Sheath disassembly in Methanospirillum hungatei strain GP1

Sheath disassembly in Methanospirillum hungatei strain G P ~ ' G. DENNISSPROTT~ Division of Biological Sciences, National Research Council of Canada, Ottawa, Ont., Canada KIA OR6

TERRYJ. BEVERIDGE Department of Microbiology, College of Biological Science, University of Guelph, Guelph, Ont., Canada NIG 2WI AND

Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by National Research Council of Canada on 02/07/13 For personal use only.

GIRISHCHANDRA B . PATELAND GIULIOFERRANTE Division of Biological Sciences, National Research Council of Canada, Ottawa, Ont., Canada KIA OR6 Accepted August 5, 1986

SPROTT, G. D., T. J. BEVERIDGE, G. B. PATEL, and G. FERRANTE. Sheath disassembly in Methanospirillurn hungatei strain GPl. Can. J. Microbiol. 32: 847-854. Sheaths of Methanospirillum hungatei are very resilient structures and consist of circumferential rings, which have been likened to the hoops of a barrel. The isolated sheaths are dramatically disassembled to the individual hoops upon treatment with P-mercaptoethanol at 90°C. Sheath disassembly resulted in a large decrease in suspension turbidity and a release of approximately 10% of sheath protein in the form of 4.6 to 7.0 kiloDalton ( m a ) peptides. These are referred to as "glue peptides" to suggest a function in linking the hoops together to form the intact sheath. The liberated hoops consisted of a protein surface array crystallized on a matrix material. Intact sheaths, or hoops largely freed of the glue peptides, could be solubilized at 90°C by ( i )a combination of P-mercaptoethanol and sodium dodecyl sulfate, (ii)by alkaline conditions of pH 12 or more, or partially (iii) by carbonate anion at pH 11. Fractionation by liquid chromatography of hoops solubilized by alkaline conditions of pH 12.6 revealed major polypeptides of about 24 and 45 m a ; a smaller peak occurred at 12 kDa. Based on the dimensions of the unit cell of the crystalline array, we suggest that two copies of the 24-kDa polypeptide form the 5.6 x 2.8 nm unit cell as a dimer, which then tends to form larger aggregates. The 2.8-nm subunit may be a dimer of the 12-kDa species. Sheaths of M. hungatei strain JF1 exhibited a similar polypeptide profile, but in this case, the 45-kDa species predominated.

SPROTT, G. D., T. J. BEVERIDGE, G. B. PATEL et G. FERRANTE. Sheath disassembly in Methanospirillurn hungatei strain GPI. Can. J. Microbiol. 32: 847-854. Les gaines de Methanospirillurn hungatei sont des structures trks rksilientes; elles sont constituCes d'anneaux circonf6rentiels qui ont 6t6 compar6s aux cercies d'un baril. Un traitement des gaines isolCes au P-mercaptotthanol a 90°C les a r6duits trks rapidement 2 leurs cercles individuels. Cette dissociation s'est traduite par une forte diminution de la turbidit6 de la suspension et par une IibCration d'environ 10% des prottines des gaines, sous forme de peptides de 4,6 a 7,O kiloDaltons (kDa). Ces peptides sont dits "collants" par r6fkrence a la fonction du maintien des cercles ensemble pour former des gaines entikres. Les cercles libCr6s sont constitu6s d'une assise de protCines de surface cristallis6es sur une basse matricielle. Les gaines intactes, ou les cercles fortement libCr6s de leurs peptides d'assemblage ont pu Ctre solubilists a 90°C: ( i ) par une combinaison de P-mercapto6thanol et de sodium dod6cylsulfate, (ii)par des conditions de pH alcalins de 12 ou plus, ou (iii)Ctre partiellement solubilis6s par I'anion carbonate a pH 11. La fractionation par chromatographie liquide des cercles solubilists sous conditions alcalines de pH 12,6 a mis en Cvidence des polypeptides majeurs d'environ 24 et 45 kDa, un pic plus petit et survenu 12 kDa. Sur la base des dimensions de I'assise cristalline par unit6 cellulaire, nous sugg6rons que deux copies de polypeptides a 24 kDa foment les unit6s cellulaires de 5,6 X 2,s nm, cornme un dimkre qui tend a former des agr6gats plus importants. La sous-unit6 de 2,s nm peut-Ctre un dimkre d'une espkce a 12 kDa. Les gaines de M. hungatei, souche JFl , ont pr6sent6 un profil polypeptidique similaire mais, dans ce cas, I'espkce ti 45 kDa a pr6domin6. [Traduit par la revue]

Introduction Methanospirillum spp. represents one of only two genera of sheathed methanogenic bacteria presently described. The sheaths surrounding these bacteria have circumferential striations and tend to fracture along these striations during isolation (Zeikus and Bowen 1975; Kandler and Konig 1978). The sheaths of these organisms can be isolated as hollow tubes following either mechanical breakage (Kandler and Konig 1978) or cell lysis at alkaline pH (Sprott and McKellar 1980), but further dismantling of the structures has proven difficult (Beveridge et al. 1985; Kandler and Konig 1985). The isolated sheath of M. hungatei strain GP1 contains on its outer surface a planar crystalline lattice having p2 symmetry (Stewart et al. 1985; Stewart et al. 19843). When Shaw et al.

