Ill

Btm homea e l Bl.pk~ ~u o 4~ ta. 1()7~ I 1991 } l 11 116 ' 1991 k.l~,c..~t:r ~clv,:¢ Publish,m, B V ~167-48~g/" ~ ~ a q~

BBAPRO z3~2~1

Physical. chemical and immunological propertit.. of the bacterioferritins of Escherichia colt, Pseudomonas aeruginosa and Azotobacter vinelandii S i m o n C. A n d r e w s ~, J o h n B.C. F i n d l a y -' J o h n R. G u e s t ~. Pauline M. Harrison ~, Jeffrey N. Keen -' a n d J o h n M,A. S m i t h ~ ' lm,J,x Imtautc [.r Bmm-/~ ~Idar Reu,an h. Department q[ r~Oll't ulur B;td.gl and ~Ymtct hm)l.I,,I, (t~le ct m'i ; t 5h£lhda. ~lliS'/~Od ~t h ; a~td ' Departmenl ;~[ Bl.lht, oll llrl. [ tlt~ ¢i'~ll! . [ la'i'dL la'ed~ I I K

~Rccet'.ed 4 September !9~q fRe~ixed m a n u ~ n p l received I!i tanuar~ 1991

Kc~ v,ords l]aclcr]ol'¢rrllln: t-~lrlIlli. ]T~n MoLlie

The 70-amino-acid-residue N-lerminal wequence o1"the hacleriolerrilin (BFR) of A:otobacter rinelandii ~as determined and simon to be higld) similar to the N.terminal ,~-,quence~ of the Ewheriehia co/i 'and .~lrobacrer [email protected] baelerioferrilins. Eleclropl~relie and immunological anal),,e~ I'urlher indicate lhat the baeterioferritins oI jr. co//, A. rinelandii and Pseudomonas aerugino,,a are do~el) rdaled. A noYd, I',~(r-,,ulmnil as,,embl) state thai I~'edeminate,, over the 24-subunil form of BFR at low pH wa,~demonstrated. The re~lll~, indicate Ihal lbe baclerioft~rilin,, form a I'amil) of proteins thai are dMincI from the ferritins of plants and animal,,.

Introduction

Bacterioferritin (BFR) is a mullimeric, iron-storing haemoprotein. The presence of an iron-storage protein in Estheru'hia coli was first suspected from M;3ssbauer spectra o1 cells grown in :?Fe-enriched media ~hich indicated the presence of a p~.31 of non-haem iron [1]. An iron-containing protein was subsequently isolated I2.31 and electron micrographs sho',xed Iha! each molecule contained an electron-dense core (of non-haem ironl surrounded by an 'electron-transparent'. spherical protein shell [3]. B', virtue of its similarity to ferritin. the iron-storage protein of eukaryotes, the protein ~'as termed a "bacterioferritin'. The cloning and nucleotide sequence of the baeterioferritin gone {f~[r) of £ ~oh K-12 have recently been rcporled [4.51. In overall ~ize. Ahbre'.ialion'.: BFR. bacler~oferri.qn; IEP. ~ : h : d n c poml; IEF, i~o¢lcctri,, locus:..mg: PAGE. polyaco!amlde gel ek~m~phore~ ~. SDS s ~ i u m d~e~'.l .,ulphat¢: A'.. ,-12otohalter ~me/a~ldl~: Ec. Eah~rch~a (¢i1~: ~ , ~,'HrO~l~let ~ln.gradsktt: Pa. Pwud, mtt,na~ ~ , r ~ l / i . s a

* the Ierm ~¢quc~¢c 'qri311~fflD. I~ u~,~2d,~ctord~ng to Ih¢ ro.O.qlmendah',m ~ff R,m.k ¢I ~l [27 I ('orrcspiindcnce: J M . A . Smilh. Du~atllllerll d~[ M~d,ct.ul,~r B~:flt~g,. & Bu'ilechn,~h~g~, I'ni~.cr.lt?. of Sheffield Sheffield. SI0 2TN. L . K

E, .d~ BFR (Ec-BFR) is similar to mammalian ferritin. cnmprising 24 protein suhunils arranged in 432 s~mmetn to form a hollo~ ~,hell v, ith an a~erage external diameter of approx t20 A in hydrated crystals I6.7[ Howe',or. unlike ferritin. BFR contains protoporphyrin IX 13] and the deduced amino acid ,,equence ~ho~;s ~irtuall~ no qrnilaritv" ~xith an: of the eukar~mtic ferritin ,equence~, 14]. It has also been shov, n [g,9] that

