INTRODUCTION

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Xylans are included within the family of plant cell wall heteropolysaccharides referred to as hemicelluloses. They consist of a -1,4-linked xylopyranose backbone, to which are often attached side groups of arabinose, (O-methyl-)glucuronic acid, ferulic or p-coumaric acid, and/or acetate, depending on the plant source (2, 43). Next to cellulose, xylans are the most abundant polysaccharides on earth (4), and it has been estimated that about 10¹⁰ metric tons are recycled annually, with the degradative arm occurring largely through the action of microbes (47).

In an effort to increase our understanding of the microbiology and biochemistry of xylan degradation, we initiated a study of the xylan-hydrolyzing system of a new, anaerobic, gliding bacterium, Cytophaga xylanolytica XM3 (12). This bacterium and other freshwater and marine strains similar to it were isolated from freshwater sediments on the basis of their ability to adhere to and dominate the fermentation of insoluble xylan particles in sulfidogenic and methanogenic enrichment cultures. Unlike the secreted xylanases found with most other organisms, the xylanase system of C. xylanolytica is almost entirely cell associated, but it can be easily extracted from whole cells by using 0.2% Triton X-100. Such Triton X-100 extracts were shown to possess activities important for xylan hydrolysis, including endo-1,4--D-xylanase (EC 3.2.1.8), -D-xylosidase (EC 3.2.1.37), -L-arabinofuranosidase (EC 3.2.1.55; referred to below as ARAF), and -D- and -D-glucopyranosidases (EC 3.2.1.20 and EC 3.2.1.21, respectively) (13). Triton X-100 extracts were also remarkably stable, retaining full xylanolytic activities for more than 6 months when stored at 4°C; however, they exhibited no activity toward microcrystalline cellulose, ball-milled Whatman no. 1 cellulose filter paper, or carboxymethyl cellulose (13). The latter observations were consistent with the inability of cells to grow on cellulose or carboxymethyl cellulose.

Efforts to resolve the nature and number of components of the xylanase system yielded several column chromatography fractions, each enriched with multiple endoxylanase (ENDOX) activities, as well one fraction enriched with a single ARAF activity. ARAFs are important because they catalyze the hydrolytic removal of arabinofuranosyl residues from hemicellulosic polysaccharides (18). Such residues are typically attached by -1,2- and/or -1,3-linkages to the backbones of arabinoxylans, arabinans, and arabinogalactans (4, 9), and their removal by ARAFs usually facilitates the attack of the xylan backbone by ENDOXs (11, 23, 35, 49). Here we report the purification and properties of an ARAF (ArfI) produced by C. xylanolytica XM3 when it is growing on arabinoxylan. Also included in this paper are the results of studies performed to (i) determine the cellular location of ArfI, (ii) examine the synergy between ArfI and ENDOXs of C. xylanolytica, and (iii) evaluate the occurrence of ArfI-like proteins in other freshwater and marine strains of C. xylanolytica. In another paper (19) we will report on the cloning and sequencing of the ArfI-encoding gene (i.e., arfI), as well as of an additional gene (arfII) that encodes an ARAF not synthesized by C. xylanolytica during growth on arabinoxylan.

(A preliminary report of the findings has been presented previously [36].)

	MATERIALS AND METHODS

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Growth of cells and preparation of cell extracts. C. xylanolytica XM3 (= DSM 6779) and other freshwater strains were grown anoxically at 30°C in rubber-stoppered 2-liter Pyrex bottles nearly filled with Na₂S-reduced, CO₂-bicarbonate-buffered freshwater mineral medium no. 2 containing 0.2% (wt/vol) oat spelt arabinoxylan (preextracted with 70% [vol/vol] ethanol) as the sole fermentable substrate (12). Marine strains were grown in a similar manner, except that marine medium was used (12).

