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Ribosomes,

md~da~ / ~v~

9~

P~s

bosom subuni and bosom of Lactococcus c6s IL1403

protons

N Limas N z o u ~ , M F Guefin, D H Hayes* Labomtoi~ ~ C h ~ Cellu~ire, ~ t u t ~ B~log~ P~Mt~-Ch~ique, 13, rue ~.et-Marie-Curie, ~ 5 Paris, ~ w e

(Recover 10 ~bma~ 1992; acceded 25 June 1992)

Summary - The preparation of ribosomes and ribosom~ subuni~ of Lactococcus lacHs and the characmriz~ion of the proteus of the subuni~ by one- and two-dimenfional polyacrylamidegel electrophore~s and ion exchange chrom~ography are descfibe~ rib~ome / pmte~ I electrophorus I ~n ~ a n ~

~mm~p~

~tmdu~n ~ S ~ of ~ e c o m ~ a b ~ econom~ impo~ance cf the ~cfic ~ b ~ f i ~ ~ r geneti~ and m d ~ ~ o g y ~ m ~ n ~ l a t ~ e ~ undeveloped. For examO~ • e phy~genetic ~ l a t i o n ~ s between ~ e membe~ of ~ g ~ u p of b ~ d a are ~ bring ~tabl~hed [1-8], and a cu~em c o m p l a i n (EMBL GenBanL June 1992) of eubac~ri~ dbosom~ RNA ~ q u e n c ~ ~ S rRNA, 266 ~quences; 16S rRNA, 836 ~quenc~; 23S rRNA, 135 sequences) com~ns v e ~ ~ w ~ s d t s concem~g Lactococci, Streptococci and P e ~ o c o c d ( ~ q u e n c ~ of 5S rRNAs of two Lac~cocci and one Streptococcus, cf 16S rRNAs of fix Lacwcocci and 13 Streptococci, and of the 23S rRNA of one Streptococci) (~quences of 16S rRNAs of seven Pediococci published in 1990 [8] appe~ not to have ~een deposited in the EMBL GenBan~. To ~ c ~ e ~ e fimited ~ r m a t i o n p ~ n t l y availab~ coccem~g the m ~ e c u ~ r ~ o g y of the lactic ~ b ~ r i a we have begun a ~udy of the prote~ sy~hefis a p p a r ~ of Lac~coccus ~ s ~ ~ctis ILl403. The d b ~ o m e and f i b o ~ m f l p r o t ~ of t ~ s ~rain a ~ charactefised [ e ~ and a detailed a n ~ of the p d m a ~ ~ m c m ~ of a DNA segment c o ~ n g one of hs fix dbosom~ RNA tran~fiption u ~ w i ~ both flanking ~ o n s wi~ be punished d ~ w h e ~ . *Co~e~e

~ d repfi~s

In ~ e course of ch~ac~fis~g the ribosom~ pro~ s of L ~ a ~ ssp ~ IL1403 ~ e y w e ~ compa~d by o n ~ m e n f i o n ~ d e ~ r o p h o ~ s with those of ~veml o ~ e r ~r~ns of Lacwcoc~ and it became evident • at ~ e fibosom~ protons of several of ~ese ~rains p~sen~d smfll b ~ sig~ficant ~ f f e ~ n c ~ in electrophoret~ behav~u~ R t h e ~ f o ~ ~emed possible ~ e~cgophoret~ comparison of theft ribosom~ protons might be a use~l ~chn~ue in ~ud~s of the ph~ogenetic ~lationships of lactic ~cid bacteri~ These an~yses w e ~ ~ e ~ ex~nded to the genera Streptococcus and Pediococcus and ~ ~ e accompany~g paper m t~s aaic~ we show ~m comparison of deetrophoret~ ~ f i b u t i o n p ~ m s of fibosom~ pro~ins of Lacwcocd, Strepwcoc~ and P e ~ o c o c d can eomp~me~ ~ehn~ues such as DNA-DNA hybrid~ation, and comparison of ribosom~ RNA gene ~stficti¢n pm~rns in ph~ogenetic studie~

M a t e r , s and m ~ h o d s Bacterial s~ain, ~lture, m a i n t e n ~ and M b ~ g ~ n ~ L ~ctis IL1403, ~ m ~ free, ~c-, p m ~ e - , re~dcfi¢nffmodification- (sff~n ~7m ~ l~) w~ o ~ n e d from A Chop~, INRA, Jou~en4os~, France. Cells were ~own ~ 30°C wi~om agitation ~ med~m MITA (compcs~on ~ g/l; ~ p ~ o n ~ ~ ~ o s o y ~ 5; yea~ ex~acL 2.5; beef e~m~, 5; Ouco~, 1~ KH:PO~ 1,78; N~HPO~.2H:O. 9.55; MgSO~. 7H~O, ~24~. This me.urn, derived ~om med~m Mi7 [101

996 buffer 3, recen~fuged un~r be s ~ e cond~ons ~ield 1~ mg wet paced ~lls~ and i ~ e ~ a t e ~ ex~act~ ~ ~ s c 6 ~ ~w. l s o ~ n ~ ~bosomes a ~ riboso~i subun~ ~H operatio~ at ~ o ~

~ e 6~ewd~ l a ~ e [1~d s ~ t a % ~ v~V~e clugs5 ~r ~d/~g~Y~~ nenfi~ growb ~ a defined synbefic me.urn [11] developed during s ~ e s with the pro~zoa Tetrahymena ~ermopM~ whichIL1403 tis w~ ~ d d t ~ ll~dmb~,a~t, i2v~e~7w~,8°~ ~ : ~ log ph~e). Chemica~ and other produc~

~ail~n~°~reo~b~s~uYxe,~tral~a~d grade beef extraetsuc~seWerewas ~ ~sUA~ A~a~t ~ l~h.~yl.e~ie~~ i ~ Eastmano~ned from ~uk~ Swi~edan~ and E~tman Kodak, USA, ~spe~ive~ were ~c~allised onc~ All ~her ~agems were analytical ~ua~]~ fa~nr~oProlab~ac ~ mi~u~France(l~5GBqoMe~k,/miliatom r -Germany" car minations were from Boehringer, German~ purified E co~ ribosom~ proteins S13 and S15 and t ~ protons of the 40S ribosom~ subu~t of Tetrahymena ~ermophila were gifts from Dr F Hayes. Bz~ers

