) 13263
+
6530 6380
+ + +
+ +
+ +
f
+ + + + +
+ + + +
+ + + +
9604 10634 14729 1x14 20030
CLConditions are the same as in Table IT. Total 0.5 ml. 55 pg of the supernatsut fraction (S 105-S) alld 23 JL~ribosomal (W-Ttih-8) protein per 0.5 ml of the reaction rnistru-e wew l~scd iI1 each case.
vol~nnc,
(i'li
corpor:~tion of t’lic germinated sports anti the vegetative ccl1 systems. The optim:ll magiicsium concentration and t8he 0ptim:il temperature is the same for hot11 xyskms. The s~xtem obtained from germiii:~t,~~tl sporw is heat-1:ihilc. At Go, the germinated spore sys’tem does not show any phenylalanine iticorr)or:tt,iorl into protein after :I I-hour incl~bntion. The concentration of 16 sRXr\ or poly I’ required to half-~:aturntc~ 2-l 21 amino xid incorporat,ion is a~)l)rosim:Ltcl?. the same in the two syst,ems. Thcreforc. 2O(i5” t,lie two nniino acid incorporating syslein~ l-1 arc essenti:illy t,hc wnc. If~towlo!/ciuts
cw/rbimlior/
f,.cl,ei,i/,lrrrls.
‘I’0
examine tlw loc:lt8ion of the defecb iti thr, incorpotxti~ig s\-st em of dorm:mt sporw, t,hr SllpFrll:~t:lnt and ribosomc fract,ioiis f’rc~nl Ihe dormant, Iic:tt~activnt,ed :urd gcrminnted sporw and from the vegfkt iw cells ww prqxux~l and tested in a scrics of liet,~w~logous comhiii;ltiolis. I’hc~rl~lal:~~~ii~~~ incorporatioil
HeaL-activatcd sp~‘rrs
IN SPOT:I<::
into
protc~in
wts
incxswwl
the J~YSY~~~ 01’ poly I! (Tsblc VI). spores hve 311 Nibosomcs fronl tlonnant~ extremely low activity. The sup(wat:tnt fraction from dormant spores is inactive and slightly inhibitory on the voget:ltive systen1. A\ftjer ~lc:lt-:ict,iv:rtiolI, ~porc rihoaomex have 10 20 ‘7 activity of the activity of ril~osomw from vegetative cells. Supernatant fix*lions from lifat 4ctiv:Ltetl sporw nw eclu:rll,~ inactive. .\ftcr tlw irliti:~t,io~l of germinntio~l, spore rihosoni(::: accpir(~ 60 “; of the :LctivitJ of t tic ribosomw from vcgetntiw cells. The supcrnatnnt fraction :tlso increww its activity to ahout 60 Y;’ of the supcrnat:uit fr:lct,ion from vcget ative cells. These results show clearly that the defects in the ~LnlilHJ acid incorpor:&g System in the dorma~lt sporw exist lwth in tlw s\lpcrn:itant fix+ tion 2nd the ri~~osomcs. irl
K?s
KOBAYASHI
AND
HAI,\‘OR.SOri
During heat-activation, activated. Amirm
acid
accepting
this ability
enzyme
is
of sKNA
pj~epa?~ed fwtja dormant spo?‘es.We determined whet,her sRKA from dormant, spores has t)he same amino acid incorporating ability as sRNA from vegehative cells. As shown in Fig. 4, dormant spore sRNA has a slightl?, lower activity for phenglalanine than sRNA from vegetative cells at low concentration; but at suturat,ing concentratjion, t h(, activity is almost the same.
a ifi07 b z
8or-----0
0
IO TIME
20
(MINI
P’IL;. 2. Time course of phenylalauyl-sKiYA synt,hetase from the dormant, heat-activated, aud germinat,ed spores aud vegetative cells. Extracts (SF II) were prepared by the method of Wood and Berg (9). For incltbatioll conditions seeRIAw~iur,s h~u -\b3rHODS. SF 11 were from: 1, dormani spores; 2, heat-activated spores; 3, spores heat-act,ivated in the presence of chloramphenicol; 4, germinated spores; 5, spores germinated ill the presence of ~hloraInph~rlico1; atrd C,, vegetative crlls.
sRNA synt,het,ase uct’ivitjy in t’he supernst,ant fraction from dormant, heat-activated, and germinated spores and vegetative ~11s. As can be seen, t’hc enzyme activit,J was essentially the same in each cast. The presence of chloramphenicol during hcatshock or germination does not have my effect on the appearance of enzyme activity. Vigure 3 shows the t’ransfer enzyme act,ivity in the supernatant’ fraction from these same extracts. The supernat’ant, fraction from the dormant spores has almost while heat-activxt’ed and no activity, germinated spores have high activit?. The presence of 100 pg chloramphen~coljml during heat-activation or germination does not affect, t,he appearance of the trnnsfcl enzyme. These results indicate that the defect in amino acid incorporating act,ivitJ of t,he supernatant fraction from t,hc dormant spores is in the transfer enzyme.
