FEMS Micrcrhiology Letters 95 (IYYZJ 207-112 1s:1092 F&ration of European Microbiological

Sociclic’s lJ~78-lt~J7/(J2/$llS.lWJ

Published hy Elvevicr

FEMSLE

IIJ’J’J~J

Isolation of a ferritin from Bacteroides fragilis

Received

II May

lYY2

Accepted IX May 101):

Key words: Bucteroiks frugilts; Ferritin; lron storage; Anaerobic bacteria

I. SUMMARY

2. INTRODUCTION

A ferritin was isolated from the obligate anaerobe Bucteroides frugiks. Estimated molecular masses were 400 kDa for the holomer and 16.7 kDa for the subunits. A 30-residue N-terminal amino acid sequence was determined and found to resemble the sequences of other ferritins (human H-chain ferritin. 43% identity; Esdtericitiu coli gert-16.5 product. 37% identity) and to a lesser degree, bacterioferritins t E. co/i bacterioferritin, 20% identity). The protein stained positively for iron, and incorporated ‘“Fe when B. fmgilis was grown in the presence of [‘“Felcitrate. However. the isolated protein contained only about three iron atoms per molecule. and contained no detectable haem. This represents the first isolation of a ferritin protein from bacteria. It may alleviate iron toxicity in the presence of oxygen.

Iron is an essential growth requirement for almost all living cells. However, in the presence of oxygen. iron is potentially toxic due to the catalytic generation of cell-damaging free radicals [I]. Consequently, excess iron is stored in ferritin, a protein originally isolated from mammalian tissues [2], but also present in plants [3] and yeast [4]. In aerobic bacteria, iron is stored in bacterioferritins. a distinct group of proteins [5] found in several species including Azotobucter tkelamfii [6], Escherichiu coli [7], Azotobucter chococcttn~ [8]. Rsettdomotzus uerttginosu [9] and Mycobucteritmt puruttrberculosis [IO]. However, a gene (getI-165) has recently been cloned and sequenced from E. co/i which encodes a protein having greater amino acid sequence similarity to human H-chain ferritin than to bacterioferritin ill: Both ferritin and bacterioferritin may not only prevent toxicity of excess iron but may also provide a source of this metal when exogenous iron is not available [2]. However, under anaerobic conditions, iron is likely to be readily available at

Corrc~pottdctrc~ a: E.R. Rocha. University Department of Btctrriology anu Immunology. Western Infirmary. Glasgow G I I 6NT. Scotlund, UK.

2O8

neutral pH as free Fe 2+. and filrthermore the absence of oxygen will remove the risk of toxic radical generation. Thus it would appear that anaerobic bacteria have little need for iron detoxification or storage mechanisms. Nevertheless. this may be an over-simplification. We have recently found that BacteroMes fragilis, an obligate anaerobe which can cause intra-abdominal infections, brain abscesses and septicaemia [12] and requires either haem, or protoporphyrin plus non-haem iron, for growth [13] may take up iron as Fe 3+ rather than Fe 2+ [14]. in this paper we report the isolation of a ferritin protein from B. fi'agilis grown under strictly anaerobic conditions, thus providing further evidence that uptake of non-haem iron by anaerobic bacteria may be more complex than previously assumed.

supernatant to a final concentration of 25% (w/v). The precipitate was removed by centrifugation and more ammonium sulphate was added to the supernatant to give a final concentration of 50% (w/v). The precipitate was recovered by centrifugation, dialysed against 25 mM Tris- HCI, 0.15 M NaCI, pH 8.8, and concentrated by ultrafiltration using an Amicon PMI0 membrane (Amicon, Stonchouse, UK). The concentrated protein was applied to a DEAE-Sephadex column and eluted with 251) mM potassium phosPhate, pH 5.8. For final purification, the sample was applied to a calibrated column (1.6 × 9(1 cm) of Sephacryl S300 HR (Pharmacia) equilibrated with 25 mM Tris. HCI, 0.15 M NaCI, pH 7.5, at a flow rate of 12 ml/h. Fractions giving a single band on nondenaturing polyacrylamide gel electrophor;,sis (PAGE) and staining positively for both protein and iron were pooled and freeze-dried.