'NRcC No. 26 333. 2 ~ u t h oto r whom all correspondence should be addressed. 3 ~ Stewart, . T. J. Beveridge, and G. D. Sprott. 1984. Crystalline order to a resolution of 4.7 A in the sheath of Methanospirillurn hungatei: a cross-beta structure. Proc. Annu. Meet. Electron Microsc. Soc. Am. 42nd, 1984. pp. 214-215.

(1985) examined the sheath of M. hungatei JF1, they found it to share the same general structural format as reported for strain GP1 (Stewart et al. 1984, see footnote 3), but the spacings and symmetry were different. The 5.6nm diffraction reflections which gave GP1 its p2 symmetry were faint and difficult to see in all electron diffraction patterns (Stewart et al. 1985; Stewart et al. 1984, see footnote 3), but obviously were real and represented some slight difference between successive rows of subunits. More recently, a comparison of the crystalline lattices of sheaths isolated from M. hungatei (strains GP1 and JFl) and Methanothrix concilii has shown that indeed all three have an identical subunit arrangement with p2 symmetry ( a = 5.6 nm, b = 2.8 nm, and y = 86") (Pate1 et al. 1986). The unusually small subunits of these arrays are arranged in a close packing format suggesting a sieving role (Shaw et al. 1985; Stewart et al. 1985). The stability of the sheath is remarkable. Resistance of isolated sheaths of M. hungatei GP1 to various denaturants was demonstrated by light scattering measurements in conjunction with electron microscopy and optical or electron diffraction (Beveridge et al. 1985). Resistance occurred at 22°C for a range

CAN. J. MICROBIOL..


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of proteases, a lipase, a cellulase, a glucosidase, P-mercaptoethanol, EDTA, 6 M urea, 6 M guanidinium hydrochloride, 1 0 M LiSCN, cyanogen bromide, sodium periodate, and sodium dodecyl sulfate (SDS). T o dismantle the sheath structure it seemed necessary t o employ 1N NaOH at 23"C, o r 3 N HC1 at 100°C. To account for the resistance of the crystalline surface array, w e predicted a covalent bonding of the subunits perhaps to an inner amorphous layer of the sheath (Beveridge et al. 1985). By electron microscopy w e have resolved two domains in each unit cell, each of approximately 2.8 n m diameter, which would b e consistent with a polypeptide of about 20 kiloDaltons (kDa) (Stewart et al. 1985; Beveridge et al. 1985). Further studies of the subunit composition requires complete dissolution and chemical resolution of the released polypeptides. In a recent comparative study of the sheaths isolated from methanogens, we found that in M. hungatei strain G P 1 (but not JF1) a combination of P-mercaptoethanol and S D S at 90°C caused the turbidity of the sheath suspension to clear (Pate1 et al. 1986). Here w e define a chemical method to dismantle the sheath into its constituent hoops which were then solubilized b y three methods to yield resolvable polypeptides.

Materials and methods Methanogens and growth conditions Methanospirillum hungatei strains GP1 and JF1 were obtained from the sources described before and grown in modified synthetic medium JM containing acetate (Pate1 et al. 1986). Cells were grown to the late logarithmic growth phase in 20-L glass carboys containing a C02-H2 (1:4, v/v) gas phase and 4 L of medium reduced with cysteine- sodium sulfide. Sheath isolation Cells of M . hungatei GP1 (2.243 dry weight) were processed exactly as described previously for cytoplasmic membrane isolation (Sproa et al. 1983). The procedure involved spheroplast formation with 16.2 mM dithiothreitol at pH 10, osmotic lysis, and sucrose gradient separations. The sheath was separated from membranous material in the first discontinuous gradient, consisting of 65 and 70% (w/v) sucrose (1 10 000 X g for 22 h). l'he pink cytoplasmic membrane band was removed from the top of the gradient by upward displacement with 80% sucrose. Sheath material banded at the 70% surface and was also distributed throughout the 70% sucrose layer (Sprott et al. 1983). Initially each of these two sheath fractions were processed separately. Each sheath fraction was dialyzed for 72 h against three 1-L volumes of water and collected by centrifuging at 13 200 X g for 15 min. Pellets were resuspended in 50 mL of aqueous 1% (w/v) SDS and heated at 90°C for 30min. After two 100-mL washes in water, each sheath fraction was loaded onto a second discontinuous gradient consisting of 65, 70, and 80% (w/v) sucrose steps. Following centrifugation for 22 h (I 10 000 X g), both preparations banded at the top of the 80% step and appeared identical upon microscopic examination. Therefore, the two fractions of sheath were pooled, washed three times in 50-mL aliquots of water and resuspended into 50 mL of water. The yield was determined by drying an aliquot to constant weight at 62°C. When required, residual spacer plugs were removed from the sheath preparation. Incubation at 23OC for up to 60 min in 0.1 N NaOH removed the plugs (Beveridge etal. 1985). The sheath was centrifuged through 50% sucrose (1 10 000 X g, 22 h), and washed extensively with water. Sheaths of M . hungatei JF1 were prepared by lysis of the cells in 0.1 N NaOH (Sprott and McKellar 1980) and subsequent SDS treatment (Pate1 et al. 1986). Electron microscopy Isolated sheath material was negatively stained with 1% uranyl acetate and viewed in a Philips EM 300 or EM 400 T microscope (Beveridge et al. 1985).