0
30
60
TIME ( MIN 1 F’lo. 3. Time course of transfer enzyme from the dormant, heat -activated, and germinated spores and vegetatiw cells. Extracts (SF II and rihosomes) were prepared hy t,he methods ol Wood and Berg (9). Twellty-folw pg of SF II, l.GO O11260 units of rihosomes prepared from the vegetative cells, and 1.80 01&,0 lulits of phenylalanyl-‘4C-sRNA (1200 c’pm) were used. For incubation conditions see RIATEIUALS AND METHODS. SF II were from: 1, dormant spores; 2, heatactivated spores; 3, spores heat-activated ill the presence of chloramphenicol ; 1, germinated spores; . sports germinated J, iIt i he preseucr of chloramphrnicol ; and G, vegetative rells
100
0
200
sRNA ( yg/O
300
400
500
I ml react mixt 1
FIG. 1. l’liet~ylalanine acceptiflg ability of sRSA f’rc~rndormant spores (0) and from vcgetat ive wlls (0). Soluble RX.4 was preparetl as ConditIescribed ill ~\IA~:ILI.~LY Am NE’~H~I)s. 1iolw for phenylalanine loading are the same as for phellylalx~~yl-sR~;A synthetasr. Sllpernatant fraction (SF II) from vegetative wlls was I~se(i. I~~c~llhatetlfor 20 mitnlites al SC,“.
To csamittc~ the nature of defects in rihosomes from spores, the characterization of rihosomes from spores and vegetative cells \v:ts undertaken. Cell-free extracts (S-30) \vcre prepared from dormant, heat-activated, :rnd germinated spores and vcget,ative cells :md :malyzed by analyt8icnl unttxccntrifugatiott
in lo-” nt AI@!+. Figure
5 shmvs
thr
of t.hc ribosomal particles dist~rihutioti during growth. As can he seen, vegetative ~~~11scontain four classes of rihosomal p:trt~icles
chwracterizcd
by
scditnettt:ttiott
constants of roughl>- 30, 33, 70, and 100s; the 30s and 50s particles arc minor cornpottents. The distribut’ion pat8tern of ribosomal particles in sporca is quite different from that of veget,ative cells. Sports contain three major particles, namely, 30, 50, and 70s. Thcl relative txtio of these particles tlocs ttot change in dorm:m~, heat-:lct,iv:tt’cd, 01’ gwtnitt:tt(~d
(6 minutes)
qmw;
hut
LX)
minut,es after the itiit~iation of germiti:ttiott, the relative amounts of XOS and 50s ittcrease. The :3OS and 50s subunits csist frw at higher magnesium coticetitrat~iott (0.05 11) and do not) form TOS ribosomcs. I’oly Kdcpendent phet~ylalnttitte incorporating activity of these rihoscrmal particles was cotnp:wed after fractionation b\. sucrow densit,?--gradient’ centrifugation. l‘he r~‘suits arc shown in E’ig. 6. Parts 9, 13, and (’ are rihoaomes from dormant and germinated sports, and vcgetwtivc cells, I’(‘xpcctivelg. Sittw crudr cstracts front dormant sports con~aiii large :Imoutits of ultraviolet-ut)sorbitlg material (III’A), th(l 30s suhuttit~ is not, shown clearly. As can bt> seen, tteit’her SOS nor 50s p&iclcs have att~ atnitto acid incorporating activity. The 70s ribosomes from dorm:ntt spores have sonic’ :tct’ivitJ.. Aftw the initiation of germitintioti, 70s ribosomcs become fully active, and the specific activity (cpmj 01&) is about twofold higher than that of vegSet:tt,iw crlls. This ittcreaw is not t,hc result of r/r UOZYJ syttt-,hesis of IWW ribosomtrs sitw t,hti rihosomw from spores germinated in the
w w
w
dormant
spare
heat-activated
11
germinated 6 min
11
germinated 20 min
11
630
KOBAYASHI
AN11 IIAI,\~ORSON
FRACTION
NUMSER
FIG. 6. Phenylalanine incorporat,ing activity of ribosomcs fractionated by sucrose derlsity-gradient centrifugation. Crude extracts obt,ained hy sand grinding and centrifugat.iotr For condit,ions at 10,000 9 for 10 minutes were layered on linear sucrose density gradiellt. see MATERIALS AND METHODS. Two-tenths ml of each fraction was added to the amino acid incorporating system described in Table II. Forly-seven pg of the preincubated sllpernatant fraction (Inc-S-105-S) from vegetative cells was used. Total vollune, 0.5 ml; incubated at C, 36” for 1 hour. A, Dormant spore extracts; B, germinated spore (20 minutes) extracts; vegetative cell extracts.