3. MATERIALS AND METHODS

3.3. Polyaclylamide gel electrophoresis and Western blotthzg

3.1. Growth of B. fi'agilis B. fragilis 20656-2-1 was routinely cultured as described previously [14]. For ferritin isolation, B. fragilis was grown in Wilkins-Chalgren anaerobe blood agar (Oxoid, Basingstoke, UK) or in tryptone soya broth (Oxoid) supplemented with 5 g/I yeast extract (Oxoid), 0.1 mg/! protoporphyrin IX (Sigma, Poole, UK), 500 mg/I cysteine-HCI and 4 m g / l FeSO 4 • 7H20. Inoculated plates and broth were incubated for 48 h and 24 h, respectively, under anaerobic conditions (80% N 2, 10% CO_,, 10% H2). Bacteria were harvested by centrifugation (3500×g) for 15 min at 4°C and washed three times with 25 mM Tris. HCI, 0.15 mM NaCI, pH 8.8. 3.2. Purification of ferritin Cells were disrupted in a prechilled X-Press (LKB, Stockholm, Sweden). Purification was carried out by" a modification of the method of Smith et al. [15] tbr the isolation of E. coli bacterioferritin. Cell debris were removed by eentrifugation and the supernatant was heated at 65°C for 15 min and then immediately cooled to 4°C. The precipitated proteins were removed by centrifugation, and ammonium sulphate was added to the

Denaturing sodium dodecyl sulphate (SDS)PAGE (15% acrylamide) was performed according to Laemmli [16]. Non-denaturing PAGE was carried out using gels containing 5% acrylamide and 1% agarose. Gels were stained for protein with Coomassie brilliant blue and for iron with Ferene S [17]. Western blotting and subsequent immunochemicai detection using antiserum raised against E. coli bacterioferritin were carried out according to Andrews et al. [18].

3.4. N-termbl,d amhto acid sequence determbtation An aliquot of polypeptide was dissolved in 35 /,tl 0.2 M 4-methylmorpholine, 0.1% (w/v) SDS and dried onto a SequeionXM-diisothiocyanate (DITC) membrane disc at 56°C for 45 min. The DITC-coupled polypeptide was then subjected to automated solid-phase Edman degradation on a MilliGen/Biosearch 6600 ProSequencer.

Other methods Incorporation of 5'JFe was carried out using bacteria grown in broth as above, containing 5 p.Ci/I [S'~Fe]citrate (specific activity 10 p.Ci/p.g; Amersham, UK). The [5'JFe]-containing proteins

were fractionated by non-denaturing PAGE and detected by autoradiography. The UV-visible absorption spectrum was obtained using a PMQ 11 Spectrophotometer (Carl Zeiss, Oberkochen/Wurtt., FRG). The iron content of the isolated protein was determined by atomic absorption spectroscopy, and related to protein content, determined by the Lowry method.

!

2

§

4. RESULTS Electrophoresis in non-denaturing gels of the 511% (w/v) ammmonium sulphate precipitate of the B. fragilis extract revealed the presence of a protein staining strongly for non-haem iron (Fig. 1) This iron-containing protein had an clcctrophoretic mobility very similar to that of the main band of horse ferritin. The protein was found in extracts of B. fragilis grown in iron-supplemented liquid medium, but not in bacteria grown on blood agar. After further purification by ion exchange and gel filtration chromatography, a single band was observed on non-denaturing PAGE (Fig. 2). The melecular mass of the protein, as determined by the gel filtration using Sephacryl S-300 HR. was found to be approximately 4110 kDa. SDS-PAGE of the protein under reducing conditions showed a major component with a molecular mass of 16.'7 kDa (Fig. 3), indicating the presence of subunits of similar size to those of other ferritins and bacterioferritins. Two minor components with molecular mass around 40 kDa and 51 kDa were also detected. Autoradiography of the 50% ammonium sulph~te fraction from bacteria grown in the presence of "~'~Feshowed a single band corresponding to the protein that stained with Ferene S (Fig. 2). It thus seems likely that this protein is a ferritin. The sequence of residues 1-3[} from the N-terminus of this protein (Fig. 4) has a strong similarity with the H subunit of human ferritin (43% identity) and with the fcrritin-likc protein (gen-165 product) from E. coil (37%). However, there was only a 20% identity between the B. fragilis protein and E. coil bacterioferritin. When conservative substitutions scoring > 0.12