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Turbidity measurements The influence of various treatments was assessed by monitoring light scattering of sheath suspensions (Beveridge et al. 1985). The standard reaction mixture in a round cuvette (1 cm path length) consisted of 0.75 mL of 0.1 M buffer, usually CHES (2(N-cyclohexylamino)ethanesulfonic acid) at pH 9.0, other test reagents, and sufficient water to bring the volume to 1.3 mL. The tube was sealed with a cork plug and placed in a heating block (usually 90°C) for 5-10 min. The assay was begun by adding 0.2 mL of sheath suspension in water. Readings at 600nm were made using buffer as a blank. An A,jO0 of 0.090 corresponded to the addition of 1.3 mg dry weight of sheath. Separation of hoops from low molecular weight peptides Methanospirillum hungatei GPl sheath (33 mg dry weight), freed of spacer plugs, was incubated 30 min at 90°C in 12 mL of 0.05 M CHES buffer, pH 9.0, with 2% (v/v) P-mercaptoethanol. The mixture was centrifuged for 30min (81 600 X g). The pellet consisting of sheath hoops was treated again with P-mercaptoethanol, recentrifuged, and washed with water to give the hoop fraction. Low molecular weight peptides were present in the supernatant fractions. High-pressure liquid chromatography (HPLC) Prior to HPLC analysis, the hoops were solubilized and dialyzed at 23°C for I h against the appropriate HPLC buffer system. Hoop (or sheath) samples were solubilized at 90°C using either an arginineKOH buffer (pH 12.6), Na2C03buffer (pH 1l), or (3-mercaptoethanol in combination with 2% (w/v) SDS (see figure legends for details). HPLC separations were performed with a Superose 12 column (10 X 300mrn, Pharmacia, Sweden) using a 30mL/h flow rate of 0.2 M NH4HC03, pH 7.8. Calibration standards were bovine serum albumin, ovalbumin, carbonic anhydrase, soybean trypsin inhibitor, cytochrome c , bacitracin, and polymyxin B sulfate. Alternatively, a TSK G2000 column (7.5 X 600rnm, LKB) was used with 50mM sodium phosphate buffer, pH 7.0, at a flow of 60mL/h. Thyroglobulin, y-globulin, ovalburnin, myoglobin, and vitamin B12 were used as calibration standards. The HPLC system was manufactured by LKB (Fisher Scientific, Canada). SDS - polyacrylamide gel electrophoresis The system of Laemmli was used as described previously for 12% gels (McKellar et al. 1981). Hoop samples were prepared for analysis by solubilizing them for 30 min at 90°C in a 0.05 M CHES buffer, pH 9.0, containing 2% SDS (w/v) and 2% P-mercaptoethanol (v/v). Analytical methods Lipids were extracted from hoop preparations by the method of Bligh and Dyer (1959). Separation of the chloroform-soluble extract was performed on a0.25 mm silica gel plate (Brinkmann Instruments, Canada) using chloroform - methanol - acetic acid - water (85:22.5: 10:4, by volume). Carbohydrate was estimated by boiling samples for 5 min in phenol-H2S04 using glucose as a standard (Dubois et al. 1956). Protein was measured by the SDS-Lowry procedure (Peterson 1977) using bovine serum albumin as the standard. For the protein analysis, hoops were first made soluble by adding an equal volume of 0.1 N NaOH and heating to 90°C for 5 min.

Results Sheath isolation During purification, the sheath separated into two fractions on a sucrose gradient, one banding at the 70% surface and the other distributing throughout the 7 0 % layer. Subsequent heating of these fractions with 2% S D S removed membrane contamination which was observed within some of the sheath tubes, and yielded fractions which then banded identically at the 80% sucrose surface. The two purified sheath fractions were pooled, therefore, yielding 14.5% of the starting cell dry weight. The purity of this preparation is indicated by the control fraction shown in Fig. 1A.