presence of chloramphenicol, have similar activities (Table III). The high specific act’ivity of ribosomes from germinated spores could be explained partly by t,he absence of natural mRKA in spores making a binding of poly U much more effective in ribosomes from germinated spores than from vegetative cells. The apparent’ defect observed in the heterologous combination experiment (Table IV) in ribosomes from germinated spores is due to t,he existence of a large amounts of nonactive 50s and 30s subunits in spore ribosomcs, while ribosomal particles in vegetative cells are almost exclusively active fOS and 100s ribosomes. Several at,tempt,s have bx~~ made to correlate the biosynt,het,ic activity of spores following outgrowth \vit)h the maturation of t,he ribosomal system. Only 50s and 70s particles have ‘been observed in spores of Bacillus subtilis (16) and Bacillus meqatwiuw (17), but, as reportjcd here, spores of
B. cewus strain T cont,ain 30, 50, and 70s pnrt(icles. Woese et al. (16) observed that during out,gron-t,h of B. subtilis spores, both ribosomal precursors as well as polvsomes were formed. In t,he present experiments, only the 70s part,iclcs in germinated spores are fully active in amino acid incorporat’ing activit,y. Prior to the onset of protein synthesis it1 vim, these must either be derived from inactive 70s part)icles or from the conversion of 508 and 30s particles to active 70s particles. From quant,it,ative considerations it appears that t,he rat,climiting step in protein synthesis during outgrowth is the synthesis of mRNA (1). Nature oj the defects in ribosotnes. What is the nature of the defects in t’he ribosomes from dormant spores? Since active ribosomes bind both aminoacyl-sR?CA and pl~er~ylxlanyl-14C-sRNA binding mRNA, with ribosomcs from spores and vegetative cells was compared. Ammonium sulfatetreated ribosomes were prepared according
0
IO
20
RlbosomesC 0Dxd0 I’llr,lr)-l:tlau?-l-sl(Nh
25m1)
bilrtlilrg
with rit)o-
632
KOBAYASHI
ANI)
P. C., Biochim. 11. SARIN, P. S., AND ZAMECNIK, Biophys. Acta 91, 653 (1964). 12. STEPHENSOX, Xl. L., AND ZAMECNIK, P. C., hoc. 1Vatl. Acad. Sci. U.S. 47, 1627 (1961). 13. LCWRY, 0. H., ROSEBROUGH, N. ,J., FAJ~R, A. I,., ANI) RANDALL, R. .J., J. Biol. Chom. 193, 265 (1951), 14. FJTZ-JAMES,
teriol. 15. HAHN,
P. C., AND YOUNG,
78, 733 (1959). F. E., in “Biochemistry
I. E., J. Bac-
IIALVORSOX (Ii. H. Spitz>- and R. Brutmer, eds.), p. 101. I’ergamon Press, Londo~l (1959). 1G. WOESE, C. Ii., LANGIUDGE, R., AND MOIU)\VJIX, H. J., J. BacleCol. 79, 777 (1960). 17. CHALOUPECKY, I-., Folia .Ilioobiol. 9, 232 (1964). 18. STEINBERG, W., HALVOI~S~N, H. (I., KEYNAN, A.,
of Antibiot,ics”
AND
(196.5).
WEINBERG,
E.,
>\‘nlure
208,
710
for a Defective
Protein
Spores
Received
October
Synthesizing
of Bacilios
System
in Dormant
cereus
11, 1967; accepted I>ecember
8, 1967
The rell-free ltmirlo xcid incorpor’:ltillg system from dormarlt spores of Bacillus cererrsis defective. After germinat.ion t,his activity rises to 50?;, of that of preparations from vegetat.ive cells. This increase is llot dependent upon either RN,4 or protein synthesis, and leads to an incorporating system essentially the same as that folmd from vegetative cells. Two defects ill protein synthesis were forlnd in dormant spores: (a) the absence, or near absence, of transfer enzyme activit,v, a11d (b) impaired bintling of mRN,4-aminoaeyl-sRNA by spore ribosomcs. The first defcrt is repaired bJ act,ivation of the spores and the second by germinatioll.
The suppression of protein synthesis during crypt,obiosis is a-e11 known. A fe\i minutes after the breaking of dormancy, :I rapid increase in protein synt’hesis is i&i:tted. The nature of the block in protein synthesis in some dormant systems is at the level of tmnscription (1 3), :md in others nt, the level of tr:mslation (4, 5). Investigations of these systems provide v:~lunble insights into t’he controls regulating the initiation of developmental changes. Hact,eri:Ll spores ;~re one of t)he most dormant, systems known. In lZacil/us cweus strzlin T, RXA synthesis commences npprosimntely 3 minutes after the initiution :md is followed sever4 of germination, minutes later by protein synthesis (1). Several lines of evidence, including the cl:rsscs of RX:1 p’csent in spores xrd the inhibition of ILKA, proteill, :md enzyme synt,hcsis by :tctinom>-tin 13, indicate that spores w-e devoid of st:thlc messenger RNA (mK.SA) :md that protein synthesis is dependent upon nctv t~ranscription (6). If the abscnco of protein synt.hesis in 1 Present address: Irlstitrtte of Applied !MicroI)iolog,v, Ulriversity of Tokyo, Tokyo, Japan. ? National Tnstitrlt es of Health Research Career Professor.
dormant and :tctivated spores is due only tu the :Lbsence of mRNA, t,hen one would prcdictj that. cell-free systems prepared from t,hcse org:misms should be devoid of basal amino acid incorporating tlctivit’y and should possess full capacity nhcn supplemcntcd n-it,h exogenous mRSA. To determine Ivhether blocks in the protein synthesizing system exist in addition to the apparent :~bscncc of mR?JA, cell-free :mlino ncid itlcorporxting syst,ems were prepared from sports of H. cer’eus str:rin T and comp:u-ed with the previously described system from vegetjativc cells of this same organism (7). The present p:tpcr deals wit,h the description of (a) the :Lctivntion of amino :Icid incorpornting activity during germination of spores of Hacillus ce~us str:tin T, (b) the char:Lctcriz:ttioll of the cell-free :unino ncid incorporating system from the germin:tted sports, and (c) the n&m-c of the defects in amino acid incorpornt,ing :IctivitJ. in dormwit spores.