Purification of ArfI. Purification of ArfI from Triton extracts was done at room temperature (RT) by a low-pressure column chromatography procedure performed with an Econo System apparatus (Bio-Rad Laboratories, Hercules, Calif.) generally operating at a flow rate of 1 ml · min¹. Eluted fractions were monitored for protein content by measuring the A₂₈₀ and for ARAF activity by using a semiquantitative microtiter plate assay (see below). Sequential column chromatography was performed by using the following steps. In step 1 Triton extract (204 ml, 430 mg of protein) was applied to a DEAE-cellulose column (2.5 by 50 cm) that had previously been equilibrated with 50 mM HEPES buffer (pH 8.0). The column was washed with 1 liter of equilibration buffer at a flow rate of 2 ml · min¹, and elution of ArfI was achieved by using a linear gradient consisting of 0 to 1 M NaCl in the same buffer. Fractions containing ArfI eluted between 50 and 200 mM NaCl; these fractions were pooled and concentrated by ultrafiltration through a type YM 10 membrane having a 10,000-molecular-weight cutoff (Amicon Co., Danverse, Mass.), and in the process the buffer was changed to 20 mM HEPES (pH 8.0). In step 2 the ArfI pool from step 1 (75 ml, 180 mg of protein) was applied to a DEAE-Sephadex A-50 column (1.5 by 50 cm) that had previously been equilibrated with 20 mM HEPES (pH 8.0), and elution was performed with a linear gradient consisting of 0 to 1 M NaCl in the same buffer. Fractions containing ArfI eluted from the column between 0.1 and 0.4 M NaCl; these fractions were pooled, dialyzed, and concentrated as described above, except that the dialysis buffer was 20 mM potassium phosphate (pH 6.5). In step 3 the ArfI pool from step 2 (75 ml, 74 mg of protein) was applied to a hydroxylapatite column (1.5 by 50 cm) that had previously been equilibrated with 20 mM potassium phosphate buffer (pH 6.5), and chromatography was performed with a linear gradient consisting of 20 to 300 mM potassium phosphate. Fractions containing ArfI eluted from the column between 170 and 225 mM phosphate and were pooled. In step 4 a 5 M NaCl solution was added to the ArfI pool from step 3 (43 ml, 7.7 mg of protein) to achieve a final NaCl concentration of 1 M, and the mixture was applied to a Phenyl-Sepharose CL-4B column (1.0 by 50 cm) that had previously been equilibrated with a solution containing 1 M NaCl in 5 mM 3-N-morpholinopropanesulfonic acid (MOPS) buffer (pH 6.5). Elution of ArfI was performed with a simultaneously decreasing linear gradient consisting of 1 to 0 M NaCl and increasing pH 6.5 to 8.0 gradient in 5 mM MOPS buffer. ArfI activity eluted between 250 and 0 mM NaCl and pH 7.5 to 8.0, and the relevant fractions were pooled. In step 5 the ArfI pool from step 4 was concentrated by ultrafiltration (as described above), and the buffer was changed to 20 mM MOPS (pH 7.5). This pool of ArfI (74.5 ml, 1.7 mg of protein) was then applied to a Macro Prep 50Q column (1.0 by 50 cm) that had previously been equilibrated with 20 mM MOPS (pH 7.5). Chromatography was performed with an increasing linear gradient consisting of 0 to 1 M NaCl in the same buffer. Active fractions eluted between 50 and 100 mM NaCl and were pooled. In step 6 the ArfI pool from step 5 was dialyzed, as described above, against 20 mM MOPS (pH 8.0) containing 0.5 M NaCl, and the dialyzed pool (91 ml, 1.6 mg of protein) was rechromatographed on a Phenyl-Sepharose CL-4B column (1.5 by 50 cm), but this time with a decreasing linear gradient consisting of 0.5 to 0 M NaCl in the same buffer. ArfI-containing fractions eluted between 25 and 0 mM NaCl and were pooled. Ultrafiltration was then used to concentrate the ArfI preparation to a volume of 28.8 ml (1.2 mg of protein); this preparation was stored at 4°C.

