Identification and purification of a calcium-binding protein from Bacillus subtilis

Biochimica et Biophysica Acta, 1080 (1991) 160-164 ~: 1091 Elsevier Science Publishers B.V. All rights reserved 0167-4838/91/$03,50 ADONIS 0167483891003188

160

BBAPRO 341~1~

Identification and purification of a calcium-binding protein from Bacillus subtilis M a r i a G r a z i a T o z z i ~, U g o D ' A r c a n g e l o ~, A n t o n e l l a D e l C o r s o 1 and George W. Ordal 2 t Dipartimento di Fisiologia e Biochimica, Laln~rato;i di Biochimica. Pt~a (hal)') and ' Department of Biochemistry, Unirersityof Illinois, Urbana (U.S.A.) (Received 25 October 1990) (Revised manuscrip! received 3 June 1991 )

Key words: Calcium ion binding protein: Chemotaxis: (B. subtilis)

A Ca 2 +-binding protein was identified in Bacillus subtilis in the log phase of growth. The molecular mass of this protein is about 38 kDa as estimated by polyacrylamide gel electrophoresis in the presence of SDS and by gel filtration. The protein was found to be resistant 10 min at 65 °C and was purified about 400 times, starting from heated crude extract, by conventional procedures. This novel protein is able to bind Ca "+ in the presence of an excess of MgCI 2 and KCI both in solution and after SDS gel electrophoresis and electrotransfer. Since an impairment of the Ca z+ intake, in Baci//us subt///s, results in an impairment of chemotactic behavior (Matsushita, T. et al (1988) FEBS lett. 236, 437-440), 38 kDa protein may be involved in the regulation of chemotaxis.

Introduction In eukaryotes, Ca :+ plays an important role in t.hP regulation of a number of motile processes such as muscular contraction [1], cytoplasmic contraction [2], and ciliary reversal [3]. Most of the effects of calcium as a regulator of cell functions are mediated by calcium binding proteins which are present in all eukaryodc cells and which undergo Ca 2* dependent conformational changes and respond to transitory increases in intraceilular Ca 2+ concentrations. Bacterial chemotaxis has been described as a primitive sensory process which enables bacteria to sense their environment, process the information, and move accordingly. There are reasons for believing that chemotaxis may also be influenced by calcium ions. In Bacillus subtilis, calcium enters the cell through a voltage dependent Ca 2~ channel [4] and this transport system is sensitive to Ca 2÷ channel blockers of animal excitable membranes [5]. Ordal et al. [6], reported that Ca 2÷ affects chemotactic behavior in Bacillus subtilis;

more recently Matsushita et al., demonstrated that Ca 2÷ channel blockers inhibit chemotactic behavior without any effect on either cell growth or motility [7]. These results suggest that calcium may play a role in regulating the motile and sensory system of Bacillus

subtilis. Several authors, searching for calmodulin-like proteins, reported the presence of caJcium binding proteins in bacteria [8-10]. In Bacillus subtilis, the presence of a heat stable calcium binding protein, which was soluble at 100% ammonium sulfate saturation, was reported by Fry et al. This protein was found to pos~ss some characteristics in common with calmodulin [11]. However, none of the bacteria calcium binding proteins was purified to homogeneity. Here we present the identification and purification of a calcium binding protein from Bacillus subtilis, which is highly specific for Ca 2+ in the presence of high concentration of Mg 2+. Materials and Methods

Materials Correspondence: M. Grazia Tozzi, Dipartin'er, to di Fisiologia e Biochimica, Laboratori di Biochimica, via S. Maria, 55. 56100 Pisa. Italy.

45CAC1 (21.6 mCi/mg) was from Amcrsham. Distilled water and all the buffers were Chelex-100 purified before use.