Spores and vegetative wlls of II. ceu:rts straifi ‘I’ were employed ill Lhese studies. Spores were ~~11ivated in rwboys containing 10 liters of modi-
Uethod
.--~
-.__.
~-._--_-
f)ry grinding Sonic oscillation Nossal cell disintegrator Mini-mill Sand grinding .____ Mickle t.issue disintegrator”
Protein
-~~~
39.9 36 .2 13.2 64.5 32.2
~-_
20.5 22.5 8.4 26.4 10.8
-.-
Ribosomes"
1.85 5.-l 2.03 Not tested 0.76 Not t.est.ed 2.38 11.8 0.97 ___---~---^- 3.6 2.34
Yield of RNA(P)I'/;)
79 87 32 102 41 100
* RNA was determined by the orcinol method. h Phosphorous value was calculated by assuming 1 ml RNA contains 9% of phosphorolts. CRibosomes were isolated from the crude ext,racts by centrifugation at 105,oOOgfor 90 minutes. .\mount was calculated by assuming 1 mg ribosomes/ml in HZ0 has an absorbance at 260 rnp of 15. d Data from Fitz-James and Young (l-1).
Density-gradiellt. analysis was carried ollt by layering about a l-ml sample over the previously prepared linear sucrose density gradient from 5 to ZO”/c (a/a) containing standard hilffer. After rentIrifugation for 4 hours at. 25,000 rpm in a Bpinco model L SW-25 rotor, 25-drop samples (aborlt, 0.8-0.9 ml) were collected, atld the absorbance at, 2G0 mp was measured. -4 portion of each fraction was used for the assay of amino acid-incorporating :irtivil.y.
Crude extra& (S-30, see Ref. 7) were spuu at 42,040 rpm at. 4’ in a Spinco model E aualyticnl \lltracentrif\lge equipped with Schiieren optics. All rums were made ill a 12.mm standard rell fitted with a Kel-F centerpicre.
Protein was determined by t.he methods of Lowry et al. (13), and RNA was calcldat,ed by assuming 1 mg RNA~rnl H,O has an absorbance at 260 rn$ of 24
IIuiformly labeled r,-phet~ylalatlil~e-L”(: (X0 &/pmole) was obtained from Schwarg Bioresearch, Inc. The soIn-ces of the other chemicals have t)ectl dcscritrd
previously
0).
by several methods of spore disruption. .4s can be seen, the Mini-mill gave optimal results, comparable with t.hat report,ed by I’it,z-,l:tmes and Young (14). About 12 mg of ribosomes was obtained from 500 mg of spores by Mini-mill disrupt,ion. The ribosomes consisted of three major particles, 30, 50 and 70s. Similar ribosomnl distribution patterns were obtained by dry grinding or sand grinding, but t,hc yields were lower (50 and 3O’X, respectively). The Nossal cell disintegrator under the conditions empIoyed resulted in poor breakage; however, with Ionger treaments and a thicker suspension, maximal yields of RNA and protein can be obtained (S. Iiodenberg, personal communicat,ion). Decelopmerlt of antill acirl-incor?)oi-atirlg systei,~.4wiq wutgwwfi~. We have reported t,hat cell-free cxt,racts from previousI> dormant, spores lack amino a.cid incorporat.ing act,ivit.y (1). To further follow the development, of the prot,ein synt,hesizing q&em, ribosomes and supernat.ant fract.ions were prepared during various st,:tges of outgrowth, and the phenylalanine-‘“(2 incorporating activit,y was dct.ermined in t,he presence or absence of poly IT (Table TI). The first, column of Table 11 s11on~s the basal activity (phenylalanine incorporat,ion in the absence of poly TJ). Ten minutes after the init,iation of gcrminat.ior1, the system has about 60 5 of the act.ivit,\. of t,he vegetat,ive cells. The
‘I’ARLI~: I)EVELOPMENT
AC~UITY
OF
II
I’ROTEIN
IJURISG
t‘stmcts
SPIXTHESIXING
OUTGROWTHS Phenylalanine incorpzrated (~pmoles,fm~ ribosomal proteini -poly
spores I)orrnsnt I feat-activated spores (:erminatrd spores 5 mitt IO min 20 mitt 40 mitt Yegetativc
cells
9 9” 6.4”
u
+tloly
u
23
GG
38
4262 3550 5210 4438
88
9185
50
u Reaction mixture contained, in fimoles/ml; 45 Tris, pH 7.8; 10 magnesium acetate, JO NI14C1, :iO KCI, 0.2 GTP, 0.8 4TP, 4 PEP, 13.6 rg PEP kinase, 40 rrterc~aptoethatlol, 1 sperimiditte, 0.01 amino acid mixture, 0.02 L-phenylalanine-1X (10 pC/pmole), tiO0 pg R. cereus sRNA; and, where indicated, 100 pg poly I-. Total volume was 0.5 ml. A quantity of 55 rg of t,he supernatartt fractiotr (S-105.Sj and 23 gg of ribosomal (W-Rib-S) protein per 0.5 ml of the reaction mixture were rtaed. Samples were incitbated at 3G” for GO mill~ttes, and the hot trichloroaretir acid-ittsolttblc radioactivity was determined. ‘j These values are a limit of detect.iott, arttl, I twrefore, for all practical prtrposw, are zero.