Fig. I. Non-denaturing P A G E of the 5{Ir~ {NIl 4 },SO; precipitate from B. fragili.~ extract. Bacteria were grown on bkmd agar Hancs I. 31 or in broth (lanes 2. 4) and stained with fA~omassie blue (lanes I. 2~ or Ferene S (lanes 3.4~. Lane 5 is horse spleen ferritin stained wii.h Ferene S.

in the mutation-data-matrix [19] were also considered, the similarities with human H ferritin, E. coil gen-165 protein, and E. coil bacterioferritin increased to 60~,, 53% and 3h%, respectively. The two fcrroxidase centre residues [211] of ferritin shown in the alignment (Fig. 4) were conserved in the B. Jhtgilis ferritin. Western blotting of B. fragilis ferritiq with anti-E, t'oli bacterioferritin antisera gave no evidence of immuno-cross-reactivity. The UV-visible spectrum of the isolated protein showed no ab-

210

klla i 66

-

45

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29

i ~ Non denaturing pAGE of the 5 ~'Ic~" tNtt 4).SO4 precip...... nes 1.3. 5) and the iron-con_ n frm,dts extract fla . ~ ,~ Otis were suuned irate from u. ~, Z ...... rification tlartcs -. ",. ~ ~,-ne" 3 4). lainingproteln alto, i1~ • ~ and Ferene ~ x,. ~ -" . -- - .7"--rn" "sic blue (lanes ~. -" , t o f B fragilis grown in ~¢ttn ~_t~t, as, . '- o f the -x~ra~- ~ • • ne 5. autoradtograpny c.. ~ . La broth containing r e .

-

18.4-

F g. %

s o r p t i o n p e a k s i n t h e v i s i b l e r e g i o n ( F i g . 5), i n d i c a t i n g t h a t l i t t l e o r n o h a e m is b o u n d , W h e n t h e 50% ammonium sulphate fraction from bacteria to non-denaturing g r o w n w i t h 5~Fe w a s s u b j e c t e d radioactive PAGE and autoradiograph~', a single ferritin was band corresponding to the B. fragilis seen. This indicates that the ferritin acquires iron

14.3"

• " "ns Lane l. molecular.mass ?~ark. 'DS.PAGE ot fern~ .- . . . . . . x 8 fragilis ~ermm. F~g. 3. S -~ - -se snleen iernun; t,x. . . . . . . . ' ers~ lane -, no. r

ZO

m

~..----,....-,-~

::"":D ~ ' E ' ~

~[ K V ~ "tt..~.....". . . . . . . . . . ills [erritin ( B I ' F T N ) Wuu._!-. are indicated w i t h s o l i d Vo~ ~ ECB~'R - . , .Amino acid sequence ol .°~,!.'.~:~iofcrritin (EcBFR)..I.°enUut~ i.~ in the rautation-data'ma'r •, ent oi" the bl-tcrmlna, educe arid E. cull uac~ _.ha substitution giving. >- • " Fi~. 4, Ahgnm . . ,.::cw..rN. eene-165-pr . ".,.-:ties ~conSe~-~ - " (t~umt'D E. colt . . ierrltm ',~" ~'~..ted with dotted boxes. e r"~,~;a O a , ~ i S b "e" ¢ ;,entre residues. SIm ti t.lt | Asterisks represent the 1 r [1¢)]) are mun..,

04"

03'

02

01

0.0 200

•

,

.

300 Wavelength

( n m )

Fig. 5, UV-visible spectrum of B. t)'agili~ fcrritin (I).lt;2 m g / n t l in 25 mM T r i s . l i ( ' l . 11.15 M Na('l, ptl ,',l.l)).

in vivo. However. atomic absorption spectroscopy gave an iron content of only three atoms of Fe per molecule.