I) Tri~HCI, 20 raM: MgCI2, 20 raM: NH4CI, 60 raM: 2-me~ captoe~an~. 6 mM (pH 7,4). 2) T~s-HCI, 20 raM: MgCI2, 20 raM: NH~CI,60 raM; ~thiot~eitol, 1 mM (pH 7.4). 3) Tris-HCl. 20 raM: MgCI~ 2 raM; NH~CI, 60 raM; 2-me~ ca~oe~an~, 6 mM ~pH 7,4). 4) T~HCI, 20 mM~ MgCI~ 2 mM~ NH~CI, 60 raM: ~thiothre~, 1 mM ~H 7~). 5)6 ) NTri~HacCel~al~~ f f l ~ a ~ ' ~n~; ~ D m ~ t % ~ t / ~ ~.4m)~! (pH 5 ~ Preparation of bacwria Un~belled bac~ria

Ceils were harvested ~om m ~ a ~ log phase c~mres by convem~n~ ~-101) or c o ~ u o u s flow (~151) centrifugation ~ 0--4°C, resuspended in ~n times the~ w e ~ of buffer 1 ~ cuhure. ~C Labelled bacteria

• 25 ml of an exponential~ growing culture ~ med~m MiTA was used ~ inoc~a~ 50 ml of s y n ~ me~um and after gmw~ o v e m ~ (A42o.~,1~) ~25 ml of ~ c~mre was added m two prewarmed (30°C) 5~ml aliquots of ~ h e t i c me.urn one of wh~h coma~ed 20 MBq of a mixture of [Ut4C] amino a c ~ Gmw~ was fol~wed by measuremem of A~2~ in the non-m~oacfive c~ture and when a v~ue of ~8 was reached c~ls were harves~d from ~e ~dioacfive c~ture by centri~gafion ~0 000 ~ 5 rain. 4°C) resuspended in 5 ml of ice~o~

UnShelled c e ~

~ h ~ o n mebo~ were u~& 1) ~ o n ~ separated ~ e and de~ed fi~somal s~un~. Frozen bac~fia (~30 ~ were gro~d ~ b ~5 t~es ~e~ w~ ~ ~ u ~ n a u~fl a fl~d pas~ w ~ obtained ~ m ~ ~e~e~xmrepa~eWascen~fug~d~ute~b~ d ~c~d000 ~ 4°C forbUfferI ~ ~ 1 ~ o p ~ c e ~ s u ~ m a ~ t c~lec~d (~2mVg c ~ A~m 1 5 0 - 2 ~ Ri~somes and free ~ s o m ~ s ~ u ~ s were ben ~ . m a t ~ i ~ cen~fug18 aofin~000 rev/m~ ~ l~'~for16 h~s(mtorS~We~x ~on t 33.~3~ 5~70 ~1~30% inearofex~actSUC~at~ ( ~ n ~ v ~ r

i ~ ~

or~torOf

tions c o m ~ n ~ ~ e 30S and 50S subu~ts ~ v e or H s~u~t~ see R e s u ~ , and 60S + 7 ~ g~somes were p o o ~ ~ne~col w~ added ~ be poo~ (10% w/v fin~ concenwat~ ~ d precaution was ~ w e d ~ ~ d ove~ ~;~) ~l~di~~ ~ c 'adconcen.tion - - ~ cen~fugati°nof100-2~ (~ 000 A ~ .~ unit ~ r ~ ~ buffer ~ 30S ~ d 50S subunits produced ~ ~ss ~ i ~ o n of ~ S ~ d 7 ~ ri~somes under bese condemns ~fived or D subun~ ~ e Resu~ were ben separated ~ ce~°n~n~ons~ bese ~ ~ ' ~ l e ~ ~ ~ 16o~)hon~33.~ sus"

10-30%Gmdi~eninearwerecN~c~SUCm~~aca~o~ain..pa~em~di~socbi~t~r30~ and 50S subunits were ~ the~ M ~ concen~ation ~ d to ~ raM, p r e c i s i o n ~ b p ~ e b ~ n e O ~ carried o~ as b e ~ and recovered su~nim we~ resus~nd~ in b u r r 4 ~ ~ n~ ~ ~ ~ ~0-a n~%~ ~ ~ b ~ s P ~ ~ 1~ u ~ s ~ ~i~ne~

~

~

r

f

i°~s~

~ { ~ ~ t ~?or~ondc~t~~ ~f~m~0~!~ = uni~ ~ I s o l ~ n ~ mix~ free and ~ v ~ ~ s o m ~ subunits. Crude c~l exeacts were p ~ p ~ d ~ a~ve u~ng ~ffer 3 ins~ ~ ~r 1, and fractionated ~ ~menmtion ~ i ~ - ~ ~ s ~ 3 ~ A2~m un~) ~ ~ 000 rev~in for 16 h on ~-ml 10-30% finear su~se grad~ms ~ p ~ d ~ buffer • Un~r • ese cond~ons 6 ~ ~ d 70S particles ~ diss~iat~ and free and ~ 3 ~ and 50S s u ~ se~mem togebe~ Grad~n~ were ~acfionated ~ d s u b u ~ were p r e ~ t e d and p~fied as desc~bed a~ve for ~ su~ ~p~t~ns ~ned ~ ~ ~Hw~be 2 mMre~d m N~m ~M in asg~ o r 3 and M subuN~" 4 replac~ ~ • 1 mM M ~ I (me~od ~ or 10raM MgC~ + 4 ~ m M N ~ I ~e$~ ~ Labelled cells

Peflets of ~4Clabeled cells (~ 0.1 g) were ground with ~umina as above and the resulting pas~ was dflu~d w~h buffer 4 (2 ml), centrifuged, and the supematant was collected as before and fracfionated by centrifugation ~ 23 000 rev/min for 16 h on a 33-ml 10-30% linear sucrose gradient in buffer 4. Gradien~ were collec~d in 30-32 ~acfions, the ~4C con~n~ of which

997 were measured by ~intiHation counting (10-~1 samples). Fractions containing 30S and 50S subunits were po~e~ and • ese par'tides were precipi~ted with polye~yleneglycol as before, resuspended in buffer 2 (100--200~1) and stored ~ -700C. The spe~fic act~iw of purified subunits was =

105 cpm/A~o,m.