Disassembly of sheaths to hoops Previously w e were unable to demonstrate a decrease in

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SPROlT ET AL.

FIG. 1 . Electron micrographs of negatively stained preparations of the M. hungatei GPI sheath (A) before and (B and C) after treatment with 2% P-mercaptoethanol (90°C, 30 min). (A and B) Bar = 500 nm; (C) bar = 100 nm. Note the presence of the surface array and the circular nature of the hoops.

turbidity of M. hungatei GP1 sheath suspensions exposed to 1% P-mercaptoethanol at 22°C (Beveridge et al. 1985). However, at 90°C the turbidity of the sheath suspension decreased with time, in a concentration-dependent fashion (Fig. 2). Comparisons using dithiothreitol (45 mM), P-mercaptoethanol, ethanol, and n-butanol (each 2%, v/v) showed that the disassembly was not simply a solvent effect and that the mercapto function of either sulfhydryl reagent was essential for

a significant decrease in A600 over a 30-min period at 90°C (data not shown). Sheath suspensions treated with P-mercaptoethanol always retained detectable turbidity. Examinations of negatively stained preparations revealed a dramatic disassembly of the sheath cylinders to the level of individual hoops (Figs. 1B and 1C). Some of the hoops appeared to retain some linkage or affinity to neighbouring hoops, but others were completely freed revealing

CAN. J. MICROBI(3L. VOL. 32, 1986

TABLE1. Quantitative distribution into hoops and low molecular

weight peptides of M. hungatei GP1 sheaths Carbohydrate

Rotein Fraction

mg

5%

mg

%

Lipid, mg

Hoops PeptidesNo. 1 PeptidesNo.2

31.55 2.34 0.741

91.1 6.8 2.1

5.53 0.22 0.074

95.0 3.8 1.3

NDa ND

1.04


-

Time at 9 0 ° ( m i n )

Nom: Shes* were prified as & m iin the legend m Fig. 2, and spacer plugs remmed witbNaUH. FoIlowlng~atmentat 90T lor 30rnin with P-rnercaptoetbanol. the preparation was centnfugcd for 30 mln { 81 600 x g). Tbe supmarant waq referred to as pe~tidesNo. I . The matment and cenoifugationsteps wcfc repented to give the fractions pephdes No. 2 (supernatant) and hoops (pellet). Law molecular weigh1 ppti&s were extenslvcly dialyzed against water and lyophrliled prior to the analyses. WD, not determined.

FIG.2. Influence of P-mercaptoethanolconcentration on the absorbance of sheath suspensions. Methanospirillurn hungatei GP1 sheath suspensionswere prepared by sphaeroplast formation, sucrose gradient purification, and SDS treatment. Spacer plugs were not completely removed. Reaction mixtures at 90°Cconsisted of sheath suspended into 50mM CHES buffer, pH 9.0. P-Mercaptoethanol (ME) was added from 0 to 2.0% (v/v) to begin the assay.

their circular, independent nature. The subunits of the crystalline surface array were clearly visible showing that P-mercaptoethanol had not disrupted the 2.8 nm subunit array. Hoops and low molecular weight peptides Disassembly of the sheath tubes to hoops by P-mercaptoethanol was associated with the release from the sheath of about 10% of the protein and 5% of the carbohydrate into a nonsedimentable form (Table 1). In keeping with previous findings showing lipid to be associated with the sheath (Sprott and McKellar 1980), about 3% of the hoop dry weight was extractable as chloroform-soluble components. When the extract was separated by thin-layer chromatography, the ether lipids found in the cytoplasmic membrane (Kushwaha et al. 1981; Sprott et al. 1983) were absent. However, a yellow band of unknown structure migrated close to the solvent front, indicating a nonpolar compound(s). The low molecular weight peptide fraction was not tested quantitatively for the presence of lipid because of a shortage of material. Dissolution by P-mercaptoethanol plus SDS Complete dissolution of the sheath and subsequent hoop structures required the simultaneous presence of both p-mercaptoethanol and SDS at elevated temperature. The reaction with whole sheaths proceeded in two stages, liberation of hoops from the sheath tube and subsequent disssolution of the hoops (Fig. 3A). At 60°C a decline in turbidity occumed. but very slowly. In the presence of P-metcaptoethanol (without SDS), only the first stage reaction occurred, Liberating hoops as shown by electron microscopy (Figs. 1B and 1C). Treatment with SDS alone at 90°C for 30min had little effect on light scattering measurements, and has been shown to have no disruptive effect on the surface array (Pate1 et al. 1986). Light scattering measurements using hoops (rather than sheath tubes) revealed that SDS and P-mercaptoethanol were both required in the reaction mixture for dissolution (data not shown). This implies a second function for P-mercaptoethanol additional to its role in releasing the hoops from the tube. Dissolution of sheaths by SDS and P-mercaptoethanol was strongly pH dependent (Fig. 3B), in keeping with a role for P-mercaptoethanol as a disulfide bond disruptant.