5. DISCUSSION In this work, an iron-containing protein was isolated from B. fragilis displaying characteristics of a ferritin. The protein not only contained iron, as detected by Ferene S, but also incorporated iron metabolically during growth, as shown by autoradiography. Furthermore, it had a similar eiectrophoretic mobility to horse ferritin, and had a molecular mass, d e t e r m i n e d by gel filtration, of 4110 kDa. Finally, on S D S - P A G E the protein had a major subunit band with molecular mass of 16.7 kDa. T o g e t h e r these properties suggest that the protein is a ferritin. Iron-storage proteins have been classified into two groups, ferritins (class I) and bacterioferritins (class II) [21)]. The B. fragilis pro,*e~n is similar in size to o t h e r bacteriofc;ritlns, which ha~:e molecular masses of 260-660 kDa and subunits of 15-20 kDa [6-11),21]. However, the 3l)-residue N-terminal amino acid sequence shows a greater identity to ferritins (class i) than to bacterioferritins (class 11). Furthermore, there was no immtmological cross-reactivity b,-twcen the B. fragilis protein and E. coil bacterioferritin, although the latter does cross-react with bacterioferritins from o t h e r microorganisms, but not with mant-

malian or plant fcrritins [5]. T h e absence of any detectable haem in the protein also suggests the B. fragilis fcrritin is more closely related to mammalian ferritins than to baeterioferritins, as the latter invariably contain h a e m [22]. Surprisingly, the iron content oI the /~. fragilis ferritin was extremely low, only about three atoms per molecule. However, the gen-165 product from E. coil, purified from an overexpressing strain, was also found to have a very low iron content (Hudson, A., Andrews, S.C., Guest, J.R. and Harrison, P.M., unpublished data). Nevertheless, the identification of the protein as a ferritin is confirmed by the conservation of the two key ferroxidasecentre residues [211] within the sequenced region of the 13. fragilis protein. The two additional weak bands seen on SDSP A G E . c o r r e s p o n d i n g to polypeptides with molecular masses of 44 kDa and 51 kDa, may represent contaminants or o t h e r ferritin subunits. However. only a single N-terminal amino acid sequence was found, so covalent dimers, as have been found in sheep ferritin [23], might be responsible for thcsc bands. From these results, it is clear that B..fragilis contains a ferritin. These findings are of interest for two reasons. Firstly. this is the first isolation of a fcrritin from an obligate anaerobe. Since such bacteria grow in the absence of o.'~gen, problems of iron toxicity, and hence a mechanism for detoxifying the metal, would seem to be unnecessary. However, we have previously shown that iron uptake by B. J)'agilis is a more complex process than simple acquisition of Fe -'~. and tha,, a F c - " ~ Fe 3+ conversion may be necessary a~ some stage [14]. This suggests that some kind of redox reaction may occur, which raises the possibility of free radical generation. Forthermore, anaerobic bacteria may at times be exposed to atmospheric oxygen, which may cause a sudden oxidation of iron with consequent increased likelihood of oxygen radical formation. The potential toxicity of these radicals may be exacerbated by the lack of any protective factors such as superoxide dismutase or catalasc, which are either present at very low levels or absent [12]. T h e presence of a ferritin which could store and detoxify any excess iron under aerobic conditions may

2t2 help to explain why B. J'ragifis can maintain its viability even after e x p o s u r e to air for 24 h or m o r e [12]. This h y p o t h e s i s is consistent with the very. low iron c o n t e n t of B..fragilis ferritin, which was isolated directly from bacteria g r o w n u n d e r strict a n a e r o b i c conditions. T h e fact that this protein was not detected w h e n bacteria w e r e g r o w n on blood a g a r also s u p p o r t s this h y p o t h e sis, as o r g a n i s m s growing on the latter m e d i u m will acquire all their iron from h a e m , and thcreftrre have no need to take up n o n - h a c m iron [14]. T h e s e c o n d point of interest is that the B. fragilis fcrritin s h o w s m o r e similarity to h u m a n H-ferritin and to the E. colt get-165 p r o d u c t t h a n to bacterioferritin. T h u s , the p r e s e n t work r e p r e s e n t s the first isolation of a ferritin (class I ironstorage p r o t e i n ) from a b a c t e r i u m . F u r t h e r w o r k will be r e q u i r e d to establish w h e t h e r fcrritins tire f o u n d in o t h e r m i c r o o r g a n i s m s , and if so, how their function relates to that o f bactcrioferritins.