Ammon~m chloride washing of ribosomal subunits (a~ operations at 0-4°C)

Puttied free 30S subunits were suspended ~ a concen~atinn of 100 A~o,m units per ml in 20 mM T~PHCI, 20 mM MgCle, IM NI-~Ch 6 mM 2-mercaptoethand (pH 7~) and ~e suspension was be~ m 4°C for 16 h before centrifugm~n for 5 h m

Preparation of ribosomal RNAs (aft operations"m 0--4"C)

~pfi~ e ~ ~ ~r c ~ a l ~ g 6 M u~a and al~wed m s ~ H to ~e~ ofional ~ be~re ~ i n g de~m-

~re~ Ion exchange chromatography of ribosomal proteins (Aft buffers filtered through 0.45 ~m membranes before us~ column elution buffers degassed after filtration.) Total pro~ins of 30S and 50S ribosomal subani~ prepared as described for two-dimensional gel elecffopboresis in [12] were dissolved in buffer 6 at a concen~ation of 3-5 mg/ml. The solutions were filtered (0.45-~m membrane), dialysed for 2 h again~ 100 vol of buffer 6 and their protein concen~ations estimated by measurement of A2~0,~. Samples containing 4-8 mg of pro~in were passed onto a MonoS-HR 10-10 column (Pharmaeia) which was then eluted at a flow rate of I ml/min with a linear gradient generated from 20 ml of buffer 6 and 20 ml of buffer 6 cont~ning 1 M NaCI. Elution was monitored at 230 nm and 0.5-ml eluate fractions were collected.

From ~tal cellu~r RNA

An ~umina ex~act prepared as described above in buffer 1 (7 ml, from 1 g cell~ was deproteinised by tremment with ~1 M and ¢thanol (2 v ~ recovered by centri~gation and ~ s~ved in buffer 5 m a concenffation of 200 A~0~ un~s/mL (Yie~ 300-400 A2~munits per g cel~.) Ribosomal RNAs were separated by centrifugation on 33-ml 5-20% finear sucrose gradien~ made in buffer 5 (SW28 rotor, 26000 rev/min, 16 h),

',

Resul~

R~osomes and ribosomal subuni~ of L ~ctis IL1403 Cent~fugation o f crude extracts o f L ~c~s IL1403 p ~ p ed in buffers c o m ~ n g 0 . 1 - 2 m M MgZ÷ or

~ved in buffer 5 m a concen~ation of 100-200 A ~ u~ts/ml and stored ~ -70"C.

From separated ribosomal subunits

Suspensions of 30S or 50S ~bosomal subonits in buffer 2 were deproteinized and released RNAs were pre~Otated, dfie~ ~dved, and stored m -70°C as above.

Reassoc~tion of ribosomal subunits M~mRs of separated s u b u ~ (one Az~m 30S + two Ae¢p 50S) ~ buffer 1 (1 ml) were incubated at 40°C for 30 mm co~ed to 0°C, and ~yered o~ 33-mi 5-20% finear sucrose grad~nts made in buffer ~ After centrifugation (16h, 4°C, 17 000 rev/min, rotor SW28) ~e ~stribufion of UV absorbing material ~ ~ e gradients was recorded and the percentage of inpm Az~,, se~ment~g m 70S was calculated.

E ¢

10

Polyacrylamide gel electrophoresis of r~osomal proteins Pro~in samp~s for one- and two-dimension~ de~ropboresis were prepared as described in [1~. On~mensional SDS/po~acrylamide gel ele~rophoresis was carried out according m Laemmli [ ! ~ as modred in [14]. For t w ~ m e n ~ o n ~ elec~opho~s~ ~e me~ods of Wada [15], Kal~chrnidt and Wittmann [16] and Zinker and Warner [17] were used, ~e firm without mo~fication, the la~er twe molded as described in [1~ (2 x 300 x 300 mm second ~mension gel ~abs~ and [14] (addition of 6 M urea m elee~ode buffers in contact wi~ pro~in sample~ or first ~mension gels). For elec~ophoretic ana~sis of matefi~ in stained zones cut ~om one- or two-dimens~nal polyaerylamide gds two procedures were used:

r gzZT ?d

'

16 ~ ~ansfe~ed m sample walls of gel ~ab~, covered wi~ ~e

5

10

15 20 25 Fractions

30

35

Fig 1. Sucrose gradient fractionation of crude extracts of

998 10mM Mg ~ + 400mM Na + on sucrose gradients made in the same buffers g~es good yi~ds of dbosomfl subunits w~h segmentation coeffic~nts identic~ to those of E coH 30S and 50S pa~ides (eg fig 1, upper pand). Howeve~ subuni~ isolated under these conditions are only partifl~ funcfionfl (see be~w). A search for a procedure ~ d i n g fully funcfion~ subu~ts was therefore unde~aken and ~d to the method de~fibed here. Figure 1 (lower panel) shows the sedim e m n o n profi~ observed after centdfugm~n of a crude extract prepare~ as described in M a ~ r ~ and m~hods, from cells harvested from a s~wly coded m~-I~e exponemifl phase cuRure of L l a ~ IL 1403. Comparison of ~ e species present in peaks 1 and 2 w~h E coli ribosomfl subun~s (sedime~ation coe~ fic~nts, RNA contents: resuRs not shown) showed ~ m • ey were 30S and 50S pa~iOes. Estimation of ~ e sedimentat~n coeffic~nts of the species in peaks 3 and 4, assuming a linear relations~p b~ween migrat~n dh~nce and ~is parame~r gave vflues of 60S and 75S (very fimihr resuRs were obt~ned using isokinetic grad~nts [18]), and as expec~d matefifls ~covered from peaks 3 and 4 (MaWria~ and me~ods) both ~ssoO~ed into 30S and 50S pa~ic~s in the presence of 2 mM Mg2+. Peaks 3 and 4 thus cont~n two forms of the 70S ribosome co~espon~ng flmo~ c e ~ n l y , though this has not been demon~rme~ to the 60-63S and 70S E coH pa~ic~s observed by several authors [ 18-22] and shown by Noll et al [23] to co~espond ~spe~iv~y to naked ribosomes and ribosomes carrying a ~agment of me~enget RNA and a p e V ~ tRNA. R~osomfl subuni~ i s s u e d from peaks I and 2 (fig 1~ and those o ~ n e d by ~ o ~ n of ~ e mmerifl present in com~ned peaks 3 and 4 (fig l) will be refe~ed to hereafteu following Noll et al [24], as native, N, and derived, D, subun~s respective~ (N30S, NSOS, D30S, D50S), Reassociation of isolated ~bosomal subunits