@ ME+SDS 0. I

no addition 0-0

0.05

pH5.0

xpH6.5

5

: 001

a

1

0.005

0001 0

I

I

I0

10

20

Time (min)

FIG.3. Absorbance changes in sheath suspensions treated with combinations of SDS and P-mercaptoethanol (ME) at various pH. Sheaths of M. hungatei GPl were prepared as described in the legend to Fig. 2. (A) Sheaths were suspended into 0.05 M CHES buffer, pH 9.0. m, No addition, 90°C; 0 , 2 % SDS, 90°C; A , 2% ME and 2% SDS, 60°C; A, 2% ME, 90°C; 0 , 2 % ME and 2% SDS, 90°C. (B) Sheaths were suspended in 2% ME and 2% SDS at 90°C. 0,0.05 M CHES, pH 5.O; x 0.05 M CHES, pH 6.5; 0,0.05 M L-arginine-KOH, pH 11.1. Dissolution at alkaline pH The sheath structure is sensitive to alkali at concentrations approaching 1 N at 22OC (Beveridge et al. 1985), and to lower concentrations at elevated temperatures (Fig. 4). Heating a sheath suspension in an arginine-KOH buffer system of pH 12.9 caused a rapid, biphasic reaction, resulting in a loss of absorbance. Dissolution did not occur at pH 11.1 in the absence of reagents such as P-mercaptoethanol and SDS (compare Fig. 3B to Fig. 4). Dissolution by carbonate Sheath suspensions were susceptible to hot sodium carbonate buffer, pH 11.0 (Fig. 5). The kinetics of turbidity loss in carbonate buffer was characterized by a short lag followed by a linear phase. After 50min about 18% of the initial turbidity remained. Electron microscopy of negatively stained preparations confirmed that the sheath tubes had been disassembled to hoops, similar to those shown in Fig. 1B. The reaction occurred

85 1


SPROTT ET AL.

Time ( m i d

FIG.4. Absorbance changes in sheath suspensions incubated in ahline buffers at W T . Sheath suspensions (see legend to Fig. 2) of M. hungarei GPI were incubated at 90°C in 0.05 M L-arginineto which KOH had been added to adjust pH. O , pH 9.9; a. pH 1 I . 1; A. pH 12.0: 0, pH 12.9. ELUTION VOLUME (mL)

FIG. 6. HPLC profile of hoop proteins and low molecular weight peptides. Isolated sheath preparations lacking spacer plugs were treated twice with P-mercaptoethanol to separate hoops from low molecular weight peptides. (A) The hoops were solubilized in 0.05 M L-arginineKOH buffer at pH 12.6 (90°C for 10 min) and dialyzed for 1 h into the HPLC buffer. A sample of 1.7 mg protein was separated on a Superose 12 column, using 0.2 M NaHC03, pH 7.8, as eluting buffer. The molecular weights shown in the figure represent averages of five separately solubilized preparations. (B) The column received 0.18 mg of low molecular weight peptides released by the first P-mercaptoethanol treatment.

0.01 1 0

i

I

20

I

I

40

I

1

60

Time (min)

FIG.5. Dissolution of the sheath structure by carbonate buffer, pH 11.O. The sheath preparation described in the legend to Fig. 2 was treated with NaOH to remove spacer plugs. Assays were begun by adding 0.75 mg sheath protein. The pH of the 0.067 M valine buffer system was adjusted to pH 11.0 with NaOH. 0 , Valine, 94°C; (7, valine plus 100 mM NaCI, 94°C; U, 50 mM Na2C03plus valine, 62OC; 0, 50 mM Na2C03plus valine, 94°C.

very slowly at 62OC, as compared with 94°C. It was dependent also on the concentration of carbonate used, giving a linear rate of dissolution up to 40 mM, and occurred more rapidly at pH 11 than at pH 10 (data not shown). Neither the valine buffer (pH 11) alone nor NaCl supplementation caused dissolution, showing that carbonate was the active agent. Since carbamate formation by reaction with amino groups would result in the generation of hydroxyl ions, we tested the effect of low concentrations of NaOH at 94°C. A decline in A6O0 of only 15% occurred after a 120-min exposure to 60 mM NaOH. Higher concentrations caused a loss of light scattering (Pate1 et al. 1986). High-pressure liquid chromatography P-Mercaptoethanol was used to isolate the hoops from the sheath tubes which had been pretreated with NaOH to remove