in dormant and hea-activnted spores was less than 10 ~~moles per milligram ribosomal protein, and approximates the level of detection of radioact’ivit> in t,he zero-time control, and is, for all practical purposes, zero. This inactivit). might be due to (a) the nonsnturat~ing :mount of mRNA in t,he spore system, (b) defects in the prot,ein-synthesizing syst’em, or (c) the presence of inhibitor(s). To test t#hefirst possibility, phenylalanine incorporntion nxs examined in the presence of saturating amount of poly U. As shown in t,he second column, t,he addition of poly I!’ to the system from germinat#ed spores or \:eget,ative cells increases phenylalanine incorporation approximately IOO-fold, but, the system from dormant and heat +&v:tt,cd spores responds very poorly. These findings indicate th)at the dormant spores, in :tddit8ion to t,he apparent lack of mRSA, have a defect’ive protein svnt h&zing systc~m. There is ;i dramatic ‘irlincorporation
crease ill amiuo acid incorporating activity. during the transit’ion stage from dormant spores to germinated spores. Five minutes after t’hr init,iation of germination, the q-stem responds to the added pol;\r IT. Is this change a11 activatjion or a rle HOW synt’hesis of some component of the incorpornt ing system? To test, this, spores were germinated for 10 minutes in the presence of 100 p,g chlor:Lmpheuicol/ml and 25 pg actinomycin II/ml to inhibit prot.ein and RN4 synthesis. Cell-free extra&s wercs prcpnrcd and amino acid incorporating &ivity ~-as measured. Table III sl~ows th(L results. The addition of the nnt,ibiotics did not, inhibit the appearance of the incorporat ing activity-, clearly she\\-ing that the appearawe of t,he activity after germination is not due to the synthesis of some component of the system. When spores QW~ germinated in the presence of chloramphenicol, higher incorporating :Ictivit,ies ww observed. It is ~~11 known (15) that, wider these conditions, I
the amino acid incorporating sgst,em in sI )orw, it is first necessaq to determine whether the characteristics are identical to those: from vcget:ltive cells. Table IV
118
s-30-s s-30-s
(CM)
s-30-s
(CM + Act. I)1
338
X8
‘I Extracts (superrtatant fraction was obtained by certtrifngat.ion at) 30,000 g for 30 minutes aittl then dialyzed overnight against standard buffer) were prepared from the spores germituted for 10 minrttcs itt the nbsenw of antibiotics (S-30-8); it1 t,he presence of chlor:tmpheilicol, lOO&ml,S-30-F (CM) ; and chloramphenicol, 100 pg/tnl; and HCtinomycirr I), 25 rg/ml, S-30-S (CR1 + Acti I)), respectively. For inctlbat ion condit,ions see Table IT. The zero-timp valt~e was sttbtrapted frl)m all samples.
TABLE
I \.
:&minct illCorI)or:LtioI1. Amino acid incorpcnw tion :rt, :31” continues more than 1 hour in both systems. Table V compnrcs other charxturistjics oi’ polo. Cstimulnt,ed phenykkmine itl-
I:syl.”
1 Complete minus NII,Cl minus GTP rninlls ATP, PEP atltl pyruvnte Itimtse rninlls merraptoet hnnol minrls spermiditre
4.3 2.6 2.1 0.57
10.X
4.5 2.t;
x,.5 7.8
2 Complete minris spernatant minrls rilwsomes
3 .3 0 0.26
10.8 0 Il.28
3 Complete minus sILNA minus poly U minus amino acitl mixt we phw ribonuclease plus deoxyrihonnclease~
3.0 0.8 0.04 3.1
8.i 0.1 0.1 7.x
0 2.5
0 7.6
6.9 X.6
0
li For inclbtion conditions see Table II. Total volume was 0.25 ml. The germinnt ed sport system contained 44 rg srlpernatarit protrill (S-105-S) and 20 ~g ribosomal protein (W-RibS). The veget at ivcl s!-stem contained 80 pg prcilrculuted s~ipcrl~atnril protein (Inc-9-105-S) and 48 rg rit)osom:il proleilr (Inc.W-ILibS). h J)ata from (7). c In both systems 10 pg rihol,rtclease or 40 pg deoxyribolillrle;se/ml rcac t ion mixtrlre were 11sed.
requirements of poly Udependent I-‘hen?-l:tlaniIle-‘4C incorporntion into prot,ein of germinnt~ed cell-free extracts and extracts of vegetative cells. The amino acid incorporat,ion is dependent upon ATP, the suJwrt~:tt;u~t, fraction, rihosomcs, sllNA,
summarizes
and
poly
t,hc
Ij.