Ec-BFR i', indi.,tingni':,hahie from the 'c,,mchrome h-l" isolated earlier h 3 Deeh and Hagcr [10J and Fujita et aL ]||l Stiefel and Wau [12j obsmed that cytochrome /',557.5 of t-_omfia(ter ~tnelamla. a protein purified earlier h,, Buicn etal. [13]. resembled ferritin. Hov, e,,er, like Ec-BFR. it wa~ found to po~,sess b-t~pe hacm at a stolchioltlutt) of 1 flaem:2 pr~qcm suhunib and ~.~a',, subsequentl) designated a BFR. Coutometric litrauons ga',e a reduction potential. E,I. of -416 mV and this led Stiefcl and Veatt [12] to postulate that A. cineland, BFR (A.,-BFR) participates in bo!h iron-storage and electron-storage. BFR ~as independently iwlated from A cme[ol~d, h~, IJ et al. 114]. X-ray analyr, is of Av-BFR c3stal'. [7] ga~,e packing diamelers (121-126 J,) ,~imilar to Ec-BFR. A number of other bacterial b-type cytochrome~ ha~e spectro~opic or other physical properties charac-

112 leristic of bacterioferritins. However. only three of these have been shown to posses~ a non-haem iron-core and may thus be designated bacterioferritins. These are cytochrome h-557.5 of A. chroococ'cum [15], cytochrome b-557 of Pseudomonas aerugmosa [16] and cytochrome h-559 of Nitrohacrer winogradsk)'i [17]. Here the N-terminal amino acid sequence of A. rmelandii BFR is reported and compared with those of the E. coil and ,V. winograd40'i proteins. The immunocrossreactivitics and electrophoretic properties of A, einelandii, E. ~oh and P. aeruginosa bacterioferritins have also been compared. It is concluded that bacterioferritins form a discrete, closely related family of proteins, resembling the ferritins of higher plants and animals. However, differences in apparent subunit size and subunit assem'~ly are found within members of the BFR family. Materials and Methods

Protein purification Ec-BFR was obtained from a BFR-overexpressing strain, JMI01(pGS281) [5]. The protein was partially purified by heal denaturation of soluble cell extracts (60-70°C, 10 rain) followed by centrifugation (20000 × g for 30 min at 4°C), and precipitation in the 28--40% (w/v) ammonium sulphate fraction. The redissolved precipitate was dialysed against phosphate-buffered saline (1.5 mM KH,PO~/8,0 mM NazHPOJ0.15 M NaCI/3.0 mM KCI (pH 7.2)) before removal of remaining insoluble matter by eentrifugation (20000 × g for 30 min). Ec-BFR was further purified by gel filtration (Sephacryl S-300) in phosphate-buffered saline followed by anion-exchange chromatography (Q-Sepharose) in 20 mM histidine buffer/0 to 0.5 M sodium chloride (pH 5.5). Purity of protein samples was assessed by electrophoresis. Samples of pure BFR isolated from A. tqnelandii and P. aerugmm'a were kindly supplied by Dr. G.D. Wall

£scherzchza colz

EscherJ¢~ia colz Azotobacter vzne]andll Nztrobacter wzno~radskyl

i~

K G

~IF

and Dr. G.R. Moore, respectively. Av-BFR was further purified by reerystallisation. Po(vaervlamide gel electrophoresis (PAGE) Electrophoresis was carried out according to Laemmli [18] under either denaturing conditions (0.l~ sodium dodecyl sulphate (SOS). 155[ acrylamide) or non-denaturing conditions (no SDS. 6.5% acrylamide). Prior to electrophoresis under denaturing conditions samples were treated at IO0°C for 10 min in approx. 40 FI of 0,1 M Tris-HCI/10% (v/v) glycerol/2.3% (w/v) SDS (pH 6,8). Gels were stained with 0,1~ Coomassie blue. Isoelectric focus.ring (IEF) IEF was performed in pH range 3.5 to 9.5 Ampholine PAG plate gels (Pharmacia LKB, Uppsala, Sweden1 upon a Macrophore horizontal bed, acce,'ding to manufacturer's instructions. The Pharmacia Broad p/ Calibration Kit (pH 3-10) was used to provide pl standardisation. Ouchterlom" douhle-immum,di//usion Ouehterlony double-immunodiffusion was carried out according to lh,,dson and Hay [19]. Antigen samples were loaded at approx. 1 mg/ml concentration. Antiserum raised in goat to Ec-BFR was kindly ,~upplied by Dr. J. Yariv. N-terminal amino acid sequence determination of A. vine. landii BFR Sequencing was performed using an automated solid-phase sequencer essentially as de~ribed by Findlay et aL [20]. In summary, lyophilised protein was dissolved in 0.2 M NaHCO3/0.25% SDS (pH 8.5), and coupled at 56°C for 60 rain under N: to diisothiocyanate glass 117 nm pore size, 200-400 mesh). After washing, the coupled protein was subjected to automated solid-phase Edman degradation. The derived anilinothiazolinones were converted at 70 °C for 20 min

tO

20

30

40

50

60

DiT

K!N ~ G b KiR L ~ N ~ V = L ~ K ~iG

v E~w

E Y E Z S I D ~ H K ff A D~IR Y~IIE .