Table I shows the resuRs of estimations of the capacRy of isolated native and derived ribosomfl subunits ofL lactis IL1403 to associate with each other in buffer 2, Since ~ has been shown that the capacity of ribosomal subuni~ to reassociate in the absence of other macromolecular componen~ of the protein synthes~ing system ~ a measure of their functionfl acfiery [23,24] we conclude that derived 30S and 50S subun~s of L lactis prepared as described here are fully or ~ m o ~ fully function~, Ribosomal RNA

RNAs prepared ~om ~olated ribosomal subuni~ of L lactis IL1403 possess the same sedimentation coe~ ficients (23S, 16SL as E coil ribosomM RNA~

L ~ ~ of ~ a t e d ~ o m ~ s~u~ 30S ~ d 50S ~bo~m~ ~ s p~pared u ~ e r ~e s ~ d conditions we~ ~ x e d ~ buffer I ~ d the m~m~s we~ ~d as ~ d ~ Ma~ria~ a ~ me~ods. ~e

Subun~ preparation me~od

1

2 3 4

% Reassociation

N30S + N50S D30S + D50S N30S + D50S D30S + N50S M30S + M50S 30S + 50S 30S + 50S

23-40 80-94 45-48 50-70 70-72 25-52 20-50

Number of preparations tested

4 4 3 3 2 4 4

E~raction of these RNAs from purified 30S and 50S ribosom~ subunRs ~ d s produc~ wh~h cont~n h~den breaks and are paR~ conve~ed ~ more s ~ w ~ ~ m e n t i n g materi~s by he~ d e n ~ u ~ t ~ n whereas the same RNAs prepared by deproteinizafion of a c~de c~l e ~ O and fractionation of totfl cell RNA on sucro~ g ~ e m s in buffer 5 are ~most com~etely refi~ant ~ he~ den~uration (resuhs not show~. Hidden breaks are ~erefore introduced into ~ e ribosomfl RNAs during purification of 30S and 50S subunRs. Simfl~ resets have been obse~ed earl~r ~5] for E coli 23S rRNA prepped from crude cell ex~acts and from h~ated 50S subu~ts. Proteins of de~qved 30S and 50S ribosomal subunits

Figures 2 and 3 show the d~tribufions of proteins of derived 30S and 50S ribosom~ subuni~ of L lactis in two of the two-dimensional electrophores~ systems used in this study. Use of the modification of the Kaltschmidt and WiRmann system described by Wada [15], which permhted the discovery of four previously undetec~d proteins in the E coH ribosome [26], reve~ed no L lactis ribosomal proteins in addition to those shown in figures 2 and 3 (resul~ not shown~ Some details of the results in figures 2 and 3 require comment a) Reduction of the duration of first and second dimen~on electrophoresis in the Kaltschmidt and Wittmann system [16] to one4hird of the ~andard v~ues showed that the subuni~ of the L lactis ribosome cont~n no proteins with higher first or second dimen~on mobilit~s than S10/ll $23 and $24; LI, L30 and L32. entb) Ainlarge high amm°~ls~nm~qs~0~ci~uP~e~Ssl~,~i can usually be seen in traces in one-dimensional SDS g~ dec~ophorefic an~yses of the proteins of D30S

999

A O

B

Firs! dimension gHs Acid~ O ~ Basic

e

I

I

First dimens~n gels Ac~ic e • Basic

I ~

I

0 I

~

Q,o

~3 .10 ~1|

. 9 ~

~

8

sg¢

2~

~22 ~+ ................................... im~+~" Im

~' ,~.,..,,

23

.i~lo~ ~, ++ -" - --~

~.

~N ~'

.

~:~a

~,"_

++30

.~.~

Fig 2. Separation of proteins of derived dbosom~ subuni~ of L iactis ssp lac~s IL1403 according to K~tschmidt and WiRmann [16]. Samples loaded on first dimension gels confined proteins derived from 5 and 7 A260n m units of D30S and DS0S subuni~ respectively. A. pro~ins of D30S subunits. B. proteins of DS0S subunits. Inset A lower ~fl: resolution of spots 20 and 21 by increasing the duration of fir~ dimension electrophoresis by 50%. Inset B lower ~fi: resolution of spots 10, 9+ 8 by doubting the duration of first dimension eleclrophore~s. Prefixes S ( s m ~ and L (large) used to distinguish 30S and 50S subunit proteins are omitted to fimplify the figure.

A (i) i

First dimension gels Acidic e • Basic I,. ,

o 2 .3

e I

a _(!) I

First dimension gels Acidic e • Basic ~

~ ~~'~. . .~r.~ ~ . .,~

-

~

2 ~ ,~, ~ ~

,o~o.,_ ~ ~, ~~ ~

O ,~

~] ~ ~

--

-

~o~

~ ~ ~

' ~~

al

Fig 3. Separation of proteins of derived fibosom~ subuni~ ofL lactis ssp lactis IL1403 according to Zinker and Warner [17]. Loading of first dimen~on gels as in figure 2. A. Pro~ins of D30S subunh~ B. Proteins of DS0S subuni~, inset B upper ~fi: resolution of spots I0, 20, 9 by doubling the duration of first dimension electrophoresis. Prefixes S ~m~l) et L (hrge) used to distinguish 30S and 50S subunit proteins are omitted to simplify the figure.

10(0)

505

30S

~ ~ Band

P~ein

Number Mr

A__,~I~_~:~ ~r

'1

51

B ..... ~

.2

32

Number Mr

Band

-~'

A

-3

32 28

B

26

4

.~.