The
systcrn
is
sensitive
t,o
ItKase but insensitive t,o DN:wc. Omission of SH ,Cl, GTI’, or qermidine led to :L loss of :lbout~ 30 50 ‘:; of the incorpor:Lt,ing :ICtivity. These those obscrwd CCllS.
ch:w:lct.cristics in extracts
are
similar
from vegct~ativc
t,o
TIME (MINI FIG. 1. l‘imr cotwse of ami11o acid incorpora1ion of cst rcwts from gcrmiri:tted spores (---j nntl vpgctitt ive cells (~ ). For incllhation cotkdit ions see ‘l’:tl)le II. Tot al vol~~mr, 0.25 ml. l’oI,- I-, 25 pg, was :tddcd. (;errniriated (5 mitlrltes) spore sgstcm contained 48 pg S-105-S protein :clrd 20 ~g ItIc-W-RilbS proteill.
Optimal bIg3’ cotIreIItratioil (mu) Optimal rewtioll tenpernture (“C) ITalf-sntrrrated cont. of sRNA (rg/ml) Half-satrlruted conic. ol poly li (hg/ml)
11
!I
3i
3li
ItiX
13ti
“4
32
Conditiotls are the same as in Table II. Total vollmrc, 0.25 ml. Germinated spore system cow tail& 44 pg sllpernnt ant fractiotr (P-105-S) and 19 pg ribosomal (W-Rib-S) proteins, alltl the vegetative cell system contained 48 pg prrillcubated srlpernntant frwtion (Inc.S-105-S) alIt 21 pg rit)osornal ITn>-W-Ribs) protrill.
I)P:F15CTT\~E t’t:OTETN
I )orma11t spores
+ + + +
+
f + + + +
+ +
+ + + + +
+ + + +
SYh’THE$TS
10700 32 2192 I(iifi 3)
7313 10530 6177 28 12 110
--c +
+
+
932 3172
+ +
131-H) 13263
+
6530 6380
+ + +
+ +
+ +
f
+ + + + +
+ + + +
+ + + +
9604 10634 14729 1x14 20030
CLConditions are the same as in Table IT. Total 0.5 ml. 55 pg of the supernatsut fraction (S 105-S) alld 23 JL~ribosomal (W-Ttih-8) protein per 0.5 ml of the reaction rnistru-e wew l~scd iI1 each case.
vol~nnc,
(i'li
corpor:~tion of t’lic germinated sports anti the vegetative ccl1 systems. The optim:ll magiicsium concentration and t8he 0ptim:il temperature is the same for hot11 xyskms. The s~xtem obtained from germiii:~t,~~tl sporw is heat-1:ihilc. At Go, the germinated spore sys’tem does not show any phenylalanine iticorr)or:tt,iorl into protein after :I I-hour incl~bntion. The concentration of 16 sRXr\ or poly I’ required to half-~:aturntc~ 2-l 21 amino xid incorporat,ion is a~)l)rosim:Ltcl?. the same in the two syst,ems. Thcreforc. 2O(i5” t,lie two nniino acid incorporating syslein~ l-1 arc essenti:illy t,hc wnc. If~towlo!/ciuts
cw/rbimlior/
f,.cl,ei,i/,lrrrls.
‘I’0
examine tlw loc:lt8ion of the defecb iti thr, incorpotxti~ig s\-st em of dorm:mt sporw, t,hr SllpFrll:~t:lnt and ribosomc fract,ioiis f’rc~nl Ihe dormant, Iic:tt~activnt,ed :urd gcrminnted sporw and from the vegfkt iw cells ww prqxux~l and tested in a scrics of liet,~w~logous comhiii;ltiolis. I’hc~rl~lal:~~~ii~~~ incorporatioil
HeaL-activatcd sp~‘rrs
IN SPOT:I<::
into
protc~in
wts
incxswwl
the J~YSY~~~ 01’ poly I! (Tsblc VI). spores hve 311 Nibosomcs fronl tlonnant~ extremely low activity. The sup(wat:tnt fraction from dormant spores is inactive and slightly inhibitory on the voget:ltive systen1. A\ftjer ~lc:lt-:ict,iv:rtiolI, ~porc rihoaomex have 10 20 ‘7 activity of the activity of ril~osomw from vegetative cells. Supernatant fix*lions from lifat 4ctiv:Ltetl sporw nw eclu:rll,~ inactive. .\ftcr tlw irliti:~t,io~l of germinntio~l, spore rihosoni(::: accpir(~ 60 “; of the :LctivitJ of t tic ribosomw from vcgetntiw cells. The supcrnatnnt fraction :tlso increww its activity to ahout 60 Y;’ of the supcrnat:uit fr:lct,ion from vcget ative cells. These results show clearly that the defects in the ~LnlilHJ acid incorpor:&g System in the dorma~lt sporw exist lwth in tlw s\lpcrn:itant fix+ tion 2nd the ri~~osomcs. irl
K?s
KOBAYASHI
AND
HAI,\‘OR.SOri
During heat-activation, activated. Amirm
acid
accepting
this ability
enzyme
is
of sKNA
pj~epa?~ed fwtja dormant spo?‘es.We determined whet,her sRKA from dormant, spores has t)he same amino acid incorporating ability as sRNA from vegehative cells. As shown in Fig. 4, dormant spore sRNA has a slightl?, lower activity for phenglalanine than sRNA from vegetative cells at low concentration; but at suturat,ing concentratjion, t h(, activity is almost the same.