E v.

E s z D E ,

K ,

~

L~tKi

70

I ig |. Alignment of lhe N-lcrminal amino acid ~qu~nce~ of hactenofertilin~ flora A Imelu,uhf. E ~oh [4 i ;rod N wtnt,grad~k;i [|71 (onscp.'ed amino aod residue~ are h~xed

113 in 30~ (v/v) aqueous trifluoroacetic acid and the correspondipg phenylthiohydantoin derivatives identified hy reverse-phase (C~) microbore ItPLC using a gradient of acetonitrile in sodium acetate lpH 4.9).

Ps Hs Pa Av Ec ..----78000 -

- . . . .

Results and Discussion

The first 70 residues of the amino acid sequences of Av-BFR were determined and aligned with the N-terminal sequences of Ec-BFR and N. winogradsk3i BFR (Nw-BFR) (Fig. 1). An optimal alignment was obtained without insertions or deletions. The sequence identities over 50 or 70 equivaleneed residues were: 74~:~;for A~and Ec-BFR'- 54% for Av- and Nw-BFR; and 60~ for Ec- and Nw-BFR. Seconda~ structure predictions for the two partial sequences and the complete 15g-residue sequence of Fx:-BFR indicate a high a-helix content. This is consistent with the formation of a 4-helix bundle by the BFR subunits which is the major structural motif of ferritin and various bacterial cytochromes [42122] Electron paramagnetic resonance and magnetic circular diehroism spectra of the haem groups in Av-. Ecand Pa-BFR are unlike those of any other haemoprotcin and they' are thought to arise from bis-methionine axial haem ligation 1231.The Ec-BFR subunit contains se~en methionine residues of which Met-I and Met-52 are conserved. Met-31 is replaced by Leu in Nw-BFR and may thus be excluded as a potential haem ligand. Residues equivalent to Met-86. Met-119. Met-120 and Met-144 in Ec-BFR. lie beyond the sequenced regions of the other bacterioferritins. Ligation to Met-I and Met-f44 or to Met-86 and Met-144 could place the haem group close to one end of the 4-helix bundle as observed in bacterial cytochromes b-562 and ~ i22]. In Fig. 2 the electrophoretie mobilities of denatured Ec-. Av- and Pa-BFR are compared with those of horse-spleen ferritin and pea-seed ferritin. Upon SDSPAGE Ec- and Pa-BFR were resolved into single band~ with nominal M, values of 185013 and 18000. respectively, whereas Av-BFR gave two bands of appro,~imately equal staining intensity. M+ 2 1 2 ~ and 21 700. The M, values for the Ec- and Pa-BFR agree ~ith previous estimates of 18500 [5] and 18000 [16]. respo.lively. Av-BFR was previously reported to gi~e either a single band corresponding to a subunit of ~1~ 170(X) [12]. or two bands of M, 19500 and 13000 124]. The latter may' represent a degradation pn~uct. Av-BFR gave a single M, 18000 band on continuous SDS-PAGE. but two bands of 21000 and 23000 on discontinuous SDS-PAGE; the 23000 band appearing only after treatment with #-mercaptcethanol [25]. No evidence of se. quence heterogeneity was detected in the present aork. Nw-BFR also showed two bands corr~pnnding to 19500 and 17000 on SDS-PAGE. but only one (.~I+ 19500) was observed in the presence of B-mercaptoethanol I171.

,-+~,

66000 4,5000

----30000

~17200 = 12300

Ftg 2 SDS-PAGE115"; |t ,~f t~ca-,ecd f:rntffi (P,I, hor,e-,p~ccn [,.:rrltln (Hs)and h3~.t¢lloferniill, ~rilm ~:~ ~.it IECl | ¢lnc/Otldlt t ~ ) arid P oeeu~mo~a +Pa!. Proteins ~,¢r¢ nl.~lrlcd ~llh ('~rnan~l¢ hl~c and the ',1, ::due, ~! standard pn+t,zm~ ,are irld~ca!ed

A h h o u g h the source of the apparent ~ubunit heterogeneity in some bacterioferritins

is uncertain,

mam-

ferrilins gi~e t~o bands upon SDS-PAGE (Fig,. 2) and these are known to correspond m chain~ of differem amino acid sequence [211. Pea-~ecd ferritm malian

Pa

O

Ec

.............I

Av

"~ BFR2~

,BFR2

ltb ( e