the spacer plugs. Hoops were then heat-treated at 90°C in an arginine-KOH buffer (pH 12.6) to release component polypeptides suitable for HPLC analysis (Fig. 6A). A large peak of AZgO absorbing material appeared in the high molecular weight excluded volume, followed by several smaller peaks. With each of five separately solubilized preparations four polypeptides wereresolved, andtheseaveraged82.0 + 1.2,44.8 1.6,23.8 k 1.0, and 12.1 ? 0.6 kDa. A hoop suspension prepared by treatment of strain GPI with P-mercaptoethanol lost turbidity upon treatment with hot carbonate buffer (see above). When subjected to HPLC analysis, three peaks were obtained in approximately equal amount corresponding to molecular weights of 20, 39, and 100 kDa. However, carbonate buffer (pH 11.0) was a less effective reagent than arginine-KOH (Fig. 6A), since most of the protein appeared in the high molecular weight excluded fraction. Isolated hoops were subjected to dissolution in the presence of 2% each of SDS (w/v) and P-mercaptoethanol (v/v) in a pH 9, CHES buffer at 90°C. The kinetics of the decrease in A600 lacked the biphasic nature seen for sheath dissolution (data not shown and Fig. 3), as expected if the first rapid phase represents primarily hoop separation from the sheath coupled with some dissolution of the hoops. Because SDS and P-mercaptoethanol absorb at 280 nm and both are present in high concentrations, it was necessary to remove most of these reagents prior to HPLC analysis. Following dialysis, an HPLC analysis revealed only peaks of molecular weight greater than 70 m a , with most of the protein present in the excluded volume of the column. This

*

852

CAN. J. MICROBI(3L. VOL. 32, 1986


and cross-linked agarose (Janson 1985) columns (Figs. 6A and 7B). The molecular weight of the protein previously referred to as 12.1 kDa was found with less certainty to be about 15 kDa with the silica column. A low molecular weight peptide fraction appearing at the end of the fractionation range (27 mL) was especially evident with the JF1 sheath.

ELUTION VOLUME (mL)

FIG.7. Comparison in HPLC elution patterns of M. hungatei JF1 sheath proteins (A) and GP1 hoop proteins (B). Hoops of M. hungatei GPl were prepared and solubilized as described in the legend to Fig. 6. Methanospirillum hungatei JF1 sheaths were isolated by the NaOHSDS method and solubilized at pH 12.6 in the same fashion as for GPl hoops. Elution profiles are shown following dialysis for 1 h into the HPLC elution buffer. Separations were performed on a TSK G2000 column using 0.05 M sodium phosphate, pH 7.0.

appears to represent a re-aggregation phenomenon, because a sampling of the same material when subjected to SDS polyacrylamide gel electrophoresis gave major bands of 12.7 and 21.5 kDa. Other bands appeared also, perhaps originating from the underlying layer of the sheath tube. Treatment of sheath tubes with P-mercaptoethanol not only caused the tubes to disassemble into hoops, but also released soluble low molecular weight peptides (Table 1) of 4.6 to 7.0kDa (Fig. 6B). These peptides appeared in only small amounts in the profile of the hoop proteins. Similarly, small amounts of hoop proteins were detected in the low molecular weight peptide fraction. Note that the peak appearing at an elution volume of about 25 rnL corresponds to residual p-mercaptoethanol.

M. hungatei JFl sheath The sheath of strain JF1 is resistant to dissolution by a combination of SDS and P-mercaptoethanol or by sodium carbonate, as monitored by A600. Also, the sheath tube is not readily disassembled to hoops by P-mercaptoethanol using the conditions found effective for strain GP1. The sheath is susceptible to alkaline conditions at 90°C, however, which allowed a comparison of the HPLC profiles of each strain (Fig. 7). The profiles for each strain were similar, except that for JF1 the major peak was of about 42 kDa rather than 22 kDa (TSK G2000 column). It is noteworthy that estimates of molecular weight for strain GPI were similar as determined on both silica