a ifi07 b z
8or-----0
0
IO TIME
20
(MINI
P’IL;. 2. Time course of phenylalauyl-sKiYA synt,hetase from the dormant, heat-activated, aud germinat,ed spores aud vegetative cells. Extracts (SF II) were prepared by the method of Wood and Berg (9). For incltbatioll conditions seeRIAw~iur,s h~u -\b3rHODS. SF 11 were from: 1, dormani spores; 2, heat-activated spores; 3, spores heat-act,ivated in the presence of chloramphenicol; 4, germinated spores; 5, spores germinated ill the presence of ~hloraInph~rlico1; atrd C,, vegetative crlls.
sRNA synt,het,ase uct’ivitjy in t’he supernst,ant fraction from dormant, heat-activated, and germinated spores and vegetative ~11s. As can be seen, t’hc enzyme activit,J was essentially the same in each cast. The presence of chloramphenicol during hcatshock or germination does not have my effect on the appearance of enzyme activity. Vigure 3 shows the t’ransfer enzyme act,ivity in the supernatant’ fraction from these same extracts. The supernat’ant, fraction from the dormant spores has almost while heat-activxt’ed and no activity, germinated spores have high activit?. The presence of 100 pg chloramphen~coljml during heat-activation or germination does not affect, t,he appearance of the trnnsfcl enzyme. These results indicate that the defect in amino acid incorporating act,ivitJ of t,he supernatant fraction from t,hc dormant spores is in the transfer enzyme.
0
30
60
TIME ( MIN 1 F’lo. 3. Time course of transfer enzyme from the dormant, heat -activated, and germinated spores and vegetatiw cells. Extracts (SF II and rihosomes) were prepared hy t,he methods ol Wood and Berg (9). Twellty-folw pg of SF II, l.GO O11260 units of rihosomes prepared from the vegetative cells, and 1.80 01&,0 lulits of phenylalanyl-‘4C-sRNA (1200 c’pm) were used. For incubation conditions see RIATEIUALS AND METHODS. SF II were from: 1, dormant spores; 2, heatactivated spores; 3, spores heat-activated ill the presence of chloramphenicol ; 1, germinated spores; . sports germinated J, iIt i he preseucr of chloramphrnicol ; and G, vegetative rells
100
0
200
sRNA ( yg/O
300
400
500
I ml react mixt 1
FIG. 1. l’liet~ylalanine acceptiflg ability of sRSA f’rc~rndormant spores (0) and from vcgetat ive wlls (0). Soluble RX.4 was preparetl as ConditIescribed ill ~\IA~:ILI.~LY Am NE’~H~I)s. 1iolw for phenylalanine loading are the same as for phellylalx~~yl-sR~;A synthetasr. Sllpernatant fraction (SF II) from vegetative wlls was I~se(i. I~~c~llhatetlfor 20 mitnlites al SC,“.