8~-,,

8/

22

E

| ~ ~ ~

~,~

1~

N I

F~ m ~

14

18

~

13 ~,]~

16 l~,S

1~21 15,19 18,20

13 12~ 12

24

11

23 17~ 10,11,22

1~5 10 ~5

~

~

|~ | ~

0

12

15

~,~

24 ~

J

K L M~

! ~

~ I~,~ ~

Q T/

~

1~, ~ ~$ ,1~

1~1~,~

la

1,~'~23,28,32 111132

~27

10

~2625,3129

8~$9

Fig • One-dimen~on~ SDS polyacrylamide gel electrophores~ of proteins of derived ribosomal subunits ofL lacti$ ssp ~cti$ IL1403: correlat~n of one- and two-&mension~ migration pauerns. Suspensions of purified 30S and 50S ribosom~ subunits (1 and 1~ A~,m units ~spective~) in buffer 2 were mixed with 2 vol of ethanol, the mixtures were held ~ -20°C for 2 h and p~cipitated subunits were recovered by centfifugation, dried bfiefly ~ vacu~ ~suspended in Laemmli sample buffer (30 ~1), bored for 3 mi~ cookd and trans~rred to samOe wefi~ Electrophoresis was carried out as no~d in Mawria~ and me~ods. Figure 4 shows ~pic~ results. Pro~in bands observed reproducibly are identified by letter on the ~ h ~de of each paUem. To ~entify proteins present in ind~idu~ bands protein samples cozening 1-2 ~g of unlabelled tot~ subunh pro~ins and 10~-10~ ~s/min of individu~ ~ a b e l l e d proteins ~uted from ~ n e d spots taken from two-dimen~on~ g~ ~abs on which ~4C labelled tot~ subu~t proteins had been separated according to Kaltschmidt and Wittmann were prepared as described in [14] and an~ysed by one-dimension~ SDS po~acrylamide.gel electrophoresis. After electrophoresis gel ~abs we~ stained dried and autoradiog~phed (20-60 days). The results of a series of such an~yses are summarized on the fight side of each paUern in figure 4 together with estimates of the m~ec~ar masses of ~e species present in each ban~ These estimates were ob~ined by comparative SDS polyacrylamide g~ elec¢ophore~s of proteins of defived 30S and 50S subuni~ of L lactis IL 1403 and the following standard pro~in~ bovine serum ~bumin Mr 66000; ov~bumin, M, 45000; glyceraldehyde-3-phosphate dehydrogenas~ Mr 36000; carbo~c anhydras~ M, 29 000; trypsinogen, Mr 24 000; trypsin inhibitor, Mr 20 100; lactalbumin, M, 14 200; E coli fibosom~ protein SI~ M, 13 000; cytochrome c, M, 12 ~00; E coli fibosom~ pro~in S15, M, 10 100; total proteins of the 40S ~bosom~ subunit of Tthermophila, Mr 71 000-8~00 [12]. The fis~d v~ues are the averages of four determinations. The band de~gnated * in the profi~ of 30S subunit prowin~ which ~ absent in profiles of ~eshly prepared 30S proteins but accumulated during the~ storage ~ -70°C, probably corresponds to a transformation product of 30S protein.

1~1 subunits (figs 4A, 5A). ~ is flso usually v~ible in two-dimensionfl anflyses of these proteins, espe~ally in heavily loaded g d s such as those shown in figure 5E, G. The corresponding spot was present in the gels ~om which figures 2A and 3A were produced but was very faintly s~ined, hs position is indicated by an open circle in both figure~ c) Proteins $20 and $21 (fig 2A) and L I ~ L9 and L8 (fig 2B) are not resdved under standard conditions but can be separated w~h 1o~ of promins $23 and $24 (fig 2A) and L30, L31 and L32 (fig 2B) by inereafing the duration of fi~t dimenfion ele~rophores~ as shown in inse~ in the lower ~ hand come~ of figure 2A, B. d) Protein $23 seen as a well defined compact spot in figure 3A appears reproducibly as a diffuse weakly ~fined spot in the Kaltschmidt and Wittmann syaem (fig 2A). Protein L31 w ~ e h forms a weakly stfined but distinct spot m the second dimenfion front in the Kaltsehmidt and Wiumann sy~em (fig 2B) stfins very weakly in the Zinker and Warner sys~m appearing as a faint are, as seen in figure 3B. e) Spots L2, L3, LI2 and L13 in figure 2B appear not to follow the system of Kaltschmidt and Wi~mann in which numbering proceeds ~om ~ to right and top to bottom of gel flabs [16]. However, thek second dimenfion mobilities vary slightly in different gel slabs and in most cases those of L2 and LI2 are the same as or slight~ lower than those of L3 and L13, respectivel~ ~ The double spot in figure 3B cont~ning proteins L I ~ L20 and L9 is usufl~ observed as a tingle slightly elongated spot. The three proteins present are distributed from le~ to right as indicated in the figure. As seen in the upper ~ ins~ ~ee flso inset in fig 2B) they can be resolved by greatly increasing the duration of fir~ dimenfion de~rophorefis.

One-dimensional SDS.polyac~lamide gelfractionation of ribosomal proteins: correlation of one- and two-dimensional gel distribution patterns For purposes such as the comparison of ribosom~ proteins of different bac~ri~ sffains (eg accompanying paper) one-dimen~on~ elec~ophoretic separation possesses obvious advantages compared to twodimension~ procedures in spite of its lower resolving powe~ Figure 4 shows the separation of proteins of derived 30S and 50S subuni~ of L lactis IL1403 by one-dimen~on~ electrophoresis according to Laemmli. Fifteen and 20 bands are generated reproducibly by separation of the proteff~s of D30S and D50S subunits respectively under the conditions specified in Mawria& and methods. The proteins present in these bands, identified as described in the kgend to figure 4 are Hs~d in the figure, together with esfim~es of the

m d ~ d ~ m ~ s of t ~ species present in each band m a ~ by c o m p ~ a t i ~ SDS polyac~lamide g~ dectrophoresis ~ a i l s in t ~ l e g ~ d ~ fig ~ .