161

Methods Cell growth and crude extract preparation. Bacillus subtilis strain OI 11185 was grown in Luria broth as described by Thoclke ct al. [12]. The cells were harvested at the end of log phase by centrifugation at 5 0 0 0 x g and washed twice with buffer A 141) mM Tris-HCI, pH 7.5, 1 mM phenylmethanesulphonyl floride, 5 # g / I leupeptin, 5 # g / I pepstatin), resuspended in the same buffer at a cell concentration of 0.2 g/ml. and disrupted by sonic treatment. The resultant extract was centrifuged 30 rain at 20000 ×g. The supernatant was then exposed 10 rain at 65°C. After cooling and centrifugation at 20000 × g , the extract was incubated 30 min at room temperature in the presence of DNAse and RNAse at a final concentration of I).114 mg/ml. DEAE-celhdose chromatography. The extract after heat and nuclease treatment was diluted 4 times with distilled water, supplemented with 5 ~M +SCa (3000 dpm/nmol) and loaded on (2 × 15 cm) DE-52 column equilibrated with buffer B (10 mM Tris-HCi (pH 7.5) containing 5 ~M 45Ca 3000 dpm/nmol). The proteins were eluted with 120 ml linear gradient 0-0.5 M NaCI in buffer B. Fractiops of 1.4 ml were collected, radioactivity was measured on 100 pl aliquots. Two peaks of Ca-binding activity were pooled and subsequently the Ca-binding protein responsible for the Ca-binding activity pool I! was suojected to further purification. Sephacryl S-200 gel filtration. The DEAE-celluiose pool was concentrated to about 6 ml. 2 ml were applied on a Sephacryl S-200 column (I.5 × 70 cm)equilibrated with buffer B. Elution was carried out with buffer B. Fractions of 1.4 ml were collected; radioactivity was determined on 100 t.tl aliquots. The apparent molecula:- weight was determined by comparison of the elution profile with those of the following calibration standards: Bovine serum albumin M.W. monomer 66 000. dimer ( 132 0001, a-amylase (200 000), horse liver alcohol dehydrogenase 185000), calf intestinal mucosa adenosine deaminase (35 000). myoglobin (16 91~J). Second DEAE-cellulose chromatography. Pooled active fractions from Sephacryl S-200 gel filtration (fractions 48-60) were applied on a (2 × 7cm) DE-52 column equilibrated and eluted as described for the first DEAE-cellulose chromatography. SDS electrophoresis and electrotransfer. PolyacDlamide gel eleetrophoresis was used both as criteria of purity and to analyze the obtained protein fractions. The concentration of the separating and stacking gels were 12 and 5%, respectively. Electrophoresis was .r~rformed according to Laemmli [13]. Protein bands were stained with Coomassie blue in the presence of formaldehyde [14]. Electrotransfer and autoradiography were performed with a modification of the method previously described by Maruyama et al. [15]. Alter electrophoresis the gel was placed on a polyvinylidenedi-

floridc (PVDF) membrane '.~o/ikcd in methanol fc~, sccond,~ before equilibration in blotting buffer (0.025 M Tris, 0.129 M glycinc in 20% methanol). Electrotransfer was performed at a cowtant voltage of 80 V for 2 h in a mini apparatus (Bio-Rad) at room temperature. The gel was stained with Coomassie blue in order to check the cffcctiveness of the blotting. The membrane was soaked in 3 changes of 10 mM Tris-HCI buffer (pH 71 containing 60 mM KCI, 5 mM MgCI 2, for 1 h, to remove the blotting buffer. Then the membrane was incubated in the same buffer containing 200/,tCi 45Ca in 200 ml for 15 rain. Finally the membrane was washed 2 min in two changes of distilled water and a few seconds in methanol and dried at room temperature. Autoradiograms of the ";SCa-labelled proteins on the PVDF membrane were obtained by exposure to Hyper Film/3-Max Amersham for 24 to 48 h at - 80 ° C. After autoradiography the membrane was stained with Coomassie blue. Ca 2 +-binding assay. The calcium-binding activity of each step of purification was evaluated with a gel filtration method [16]. 100 txl of sample were applied on a 10.9 × 24 cm) ScphacD'l S-200 column equilibrated with buffer B. Where indicated tb same column was equilibrated with buffer B s,~,plemented with I mM MgC~_, and O0 mM KCI. -,he elution was carried out with equilibration buffer, 25(1 , I fractions were collected and radioactivity was measured on 100 pl aliquots.

Results and Discussion Fig. I shows the calcium binding activities in BacilhL~ subtilis analyzed following the metht~l previously described by Maru: area et al. after SDS-electrophore-

66k

--

_

4sk-36k--

'q~

,rob

P

24k--

14k --

1

2

3

4

S

6

Fig. I. AutoradioRraphy after SDS-electropboresis. electro4ransfer and 45Ca incubalion ( ~ e Materials and Methods). ( l ) Crude extract of log phase bacteria: (2) crude extract of stationary phase bacteria; (3) as 1 after heat treatment; (4) as 2. ~fter heat treatment: (5) as 3 after nuclease treatment: and (61 as 4 after nuciease treatment. Each line was loaded with 70 ~g of proteins.