Discussion The isolated sheath tubes of M . hungatei strain GP1 are resistant to many denaturants and enzymes commonly found effective in disrupting other bacterial surface arrays (Beveridge et al. 1985). However, P-mercaptoethanol caused a dramatic disassembly of the tubes into their constituent hoops by breakage along the circumferential striations seen in negatively stained preparations. This disassembly (Fig. 1) demonstrated unequivwally that individual circular hoops are stacked to form the sheath tube, as predicted from occasional sighting of short segments of sheath (Stewart et al. 1985). The P-mercaptoethanol effect was temperature and pH dependent explaining why previous results with P-mercaptoethanol were negative (Beveridge et al. 1985). Possible solvent effects by P-mercaptoethanol were ruled out by showing that ethanol and n-butanol were ineffective; the mercapto function was essential for the reaction. During hoop isolation low molecular weight polypeptides were released (4.6-7.0kDa), suggesting a covalent linkage between the hoops via low molecular weight peptides ("glue peptides") containing disulfide linkages. This concept is illustrated diagramatically in model form (Fig. 8). Such covalent linkages, although still hypothetical, may help to explain the extreme stability of the sheath structure. Also, the tendency of the liberated hoops to retain contact with neighbouring hoops (Fig. 1E) may be explained by the incomplete release of the "glue peptides" (Fig. 6). The planar lattice resides on the surface of the sheath (Stewart et al. 1985) and is closely associated with an inner, amorphous portion which seems to make up the bulk of the sheath thickness (Beveridge et al. 1985). It has been difficult to determine the depth of the circumferential hoap sttiations in intact sheaths by electron microscopy, but their fracturability by P-mercaptoethanol at high temperature and pH suggests that the amorphous matrix is not continuous between hoops. Each hoop, then, is a separate structure consisting of outer lattice plus inner matrix which is cemented to its nearest neighbours by the "glue peptides." Methanospirillum hungatei strain JF1 and Methanothrix concilii also have sheaths with a 2.8 nm crystalline array (Pate1 et al. 1986). Differences among the sheaths were seen with respect to amino acid composition, sugar constituents, and (or) metal contents. Also, the sheaths were quite different with respect to dissolution by NaOH, indicating that Methanothrix concilii sheaths were most resistant and JF1 sheaths intermediate. Under conditions used in the current study, P-mercaptoethan01 caused significant release of hoops from sheaths of only strain GP1 of M. hungatei. Following treatment of the sheath with P-rnercaptoethanol at W C , the hoops could be easily separated from the "gIue peptides" by sedimentation. Disassembly of the hoops into resolvable polypeptides again required heating and 90°C was used routinely. First, hoops could be disassembled at pH 1 1 by the concerted action of P-mercaptoethanol and SDS. Evidently, once hoops had been formed, SDS allowed access of P-mercaptoethanol to disulfide bonds which were normally buried. Previously we suggested a covalent linking of the surface array


SPROlT ET AL.

I i

u Hoop

FIG.8. Model of a M . hungatei GPl sheath cylinder illustrating the crystalline surface array of 2.8 nm subunit repeats and the hypothesized peptide linkages between the underlying matrix layer of the hoops.

to the underlying matrix to explain the extreme resistance of the array to various reagents (Beveridge et al. 1985) and these present results are consistent with this. A second method effective to disassemble the hoop structure at 90°C was the use of basic pH >11. In contrast to the disassembly of isolated hoops by the above methods, the disassembly of the sheath tubes exhibited biphasic kinetics. The first phase was attributed to the release of hoops from intact sheath, and the second, to disruption of the released hoops. During preliminary experiments we were surprised to find that the sheath disassembled to hoops when exposed to sodium carbonatebuffer at 94OC. A further solubilization of the hoops to polypeptides occurred to a limited extent. The formation of hoops was attributed to the carbonate anion, and was shown to occur more rapidly at pH 11 than pH 10. Dissolution via hydroxyl ion formation during reaction with amino groups was discounted. Disruption probably occurred through the breakage of critical peptide bonds, aided perhaps by a repulsion of negatively charged carbamate groups. HPLC proved especially useful to separate the polypeptides released upon dissolution of the hoops by either alkaline conditions (pH 12.6) or by carbonate (pH 11). Both treatments released predominant polypeptides of about 24 and 45 kDa. Estimation of he molecular weight of a polypeptide which would fill the 2.8 nm subunit suggested a 20-kDa protein (Beveridge er a/. 1985). T h s fits remarkably well with the major 24-kDa polypeptide detected by HPLC analysis (Figs. 6 and 7). Two similarly sized subunits appear to be closely associated with one another and comprise the unit cell (Stewart er al. 19851, which could explain the presence of the 45-KDa species as a dimer of the smaller polypeptide. The formation of a still larger aggregate could represent a tetramer, yet, it is also possible that these other polypeptides represent the component parts of the underlying matrix on which the subunits rests. So far, we are uncertain as to the extent to which the planar array was separated from the matrix. Presumably, the matrix corresponds to the high molecular weight fraction appearing in the excluded volume of the gel filtration columns. Dissolution of the hoop structure at pH 12.6 was associated also with the release of a 12.1-kDa species. A main polypeptide fragment of 12 kDa was released also from sheaths of another strain of M. hungatei upon boiling for 3 min in 1 N NaOH