To csamittc~ the nature of defects in rihosomes from spores, the characterization of rihosomes from spores and vegetative cells \v:ts undertaken. Cell-free extracts (S-30) \vcre prepared from dormant, heat-activated, :rnd germinated spores and vcget,ative cells :md :malyzed by analyt8icnl unttxccntrifugatiott
in lo-” nt AI@!+. Figure
5 shmvs
thr
of t.hc ribosomal particles dist~rihutioti during growth. As can he seen, vegetative ~~~11scontain four classes of rihosomal p:trt~icles
chwracterizcd
by
scditnettt:ttiott
constants of roughl>- 30, 33, 70, and 100s; the 30s and 50s particles arc minor cornpottents. The distribut’ion pat8tern of ribosomal particles in sporca is quite different from that of veget,ative cells. Sports contain three major particles, namely, 30, 50, and 70s. Thcl relative txtio of these particles tlocs ttot change in dorm:m~, heat-:lct,iv:tt’cd, 01’ gwtnitt:tt(~d
(6 minutes)
qmw;
hut
LX)
minut,es after the itiit~iation of germiti:ttiott, the relative amounts of XOS and 50s ittcrease. The :3OS and 50s subunits csist frw at higher magnesium coticetitrat~iott (0.05 11) and do not) form TOS ribosomcs. I’oly Kdcpendent phet~ylalnttitte incorporating activity of these rihoscrmal particles was cotnp:wed after fractionation b\. sucrow densit,?--gradient’ centrifugation. l‘he r~‘suits arc shown in E’ig. 6. Parts 9, 13, and (’ are rihoaomes from dormant and germinated sports, and vcgetwtivc cells, I’(‘xpcctivelg. Sittw crudr cstracts front dormant sports con~aiii large :Imoutits of ultraviolet-ut)sorbitlg material (III’A), th(l 30s suhuttit~ is not, shown clearly. As can bt> seen, tteit’her SOS nor 50s p&iclcs have att~ atnitto acid incorporating activity. The 70s ribosomes from dorm:ntt spores have sonic’ :tct’ivitJ.. Aftw the initiation of germitintioti, 70s ribosomcs become fully active, and the specific activity (cpmj 01&) is about twofold higher than that of vegSet:tt,iw crlls. This ittcreaw is not t,hc result of r/r UOZYJ syttt-,hesis of IWW ribosomtrs sitw t,hti rihosomw from spores germinated in the
w w
w
dormant
spare
heat-activated
11
germinated 6 min
11
germinated 20 min
11
630
KOBAYASHI
AN11 IIAI,\~ORSON
FRACTION
NUMSER
FIG. 6. Phenylalanine incorporat,ing activity of ribosomcs fractionated by sucrose derlsity-gradient centrifugation. Crude extracts obt,ained hy sand grinding and centrifugat.iotr For condit,ions at 10,000 9 for 10 minutes were layered on linear sucrose density gradiellt. see MATERIALS AND METHODS. Two-tenths ml of each fraction was added to the amino acid incorporating system described in Table II. Forly-seven pg of the preincubated sllpernatant fraction (Inc-S-105-S) from vegetative cells was used. Total vollune, 0.5 ml; incubated at C, 36” for 1 hour. A, Dormant spore extracts; B, germinated spore (20 minutes) extracts; vegetative cell extracts.
presence of chloramphenicol, have similar activities (Table III). The high specific act’ivity of ribosomes from germinated spores could be explained partly by t,he absence of natural mRKA in spores making a binding of poly U much more effective in ribosomes from germinated spores than from vegetative cells. The apparent’ defect observed in the heterologous combination experiment (Table IV) in ribosomes from germinated spores is due to t,he existence of a large amounts of nonactive 50s and 30s subunits in spore ribosomcs, while ribosomal particles in vegetative cells are almost exclusively active fOS and 100s ribosomes. Several at,tempt,s have bx~~ made to correlate the biosynt,het,ic activity of spores following outgrowth \vit)h the maturation of t,he ribosomal system. Only 50s and 70s particles have ‘been observed in spores of Bacillus subtilis (16) and Bacillus meqatwiuw (17), but, as reportjcd here, spores of
B. cewus strain T cont,ain 30, 50, and 70s pnrt(icles. Woese et al. (16) observed that during out,gron-t,h of B. subtilis spores, both ribosomal precursors as well as polvsomes were formed. In t,he present experiments, only the 70s part,iclcs in germinated spores are fully active in amino acid incorporat’ing activit,y. Prior to the onset of protein synthesis it1 vim, these must either be derived from inactive 70s part)icles or from the conversion of 508 and 30s particles to active 70s particles. From quant,it,ative considerations it appears that t,he rat,climiting step in protein synthesis during outgrowth is the synthesis of mRNA (1). Nature oj the defects in ribosotnes. What is the nature of the defects in t’he ribosomes from dormant spores? Since active ribosomes bind both aminoacyl-sR?CA and pl~er~ylxlanyl-14C-sRNA binding mRNA, with ribosomcs from spores and vegetative cells was compared. Ammonium sulfatetreated ribosomes were prepared according
0
IO
20
RlbosomesC 0Dxd0 I’llr,lr)-l:tlau?-l-sl(Nh
25m1)
bilrtlilrg
with rit)o-
632
KOBAYASHI
ANI)
P. C., Biochim. 11. SARIN, P. S., AND ZAMECNIK, Biophys. Acta 91, 653 (1964). 12. STEPHENSOX, Xl. L., AND ZAMECNIK, P. C., hoc. 1Vatl. Acad. Sci. U.S. 47, 1627 (1961). 13. LCWRY, 0. H., ROSEBROUGH, N. ,J., FAJ~R, A. I,., ANI) RANDALL, R. .J., J. Biol. Chom. 193, 265 (1951), 14. FJTZ-JAMES,
teriol. 15. HAHN,
P. C., AND YOUNG,
78, 733 (1959). F. E., in “Biochemistry
I. E., J. Bac-
IIALVORSOX (Ii. H. Spitz>- and R. Brutmer, eds.), p. 101. I’ergamon Press, Londo~l (1959). 1G. WOESE, C. Ii., LANGIUDGE, R., AND MOIU)\VJIX, H. J., J. BacleCol. 79, 777 (1960). 17. CHALOUPECKY, I-., Folia .Ilioobiol. 9, 232 (1964). 18. STEINBERG, W., HALVOI~S~N, H. (I., KEYNAN, A.,
of Antibiot,ics”
AND
(196.5).
WEINBERG,
E.,
>\‘nlure
208,
710