ACKNOWLEDGEMENTS W e thank Dr. R. Parton, D e p a r t m e n t of Microbiology, G l a s g o w University, for use of the X - P r e s s and Dr. D. Halls, D e p a r t m e n t of Biochemistry. G l a s g o w Royal Infirmary, for p e r f o r m ing the atomic a b s o r p t i o n spectroscopy. S.C.A. was s u p p o r t e d by T h e W e l l c o m e T r u s t , and E.R.R. by the C o n s e l h o N a t i o n a l de Desenvolvim e n t o Cientifico e T e c n o l o g i c o - C N P q (Brazil) and T h e British Council.

REFERENCES [I] tlalliwcll. B. and Guttcridgc. J.M.C. 119841 Biochcm. J. 219. 1-14.

[2] Thiel. E.C. (19871 Annu. Rev. Biochem. 56. 289-315. 131 Laulhcre, J,P,, Lescuric, A.M. and Briar, J,F, 11¢1881 l. Biol. Chem. 263. 1(1289-10294. [4] Raguzzi, F.. Lesuissc. E. and Crichton. R.R. (19881 FEBS Left. 231. 253-258. [5] Andrews, S.('., Finldlay. J.B.('.. Gucsl. J.R.. Ilarri:,on. P.M.. Keen. J.N. and Smith, J.M.A. (1991) Biochinl. Biophys. Acta 11178. I I I-116. [6] Stiefel, E.I. anti Walt. G.D. (19791 Nature 279. 81-83. [7l Yariv, J,, Kalb, A.J., Spcrling. R,, Baumingcr, E,R., Cohen. S.(J. and Ol~:r. S. (19NI) Biochcm. J. 197. 171-175. [81 ('hen. M. and Cricht~m. R.R. 119K21 Biochim. Biophys. Acta 7117. I-6. 19] Moore, G.R,, Mann. S, anti Bannister. J.V. (19~61 J. Inorg. Biochcm. 28, 329-336. [l()] Brooks. B.W.. Young, N.M.. Walson. D,('.. Robcrtson. R.H, Stlgc.lcn. E.A.. Niclscrl. K.II. and I-~c~ker. S.A.W.E. ( 1991 ) J, Clin, ~.,!iciohs~l. 2~,'. Ih52-1(158, 11I] Izuhara, M.. Takar~2u~lc, ~.. and Takala. R. (1991) Mol. GerL (;t:wcl. 225. 51t~ : i3. [12] Finegold. S.M and George. W.L. (198~," Anaerobic Infections in [{umans. Acadc, ;c P~css, San Diego, ('A, [13] Otto. IS.R, Sparrius, M., Vc~wcij-van VughL A.M.J.J. ;rod M;~cl,z~ren, D.M. (t990~ 1n|¢¢1. Immun. 58. 3954395.-~. [141 Rocha, t:.R., tie tJzeda, M and Brock, J.H. (19911 FEMS Microbiol. Lclt. 84.45 511. [15] Smith. J.M.A., Quirk. A.V.. Phmk, R.W.II.. Difl'iul, F.M., Ford, G.('. ~md Harrison. P.M. (19881 Biochcm. J. 255. 737-7a0. [1~] Lacmmli. U.K. (197111 Nature 227, 6811-685. [I 7] Chung, M.C.M. (1985) Anal. Biochem. 148. 498-5112. [18] Andrews. S.C., Harrison, P.M. and Guest. J.R. 119891 J. Bacterit)l. 171, 39411-3947. [lt)] Schwartz. R.M. and Dayhoff. M.O. (1979) In: Atlas of Protein Sequence and Structure (Dayhoff. M.O., Ed.), pp. 353-358. National Biomedical Research Fundation, Washington, DC. [211] Andrews, S.C.. Smith, J.M.A.. Yewdall. S.J.. Guest, J.R. and Harrison, P.M. 11991) FEBS t,cit, 293, 164-168. [21] Kurokawa. T.. Fukumori. Y. and Yamanaka. T. (19891 Biochim, Bioph~,s. Acta 976, 135-139, [221 Fatemi, S.J.A., Kadir. F.It.A.. Williamson. D.J. and Moore. G.R. (19911 Adv. hloo'g. ('hem. 36. 409-448. [23] Mcrtz, J.R. and Thicl, E.C. 119831 J. Biol. Chem. 258, 11719-11726,