162 3s7- ...................................... I

I

~,

3~,

G4

~

.

.I

.M i

.........

.

.

.

/

i

~.

V /

E

~05

=

i

~= 404 ] _

"7, o

200k 132k85k66K 35k

,o 0

iill

"-" 10 20

40 fraction number

60

80

Fig. 2. DE-52 chron]atography of Bacill,s subtili~ crude :xtract after heat and nuclease treatment. 5 ,aM ~'Ca 3000 dpm/nmol ~as present in the buffer (buffer B. see Materials and Methods). (c',) radioactivity. ( . . . . . . ) absorbance at 28f) nm. ( ) NaCI P(~fl 1. fractions 42-511; P(~)l II. fractions 56-62. The inset shows autoradiography after SDS electrophoresis, electrotransfer and ~('a incubation of DE-52 pt~.rl 11,

sis [15]. The crude extract prepared from cells harvested in the log phase of growth showed a 38 kDa band which was able to bind calcium in the presence of a high concentration of Mg-" and which appeared to be resistant to heat and nuclease treatment. The same activity seemed to be absent in the crude extract obtained from cells harvested in late stationary phase. Crude extracts contained about 9 mg/ml of protein, exposition to 65°C reduced protein concentration to about 20% and nuclease treatment left protein concentration unchanged. Fig. 1 demonstrates that heat treatment resulted in an enrichment of 38K Ca :~ binding acti;'ity, on the contrary, the incubation at room temperature with nuclease caused a decrease of the activity. probably due to proteolytic degradation. Despite this observation, nuclease treatment is necessary to remove large o m , ' - m ! of nucleic acids whose presence may compromise the possibility to evaluate Ca "-+ binding activity of proteins in our preparations. The purification of the Ca 2- binding protein (CaBP) has been followed by means of 45Ca added to the buffers. In these conditions, the Ca: +-binding activities emerge from the column as peaks ~;f radioactivity, Fig. 2 shows the proteins and radioactivity elution profile from a DEAE-cellulose column. After absorption of the crude extract on the column, a decrease of radioactivity in the buffer could be observed, thus indicating tbat a Ca -'+ binding activity was retained on the column. After the application of the gradient, two peaks of radioactivity could be observed. The i n ~ t of Fig. 2 shows that the pooled fractions of peak II contained the 38 kDa CaBP. Pool I, on the contrary, did not show any protein with a binding highly selective for Ca -'+ over the Mg 2+ (results not shown). The 38 kDa CaBP was further purified by gel filtration on a Sephacryl S-200 column. Fig. 3 shows that the major peak of Ca2+-binding activity emerged from the column at a

t!

/ / \ ~)~"

,

o_

17k

l ,

30 20

O

]1 [

50

!

;

=o

/ /

\

,

+'/'/~, , /

: \

!0 0

.,. ~-,, _, ,,;, 30

¢N

"~ o co

~

40

5~0

fraction

number

Fig. 3. ScphacDl S-200 gel fihration (if DE-52 pool il, 5 ~M 4~Ca 30iX) d p m t n m o l was present in the buffer (buffer B, see Material,',

and McthodsL (~) radioactivity. ( . . . . . .

) absorbance at 28(I nm.

mo!ecular mass around 4 0 kDa. The heterogeneity of the Ca-"+ binding activity showed in Fig. 3 may be due to the presence of a residue of nucleic :~cids after nuclease treatment. The final step of purif;cation consisted in a second DEAE column (Fig. 4). The amount of radioactivity and the proteins measured with the Coomassie method were proportional between fractions 3 0 - 4 0 . This result was verified by Coomassie blue-stained SDS electrophoresis (Fig. 5). The molecular weight of the native protein (40000) and the SDS treated protein (3801)0) are consistent with a monomeric structure. The purification table (Table 1), shows that a 400 fold purification with a yield of about 4% was obtained. The Ca-binding activity of each step was determined with the gel filtration method using 4~Ca and measuring the area of the peak of radioactivity. The calcium binding activity of the final step of purification was finally measured with the gc! f:,ltrati~n met!-od in the presence of 5 # M 45Cac12 1 mM MgCI 2 and 60 mM KCI (Fig. 6). Under these conditions,the protein appeared to bind 4 mo of Ca-"+/mol of protein.

ri

~I 3o

1°." 1o,

!S

\

.-

ol 0

10

/

',",i\

i

........... .. , . . . .