Differences between ~e protein complemen~ of native and der~ed 30S subunits One- and two-dimens~n~ e l e ~ r o ~ o r e t ~ c o m p ~ s o n ~ the pro~in comp~mems of n ~ v e and ~rived 50S subun~s Mowed them to be ~ m t i v e l y i ~ m ~ (~su~s not shown) ~ t ~ v e ~ e d ~g~ficam d i ~ ences between those of n ~ v e and d ~ v e d 30S subu~ts ~ g 5). M~or d i e . r i c e s ~ t w ~ n the onedimen~on~ e l e c t r o ~ o r e ~ distribution pm~ms of p r o ~ n s ~ N30S subunhs (fig 5A, ~fl t r a c ~ and D30S s u b u ~ (fig 4 ~ ~ : i) The p~sence in the f o ~ ~ numerous a d d i t ~ n~ bands. Most of these ~ ~ n t and c o ~ s p o ~ ~ther to traces ~ comaminant non-ribosom~ protons ~ n o w s 1 ~ , fig 5 ~ or to s m ~ amounts of 50S s ~ u ~ t p r o ~ n s ~ands i ~ by ~ o w s 5 and 6 in fig 5A have the same ele~rop~ret~ m o ~ s as prominem 50S protein bands E ~ d I in fig 4 ~ . Howeve~ the r e l ~ v ~ y strong b ~ d s in.creed by an~ws 4 ~ d 7 in figure 5A ~ l o n g to nehher of the ~ o v e m e ~ o n ~ categories, since ~ e y a ~ presem in sig~ficam amoums and possess electro~oretic mo~es which di~rentiate them from ~l 5 ~ subu~t pro~ins. ~ ~difion, these two bands ~ e c o m e . e l y ~ina~d (fig 5A, rig~ ~ack) ~ was~ng N30S s u b u ~ ~ l M A m ~ (MaWria~ and m e & o ~ whe~as ~ the other comaminam bands ~ u d i n g those con~ 50S s u b u ~ protons are ~ Last p ~ i ~ ~ant to this treatmenL It may be nmed th~ the i n ~ n ~ of 30S s u b u ~ bands A, F and M is cons~e~ ably ~duced by w ~ n g the subun~s w~h 1 M Am~. 2) In the p r o ~ n p ~ m of N30S s ~ u n ~ band A is ~ p r o d u ~ y m u ~ m o ~ ~ron~y ~ n e ~ and bands B and C ~ e ~ p ~ d u c i ~ y much ~ss ~ron~y ~ n e d than the same bands in the p ~ m of D30S subun~s ~ompare fig 5A ~fi ~ack, and fig 4A~ The ~ s ~ t s of c o m p ~ s o n of t ~ proteins of N30S and D30S s u b u ~ by two-dimens~n~ ~ e c t r o ~ o ~ t ~ an~y~s acco~ing ~ K ~ t ~ h m ~ t a ~ Wi~man (figs 2A, 5B, D, E) and ~ e r a ~ Warner (figs 2B, 5C, E G) p e ~ the ~ n g con~u~ons: i) p r o ~ n S I is p ~ in larger a m o u r s and protein $2 in s m ~ e r amounts in N30S s u b u ~ than in D30S subu ~ s ; fi)N30S s u b u ~ do not c o ~ n p r o ~ n $3. ~ e s e d i ~ n c e s co~e~ond to the ~creased in~nsi~ of band A and the ~duced intensities of bands B and C in figure 5A ( ~ ~ack). ~ e p~sence ~ reduced a m o u r s ~ protein $2 and the ~ s e n c e ~ proton $3 in N30S s ~ u ~ s is not due ~ p a ~ or c o m m i e ~ s ~ r of these specks to NSOS subun~s when 70S

1002 B

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!

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Figure 6 shows the resu~s of ion exchange chromatography of proteins of 30S and 50S subunits prepared using buffers containing 2 mM MgCI~ for ce~ ex~action and sucrose gradient fractionation of extrac~ (mixed or M subunits containing both N and D forms). The proteins present in the various peaks in each elution profile were identified as described in the legend to figure 5. The chromatographic system used (see above) perm,s rapid and relatively efficient preparative scale fractionation of ribosomal proteins. h may be noted that the additional proteins $8' and S 17' present in N30S subuni~ could be early ~olated by this procedure. Discus~on

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Fig 5. Electrophoret~ analysis of proteins of N30S ribosom~ subunhs. A. One-dimensionfl SDS gel e~ctrophoresis of proteins derived ~om I A:~,., unit of purified N30S subun~s before (~fi lane) and after (right lane) washing with I M NH4CI. B,C. Two-dimensionfl gel electrophores~ First, om nine dimension Az~,.,~ u~lt: o ~ ~ c ~ e ~ : ~ ) ~ l r ~ ~ r / ~ e e t ~ hands5,s6,Secfi°nSS7,$8, °f 7~, s~5~ c ~ r ~ : i ~ : ~ t ~ ~, ~geis~;~: D,E. Comparison of acidic protons of purified N30S and D30S subuni~ by two-dimensional electropho~s~ according to Kaltschmidt and Wittmanm First dimension gels loaded as above (B,C~ To facilitate comparison first dimen~on gels contorting N30S and D30S p ~ i n s were transf e ~ d to the same second dimension gel ~ab. D. N30S subunit acid~ proteins. E. D30S subun~ acidic pro~in~ KG. As for D,E but second dimen~on elect~pho~sis in the p~sence of SDS acco~ing to Zinker and Wame~ F. N30S subunit acidic protein~ G. D30S subun~ acidic pro~in~ ribosomes dissociate in vivo fince proteins $2 and $3 are not found in isolated N50S subuni~ (results not shown~ 3) N30S subuni~ cont~n two additional weakly bafic proteins designated $8' and Sl7' in figure 5C,D. These spots are absent in the two-dimensional pat~rns of proteins of ammonium chloride washed N30S

Of the four procedures ~ s ~ d for ~olation of ribosomal subuni~ from L lactis the most satisfactory, as judged by the r e a s s o ~ i o n capacRy of the p r o d u ~ is th~ described by Nofi et al [23] for preparation of native and derived ribosomal subuni~ from E co~ (m~hod I ). Whereas we have shown previoufly [27] thin highly act~e subuni~ can be obtained by dissociation of E coE ribosomes in the presence of 10 mM Mg2÷ and 400 mM Na÷ (method 4) this procedure y ~ s severely inactivated subunim from ribosomes of L lactis IL1403 0able D. D i s s o ~ i o n of ribosomes in crude c~! extra~s prepared in the presence of 2 mM Mg2÷ (method 2) also probably yields active subun~s. This conclu~on can be deduced as follows. Fractionation of small amounts of crude c~l extract (20-30 A2~ ~m)under the conditions used to obtain the results in the lower pan~ of figure 1 (m~hod 1) yields profits containing four well resolved peaks of native 30S and 50S subuni¢ and 60S and 75S ribosomes from which ~ can be estimated thin these two fractions contain about 40% and 60% r e s p e c t ~ y of the total cell con~nt of ribosomes. Assuming th~ extracts prepared in the presence of 2 mM Mg2÷ contain native and derived subuni~ in the same relative amount~ thin theft reassociation activ~ks are the same as those of subuni~ prepared by method 1, and th~ derived subunits reassociate preferentially with each othe~ the reassociation activ~y of the m ~ e d subuni~ can be estimated to be about 70%. This value agrees w~l w~h the experimentally de~rmined resuR indicming thin these assumptions are probably co~ect. The general charac~ristics of the ribosomal subun~s of L lactis IL 1403 described here resemble