Partial purification of ENDOXs. Components from three major electrophoretically separable zones of ENDOX activity (i.e., the top, middle, and bottom zones after native polyacrylamide gel electrophoresis [PAGE] of Triton extracts [see Fig. 2A, lane 1]) were partially purified from a Triton extract by using a four-step procedure. The first three steps involved sequential column chromatography with the following matrices and eluants by using the conditions described above for ArfI: (i) DEAE-cellulose and NaCl (step i); (ii) hydroxylapatite and PO₄³] (step ii); and (iii) Macro Prep 50Q and NaCl (step iii). A significant amount of ENDOX activity in the major peak eluting in step i did not bind to the hydroxylapatite column in step ii and eluted in the void volume. This material (referred to as ENDOX II) was enriched with ENDOX components that migrated in the middle zone of native PAGE gels and was saved. Steps ii and iii were effective in removing some additional contaminating proteins from the remaining ENDOX activity (which consisted of components from the top and bottom zones of native PAGE gels), but did not resolve these proteins. Therefore, an additional step, step iv, was included, which involved preparative native PAGE of the remaining ENDOX activity on a model 491 Prep-Cell column (Bio-Rad) containing a 2-cm (4% polyacrylamide) stacking gel and a 10-cm (10% polyacrylamide) resolving gel (7). Electrophoresis was done according to the manufacturer's recommendations, and fractions were screened for ENDOX activity by measuring the release of reducing sugar from xylan (see below) and on native PAGE slab gels subsequently overlaid with oat spelt arabinoxylan zymogram gels (see below). Step iv separated the remaining ENDOX components into a group of activities that migrated slowly through the gel and represented the top zone from native PAGE gels (referred to as ENDOX I) and a group of activities that migrated rapidly and represented the bottom zone (referred to as ENDOX III).

Enzyme assays. ARAF activity was determined semiquantitatively by a modification of the Bachmann-McCarthy method (3), as follows. Portions (1.0 µl) of enzyme (e.g., column chromatography fractions) were added to separate wells of 96-well microtiter plates that also contained 90 µl of a 4-methylumbelliferyl--L-arabinofuranoside (MU-AF) solution (10 µg · ml¹) per well. After various periods of incubation (from 10 min to 24 h at RT, depending on the relative activity), the microtiter plates were placed on a UV transilluminator, and active fractions were identified by the intense fluorescence of liberated methylumbelliferone.

1

1

Determination of pH and temperature optima and stability. The pH optimum for ArfI activity was determined by using reaction mixtures buffered with the following compounds (pHs were adjusted in 0.5-pH unit increments): sodium citrate, pH 3 to 6; MES, pH 5.5 to 6.5; and MOPS, pH 6.5 to 8.0. Prewarmed (45°C) reaction mixtures (final volume, 4 ml) contained 3.575 ml of 50 mM buffer and 400 µl of 10 mM pNP-AF, and the reactions were initiated by adding 25 µl of ArfI (0.026 U in 2 mM MES [pH 6.0]). Then 1-ml aliquots were removed at 2-min intervals for the p-nitrophenol assay (see above). A similar procedure was used to determine the temperature optimum for ArfI.

Electrophoresis and zymograms. Sodium dodecyl sulfate (SDS)-PAGE was performed by using 4% (wt/vol) polyacrylamide stacking gels and 12.5% (wt/vol) polyacrylamide resolving gels (22). Native PAGE (4% [wt/vol] polyacrylamide stacking gel; 10, 12, or 7.5 to 18% [wt/vol] polyacrylamide gradient resolving gel) was performed without SDS and 2-mercaptoethanol and without preexposure of sample proteins to 100°C (3). Unless otherwise noted, all native and SDS-PAGE gels were stained with a Silver Stain Plus kit (Bio-Rad) according to the manufacturer's instructions. Isoelectric focusing (IEF) of proteins was performed with a model 111 mini IEF cell (0.4-mm-thick minigels; Bio-Rad) and pH 5 to 7 ampholytes according to the manufacturer's instructions. Unless otherwise noted, all electrophoresis gels were prepared with GelBond PAG films (for native PAGE and SDS-PAGE; FMC BioProducts, Rockland, Maine) or Gel Support films (for IEF; Bio-Rad) to facilitate handling. The gels used for detection of nucleic acids were prepared and electrophoresed by using standard methods (40).