-_to

20

50

fraction

30

40

~

o,

{,o

number

Fig. 4. Second DE-52 chromatography. 5 ~.M 45Ca ."~O~ dpm/nrno| was ~)rcsent in lhe buffer (buffer B, ,~ee Materials and Methods). ( 0 ) radioactivity,. ( . . . . . . ) protein concentration according to Bradford, (.... ) NaCI.

l ¢~3

66k~

45k~ 36k--

24k-1

2

3

Fig. 5. ('~x)massie-blue stained S D S g~:l elcclroph.rc.is ol the a c t i w fractiom, after sCeOlld DE-52 chromalograph,,. I in¢ I. haclion 34: line 2, fraction 36: line 3. " a c t , m IS

TABLE I

('aBP purilhanon tabh" Steps

(1) Crude extract after heat anti nuclcase healnlenl (2) I DE-52

Total protein (rag)

Acnvity ( Units ~ ~

Y~cld U; )

Purifi tati.n (totd)

I IO0

57.2

lOq

I

4H

16.2 43 2.5

(3) SephacDI S-2l~1 (4) II DE-52

"B 0.;13

'9 "r q ~a

s 25 ~-~

* O n e unit is the amount o1 prolcin v.h~ch ~-a. T.,uid l!'" D P M of radioactivity. 13 r. . . . . . . . . !

?

12

9

L

':< ................................. 014

30

40

SO

traction

60

70

80

number

Fig. 6. Sephacrl,'l S-21N} gel filtration of the purified protein. IS u g of purified protein were loaded on a small ¢oltJmll a , des~.'ribed in Material and Me~ht~,ls, the elution buffer contained 5 m M Mg('l ,. ~ t r a m K(3 and 5 ,u M ~ ( ' a ( ' l ,.

()~.It allcnlpI'- ~o (~b',lin il Icpl~)dm.ihlc aiHoladiu,:rapIl) oI (;i-binding actbiI) o| ihc pLfflficd pIotci:'. afI~.t S[)S-clcdml)horc~,is and blolfing ~vcr? un,,t~cccsful. als~ shortcning the sample c x p m u r e at llltl:( ". Ho~w~cr. in thb, regard. ~ e must point out that con> pletc and inrevcrsible d e n a t u r a t i o n is a normal feature for a protein lreatcd fnr SDS clcctrophoresis: the rco.p,c U ,of the acli,.it} ~ c obtained with the palliaily purificd samplcs, probably duc to the protective cffect exerted by the high protein o m c e n m t t i o n , is an exceedingly useful cxcepuon to this rulc. On the other hand. the results that definitively d o c u m e n t the purification of the C a - " - b i n d i n g protein may bc summatizcd as Mlows: (I) we present evidence that a 3SO(XI molecular weight protein i,, tile only active spccie,, prcsenl in logarithmically growing Baciflu~ ~uhtill~- crude ,',:triter after heat a n d nuclcase t r e a t m e n t (Figs. 1 and 2). (2) During the la';t purification step. v.e isolated only onc disccrnable protein of 38tRRI molecular weight which coclutcs with Ca-binding activity and its concentration can account h~r the binding of 4 tool of C a : per tool (Fig,',. 4-6L An.~ c o n t a m i n a n t would bc t~u~ diluted ol t ( ~ small to account fi~r such an activity. O) The final preparation contains a C : , : ' - b i n d i n g actMty with high specificity m e r Mg~'" ions {Fig. 0). l h i s specificity is a c o m m o n feature for the ('at-' "-hinding proteins. in conclusion. Bacillus suhtilis Ix~s,,esscs a 38 kDa protein which binds calcium ions with a high selectivity over Mg: ", which is explessed in the logarithmic phase of growth. F u r t h e r studies and an improvement in the .~ield ot the purified prolein are necessary to assess the _t. _ i tn~p,~t.,a p m p c r t i c ~, ;Hld the C a " - b i n d i n g constant ~,f ~hi', 3 g, k['}a ( ' a B P w h i c h may repn,~,cet~ tl~,: an,.:,,'*H~-y Ca:--binding p r o t e i n , l l c w e v e r o u r results d c m o n -