1003

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Materia~Prot totaleainndpresemP M3OSmet sOf roteins ndu~e ofufing hodsi peaks sa~nplesand were M50S dbosom~containdin7"8 gibY dentmifiegcoprecipitatingSU 48.bIOunitgmangdeXChangerespectivelY(aceton~vol Chr)°m OftO at°gt~rapsampl hY5 esPrO " of peak~fM5OSi altschmdan dgn~subt i'n~iaudnit~it~a~PnP~O~ro~ids stniodc ° ~ " a l o t4-5 s gl ~ggt ~of° pprotein n hatnpeaKt ' hs ~ n(A230 e ~a l; y~nm)and act sJi~ppre0~p-i~t~s-ons cgn'30 r°aptp~epg hard~~of i ~kW -~totM lr~b~ f ru~,,~,,,=vs---~----Pr°mins a c t i ° nofs M30S subunitsor 40 gg of totM pro~ins

Fig carried I°n M30S exchange chromatographYanda s M50sdescdbedsubunRs.in

~Mnedspots.

1004 t h o ~ of E coH dbosomfl subunits: ~ m e m a t i o n coeffidems of the subunits and thek RNAs, number and molecul~ m ~ s range of thek protein~ Howeve~ two ~ f f e ~ n c e s have been o b ~ r v e d in the behav~ur of ribosomes and fibosomfl s u b u ~ of ~ e two bactefifl s~a~s. As noted abov~ ~ o c i a t i o n of ~ e k ribosomes in ~ e p ~ n c e of 10 mM Mg2~ 400 mM N ~ yields active (E coil) and ~acfive (L ~ctis) subu~ts. ~ add~on, as shown ~ ~ e accompan~ng pape~ M30S and MSOS subunRs of L ~ctis and E coil ~ffer g~atly ~ thor capacRy to form h~emgeneous 70S part~les. It seems posfi~e th~ the ~ f f e ~ n c e s obse~ed ~ • e proton c o m O e m e ~ of N30S and D30S subunits of L ~ c t ~ IL 1403 may conespond ~ ~ast in part to ~ e p ~ n c e of pro~in sy~hefis in~ation h ~ o r s on • e ~rme~ Weakly bafic proteins $8' (M~ 10 500) and S17' (M~ 22 500) p ~ ~ N30S subunits and ~ m o red from them by w ~ n g w~h 1 M AmCI (fig 5) ~m~e ~ these ~ s p e c ~ E coil initiation factors IFI and IF3 w ~ c h are weakly basic proteins with m ~ e c ~ ~r m~s of 8119 ~8], 20 668 and 19 997 ~9] (IF3 occurs ~ two forms; a ~ng ~ r m containing 181 am~oadds, and a sho~ ~ r m l a c i n g ~ e six N ~ r m ~ f l aminoacids of the ~ n g ~ r m [31]) and ~ e ~ a s e d ~om E coil 30S fibosomfl s u b u ~ in the p~sence of 1 M AmC1. In addit~n, the e l e ~ r o p h o ~ t ~ mob~ties of $8' and S17' ~ ~ e K f l ~ c h m ~ t and WiUman two~menfionfl sys~m c ~ s ~ y ~ s e m b ~ those of E coil initiat~n ~ c ~ r s IF1 [3~, and IF3 [31], ~ s p e c t i v ~ No hyp~hefis can y ~ be proposed to accoum ~ r the absence of a c ~ protein $3 (M~ 25 000) ~ N30S subunRs. T ~ s protein is confide~d to be a fibosomfl protein rather than an e~raribosomfl f a c e t ~ v ~ v e d ~ protein s y ~ h ~ because s p ~ $3 ~ seen ~pmducibly in t w o ~ i m e n s ~ n f l electropho~tic anflyses of proteus of D30S subunits ~ a m o u r s wh~h, as j u ~ ged by ~s ~ n g ~ n f i ~ are c o m p ~ a b ~ ~ those ~ s p a s $2, S I ~ and S l l (figs 2A, 3A), and because k ~ p ~ s e m though ~ ~duced amounts ~ anflyses of pro~ins from D30S s u b u ~ washed ~ 1 M NH~CI (~sults not shown~ A protein of unknown ~ncfion, the m ~ e c ~ mass and electropho~t~ prope~ies of w ~ c h resem~e ~ o s e of protein $3 has p ~ f i o u s ~ been o b s e ~ e d D2] in large amounts ~ 30S s u b u ~ ~ a t e d from E coil har~es~d in ~ationary phase and ~ smafi amounts in 30S subunits p ~ p ~ e d ~om exponentially growing ce~s.

Re~n~s

1 Ouog~li G, G ~ I, Dellagl~ F (197~ Taxonomic ~lationships between Strepmcoccus thermophilus and some other St~ptococci.J Dai~ Res 46, 127-131 2 Garv~ EL Farrow JAE (1981) Subd~isions within the genus Strepwcoccus us~g deoxyribonucleic acid/dbo~m~ dbonucleic ac~ hybridization. Zbl Bah Hyg 1 Abt Or~ C2, 299-310 3 Jarv~ AW, Jarv~ BDW (1981) Deoxyribonucleic ac~ homology among lact~ Streptococci. Appl Environ Micro~ d 41, 77-83 4 Ki~pe~B~z R, ~scher G, ScOffer KH (198~ Nucleic ~c~r ~ / ~ c ~ o d ~ i ~ ~ p N and group D Streptococci. 5 MagmmLUdwW,L, ~ ~eoe~l~RE,,F~l~(-~:k~rSanC~e~9~i 6