853

(unpublished data of E. Conway de Macario and H. Konig in Randler and Konig 1985). SDS - gel electrophoresis of hoops solubilized with SDS and P-rnercaptoethanol separated polypeptides of 12.7 and 21.5 kDa. Other molecular weight species were found also, perhaps indicative of matrix proteins. A point for future consideration is the possibility that the 24-kDa species {by HPLC analysis) may represent two equally sized polypeptides, or that the 12.1-kDa polypeptide may represent part of the 2.8-m subunit covalently linking the 24-kDa species to the matrix. Such a covalent iinkage has been suggested (Beveridge eta!. 1985). The sheath of M . hungatei GP1 contains metals and is especially high in calcium (Patel et al. 1986). The possibilityof ionic bonding within the sheath structure should be considered, since self-assembly of other bacterial regularly structured surface arrays can depend on bivalent cations (Beveridge and Murray 1976; Sleytr and Messner 1983). A sheath structure composed of circumferential hoops containing a small 2.8-nm subunit, regularly structured surface array has, so far, been described only for Methanospirillum hungatei and Methanothrix species (Stewart et al. 1985; Stewart et al. 1984, see footnote 3; Shaw et al. 1985; Pate1 et al. 1986). Such a structure should provide the cell with the ecological advantages of an effective sieving barrier and of mechanical protection. Because the hexagonal format of the end plugs is more porous than the sheath array (Shaw et al. 1985), increases in the filament length may provide a mechanism to decrease the rate of penetration of toxic substances. Indeed, M . hungatei extends into long filaments when the growth rate is decreased (Patel et al. 1979), which would vastly decrease the proportion of end plug to total surface area. Acknowledgements The expert technical assistance of Kathleen Shaw is gratefully acknowledged. We are indebted to R. E. Williams for discussions on the mechanism of carbonate action. The research undertaken in T.J .B .'s laboratory was funded by grants from the Medical Research Council of Canada and the Natural Sciences and Engineering Research Council of Canada. T. J., and R. G. E. MURRAY. 1976. Dependence of the BEVERIDGE, superficial layers of Spirillum putridiconchylium on Ca2+ or S?'. Can. J. Microbiol. 22: 1233-1244. BEVERIDGE,T. J., M. STEWART, R. J. DOYLE, and G. D. SPROTT. 1985. Unusual stability of the Methanospirillum hungatei sheath. J . Bacteriol. 162: 728-737. BLIGH, E. G., and W. J. DYER.1959. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37: 91 1-917. DUBOIS, M., K. A. GILLES, J. K. HAMILTON, P. A. REBERS, and F. SMITH. 1956. Colorimetric method for determination of sugars and related substances. Anal. Chem. 28: 350-356. JANSON, J.-C. 1985. Recent applications of high-performance liquid chromatography to biochemistry. Part I. Recent advances in high-performance liquid column technology. Biochem. Soc. Trans. 13: 1049-1052. KANDLER, O . , and H. KONIG.1978. Chemical composition of the peptidoglycan-free cell walls of methanogenic bacteria. Arch. Microbiol. 118: 141-152. 1985. Cell envelopes of archaebacteria. In The bacteria. Vol. 8. Edited by C. R . Woese and R. S. Wolfe. Academic Press, Inc., London. pp. 413-457. KUSHWAHA, S. C., M. KATES, G. D. SPROTT, and I. C. P. SMITH. 1981. Novel polar lipids from the methanogen Methanospirillum hungatei GP1. Biochim. Biophys. Acta, 664: 156- 173. MCKELLAR, R. C., K. M. SHAW, and G. D. SPROTT. 1981. Isolation


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and characterization of a FAD-dependent NADH diaphorase from Methanospirillurn hungatei strain GP1. Can. J. Biochem. 59: 83-91. PATEL,G . B., L. A. ROTH, and G. D. SPROTT.1979. Factors influencing filament length of Methanospirillurn hungatei. J. Gen. Microbial. 112: 41 1-415. PATEL, G. B., G. D. SPROTT,R. W. HUMPHREY,and T. J. 1986. Comparative analyses of the sheath structures of BEVERIDGE. Methanothrix concilii GP6 and Methanospirillurn hungatei strains GP1 and JF1. Can. J. Microbial. 32: 623-631. G. L. 1977. A simplification of the protein assay method of PETERSON, Lowry et al. which is more generally applicable. Anal. Biochem. 83: 346-356. SHAW,P. J., G. J. HILLS,J. A. HENWOOD,J. E. HARRIS,and D. B. ARCHER.1985. Three-dimensional architecture of the cell sheath and septa of Methanospirillurn hungatei. J. Bacteriol. 161: 750757.

SLEYTR,U. B., and P. MESSNER.1983. Crystalline surface layers on bacteria. Annu. Rev. Microbial. 37: 311-339. SPROTT,G. D., and R. C. MCKELLAR.1980. Composition and properties of the cell wall of Methanospirillurn hungatei. Can. J. Microbiol. 26: 115-120. SPROTT,G. D., K. M. SHAW,and K. F. JARRELL.1983. Isolation and chemical composition of the cytoplasmic membrane of the archaebacterium Methanospirillurn hungatei. J. Biol. Chem. 258: 40264031. STEWART, M., T. J. BEVERIDGE, and G. D. SPROTT.1985. Crystalline order to high resolution in the sheath of Methanospirillurn hungatei: a cross-beta structure. J. Mol. Biol. 183: 509-5 15. ZEIKUS,J. G., and V. G. BOWEN.1975. Fine structure of Methanospirillum hungatei. J. Bacterial. 121: 373-380.