strafe thai lhis cytopi,~smic p,Hcii~ can bind ( a : a~ the veU Iov, concentration in which it is present in the cell in fivo and in the r~rescncc of high concentration ol magnesium. Since calcium ion may influence chemotactic behavior. at te~st in i¢acillm subtihs, it ~ill be of great interest t:) cxatnine variote, dlcmotaxis mutants to see '~hcthc: any are missing thix novel protein. We also h~pc to clone and inactivate, if possible, the corresponding gone and to assess the effect on ehemotaxb,. in order to begin to u n d e r s t a n d its role in the p h y s i o l o g y of bacteria. During the revision of this manu.,cript, a p a p e r describing the purification and propcrfics of a ('a z ~-binding protein from sporulating B. subtilis was published [17]. This finding :s of great interest to all the r e a s e a r c h e r s involved in the sludy ol bacterial mctatx)lism. To our knowledge. C a B P does not show any caractcristic in c o m m o n with the calmoduline-like protein purified by Fry* et al.

I fi4 Ackn~,w!edl~,,rnenis

W c t h a n k l ) r . Rossan3 Pcsi ( i ) i p a r t i n l c n l o th I.i:,i.. olllgia C [ l i o c h i m i c a ) l i f t l c c l n l i c a l a'~MMillltl: T h i s w o r k was ~ l i p p o r t c d by ~ g r a n t f r o m I l a l i a n ( , N R and by ('Nit r . i r l t c ! P r i l l c c i ) l i I ] i o l c c h n o h l g y and I l i o i l l s l r t l illc';]l~il hill

References I Ebm, hi, S. anti [tndo. M. (1068). Prog. Biophys. Mol. Biol. I,'.l, 123 -183. 2 Ta~clor. D.L. ('ondeelis. J.S,. Mixir¢. P.L. and Allen, R.I). (IW3) J. ('ell. BioL 59. 378-3tJ4. 3 Schmkll. J A . alld I!¢kcrt, R. 11076) Nalur¢ 2~2, 713 715 4 I)c Vru, 'W. Ihthhui,., R . P~l'~hn,i I and K~milig~.. W N II1~5) J Ilal;ltrrlol 16-t. t294 13011 5 Ku~,
13-12

{} ()rdal. (;.W. t I{f~7) Nature 271L {)l'~ {~7. 7 ~|;ll%tlnhil~l, | , Ililala, I I , Kll',~tka I (10~8~ |:I I]S I.¢IL Z36. .1~'7 .till Y, II.ilnlon. A ( ' , Ptaqlel. I ) , ('olnlk+l. M J . ( I q I $ i ItiLichein Itioph~'~, Itt'~ ('llnimllll> 127. Jl 36. 9 InlnlW, ,%.. I larada. W.. Zt,.liian. O., Inolyt'. M. (1981)J. BacieHid 14i'I, fl7,"4-6;"13. Ill lilOl,I)t'. ,%, I:rlllltt.'M2hJnJ. "[', IIIOll~%'. M, {ItJbl~) Proc. Nail. Acad, Sci. [JSA bill. hbI29 6)433 II t'ly. IJ., Villa, L. Kuehlt. (i 1).. ![agcrnan, L I I . (1{J,~6)Bioehem. Blophys. les. (',)nlllllln, 134, 212.+217. 12 ]'hoelke, M,S., Bedalc W . A . Ncliellon, D.O,, Ordal, G.W. 0 9 8 7 ) J. Biol. ('hem. 262, 2811-2816. 13 LaemmlL U.K. (197l)) N,llurc 227. 680-685. 14 Burk, RR.. EschenbruclL M~, Leuthard. P. and Sleek, G. (i983) Mclhods |Enzymol. 91,247- 254, 15 Marnyama. K . Mikawa. 3". and [?.,ashi. S. (1¢J144) J Biochcm. 05, 511 51q. 16 Ku/nicki. J. ami Filipck. A. (19~7) Biot'hem. I. 247. 663-(1tl7. 17 Fry. IJ.. Bcckcr-Ilapak, M, and I ldgelnall. ,I.I1. II'){ll)J, Baderiol, 17t, 25111i 2513,