and ~lated Strep~coc~ ~ the genus Lac~coccus gen nov. System Appl Microbiol ~ 183-195 7 Coil,s MD, Ash C, Farrow ~A~ Wal~anks $, Williams AM (198~ 165 ribosomfl ribonucleic acid ~quence an~ ~ s of Lactococci and ~lated tax~ De~ription of Vagococcus fluv~lis gen nov sp nov. J Appl Bact 67, 453-460 8 Co~ns MD, Wi~ams AM, W ~ a n k s $ (199~ The phygenococcus gen nov. FEMS Microbiol Len 70, 255-262 9 Choon A, Choon MC, Moffi~Batt A, Langella P (198~ Two Oasmid de~rmined ~striction and m o d ~ c ~ n sy~ terns ~ Strep~coccus ~ctis. Plasm~ i 1,260-263 10 Terzaghi BE, Sandine WE (1975) Improved me,urn for lactic Streptococd and the~ bacteriophage~ Appl Microb~l 2~ 807-8 13 II Sripati CE (198~ An improved chemically defined m¢~um ~r mass c~m~s of Tetrahymena: Nutrient u~ake and growth ~g~ation. J Gen Physiol 133, 2581-2588

12 ~)~:~alB,s~s~dG~x~om~alF, p~ 13 14 15

16 Acknowledgments

The authors thank Bruno Sav~fi for ~ c h n ~ as~ance with ion exchange column chromatography. Finan~ support for this study was provided by the CNRS and La Fondation pour la Recherche M6dic~e Fran~s~ N Limas Nzouzi thanks the Government of the R~publique du Congo for a po~graduate fellowship.

17 18

Fo~(1983'Tetra.

hymena thermophila. Effect of ~e p~sence of ~doaceF amide during ribosome ex~action on the pmpe~ies of the subunit~ Eur J Biochem ! 35, 425-434 Laemmli U (1970) Cleavage of structural proteins during the as~mb~ of the head of bac~riophage T4. Namw 227, 680-685 Petridou B, Guerin M~ Hayes ff (198~ PmteiwRNA ~lt~ah~einna t t l t h : ~ . ~ i ~ / ~ t ~ n ~ c ~ ; ; s o m e Wada A (198~ Analy~s of Escher~h~ coil Hbosom~ p m ~ s by an improved two-dimen~on~ ~ec~opho~sis. I. Deletion of ~ur new protein~ J Biochem (Tokyo) 100,~83-1594 Kaltschmidt E, Wittmann HG ( 197~ Ribosom~ pmtein~ VII. Two-dimensional p~yac~lam~e gd elec~ophoresis ~r fingerp~nt~g of ribo~m~ proteins. An~ B~chem 3~ 401-412 ~nker ~ Warner JR (1976) The dbosom~ pmte~s of Saccharomyces cerev~iae: Phosphorylated and exchangeable proteins. J Biol Chem 251, 1799-1807 No~ H (1967) Ch~acterization of macrom~ecu~s by con~ant vdocity segmentation. Nacre 215, 360-363

10~ 19 Ron EZ, Koh~r RE, Davis BD (196~ MagneMum ion dependence of free and polysom~ ~bosomes from Escherich~ co~ J Mol Bio136, 83--89 20 Kaempfer R (1970) Di~o~afion of ~bosomes on p d ~ peptide eh~n ~ r m ~ i o n and o ~ n of ~n~e fibosomem Namm 228, 534-537 21 Hemog A, Ghy~n A, Bo~en A (1971) Sens~ity and ~sistanee ~ streptomycin ~ relation wi~ factor me~amd dissociation of ribosomes. FEBS Len 15, 291-294 22 Spirin AS (1971) On ~e equflib~um of ~e ~soeiat~n~ssociation ~action of ~bosom~ subparticles and on ~e e x ~ n c e of the so-called '60S interme~ate' ('swd~n 70S') du~ng centri~gation of the equ~brium m~mm FEBS Leu 14, 349-353 23 No~ M, Hapke B, Schreier MH, Ndl H (1973) ~ m ~ M dynamos of bactefi~ dbosomes ~ Ch~actefization of vacant couples and thek ~lat~on ~ ¢omp~xed ~bosomes. ~ Mol Bio175,281-294

d o ribosomes and s u b u ~ active ~ ~g tranflation of natural messenger RNA. J Mol Bio180, 519-529 25 Gue~n M~ Hayes DH (1983) Concerning the therm~ s m b ~ of E co~ 23S ~bosom~ RNA. B~chim~ 65, 345-354

26 Wada A (198~ Anfly~s of Escherichia coli fibo~m~ ~oteins by an improved two-$mensional electrophoresis. ~ Ch~actetization of ~ur new p ~ J B~chem (Tokyo) lO0, 1595-1605 27 Expe~-Bezanfon A, Guefin M~ Hayes DH, Legadt ~ Thibau~ J (1974) Prep~ation of E co~ ~bosom~ s u b u ~ wi~out ~ of biolo~e~ a c t i ~ . B~chim~ 5~ 77-89 28 PortC, Wiumann-Liebold B, Gu~er~ C (197~ ~ru~ure~ncfion r e l ~ n s h ~ s ~ Escher~h~ coli ~ i a t ~ n ~ctors. H. Elue~ation of the primary ~m~ure of ~ a t i o n factor IF-I. FEBS Lett 101,157-160 29 Brauer D, Wiamann-Leibo~ B (1977) The p~mary struc~ of ~ e ~ a f i o n ~ o r I~3 from Escherich~ co~. FEBS Lett 79, 269-275 30 Pawl~ RT, Liu~fie~ J, Pon C, Gu~e~i C (1981) P u ~ f i c ~ n and p r o p e ~ of Escherich~ co~ ~an~ati~ n~ initiation factors. B~chem lm ~ 421-428 31 Su~anaryana ~ Su~amanian R (i 977) S e p a r ~ n of two forms of IF-3 ~ Escherich~ co~ by tw~dimen~on~ gd ele~mpho~sis. FEBS Lett 7~ 264-268 32 Submman~n AR, Haa~ C, G~sen M (1976) ~ a t i o n and charae~rization of a growt~cyc~-refled~g, high m ~ e c d ~ w e ~ proton a~o~a~d wi~ Escherich~ coli ~bosomes. Eur J Biochem 67, 591-601