1

1

Fractionation of cells for ArfI localization. Cells from a 2-liter culture were harvested by centrifugation at 10,000 × g as described above, and the supernatant fluid (secreted enzyme fraction) was pooled and concentrated (10,000-molecular-weight cutoff membrane). One-half of the cell pellets (containing cell-associated enzymes) were pooled by resuspending them in 15 ml of 10 mM Tris buffer (pH 7.6) and subjected to sonication (see above), followed by centrifugation (30,000 × g, 30 min, 4°C), which yielded a particulate enzyme fraction and a supernatant (i.e., soluble cytoplasmic-periplasmic) enzyme fraction. The other half of the cell pellets were subjected to an osmotic shock procedure similar to that described by Godchaux and Leadbetter (10). First, the pellets were pooled by resuspending them in 5 ml of ice-cold 10 mM Tris buffer (pH 7.6) containing 0.3 M NaCl and then rapidly warmed to 25°C in a 40°C water bath and incubated at 25°C for 5 min. The suspension was next chilled on ice, and 0.75 mg of lysozyme (75 µl of a 10-mg/ml stock solution) was added with rapid stirring. The lysozyme-treated cells were then subjected to a slow addition of 10 ml of ice-cold 10 mM Tris buffer (pH 7.6) and incubated for 2.5 min on ice. The suspension was then warmed as described above to 25°C and kept at that temperature until more than 90% of the cells were converted into spheroplasts (as determined by phase-contrast light microscopy). The spheroplasts were centrifuged (30,000 × g, 30 min, 4°C), and the supernatant (containing periplasmic enzymes) was removed and saved. The spheroplast pellet was then resuspended in 10 ml of 10 mM Tris buffer (pH 7.6), sonicated (see above), and recentrifuged to yield a second supernatant containing soluble cytoplasmic enzymes. A portion of the remaining pellet was treated with 0.2% Triton X-100 for 2 h at room temperature and then recentrifuged, which yielded a third supernatant containing Triton X-100-extractable enzymes associated with particulate spheroplast material.

Antibody production and Western blotting. Polyclonal anti-ArfI antiserum was produced by injecting a female New Zealand White rabbit with 100 µg of purified enzyme emulsified in TiterMax (CytRx Corp., Norcross, Ga.) by using the method of Harlow and Lane (14). A booster injection (100 µg, prepared as described above) was administered after 28 days, and serum was collected 14 days later. The anti-ArfI antiserum titer was determined by using 1 µg of purified ArfI per well in a dot blot apparatus (Bio-Rad) containing an Immobilon P^SQ nylon membrane (Millipore Corp., Bedford, Mass.). The preparation was developed with a goat anti-rabbit immunoglobulin G (H + L) alkaline phosphatase immun-blot assay kit (Bio-Rad) according to the manufacturer's instructions. Western blotting was performed by using 10% polyacrylamide native PAGE gels without GelBond film, and the blots were blotted onto Immobilon P^SQ nylon membranes with a Trans-blot Cell apparatus (Bio-Rad) by using the method of Matsudaira (29). ArfI was detected as described above with anti-ArfI antiserum diluted 1:20,000. Preimmune rabbit serum (1:1,000 dilution) was used to screen a duplicate blot for nonspecific binding.

Other analytical procedures. The native molecular masses of purified ArfI and each partially purified ENDOX pool were determined by size exclusion chromatography with a Pharmacia fast protein liquid chromatography (FPLC) system equipped with either a Superose 6 or Superose 12 HR 10/30 column (Pharmacia Biotech, Uppsala, Sweden). The columns were preequilibrated with 20 mM MOPS (pH 6.5) containing 50 mM NaCl and were run at a flow rate of 0.5 ml · min¹. The column effluents were monitored for A₂₈₀, as well as for enzyme activity, by using the MU-AF plate assay (for ArfI) or the reducing sugar assay and native PAGE zymograms (for ENDOX pools). The size exclusion chromatography standards used for the Superose 6 column included blue dextran (2,000 kDa), bovine thyroglobulin (670 kDa), ferritin (440 kDa), catalase (232 kDa), and aldolase (158 kDa). The size exclusion chromatography standards used for Superose 12 columns included thyroglobulin (670 kDa), gamma globulin (158 kDa), ovalbumin (44 kDa), myoglobin (17 kDa), and vitamin B₁₂ (1.35 kDa).

Chemicals and other materials. Oat spelt xylan, arabinogalactan, xylose, p-nitrophenyl derivatives, 4-methylumbelliferyl derivatives, DEAE-Sephadex A-50, NAD, DL-glyceraldehyde 3-phosphate, KH₂AsO₄, dithiothreitol, and bovine thyroglobulin were obtained from Sigma Chemical Co. Phenyl-Sepharose CL-4B, blue dextran 2000, ferritin, catalase, and aldolase were obtained from Pharmacia Biotech. Low-molecular-weight SDS-PAGE standards, IEF standards, FPLC size exclusion standards, pH 5 to 7 ampholytes, hydroxylapatite, and Macro Prep 50Q were obtained from Bio-Rad Laboratories. DEAE-cellulose was obtained from Whatman Specialty Products Inc. (Fairfield, N.J.). Xylobiose, xylotriose, xylotetraose, xylopentaose, rye and wheat arabinoxylans, and sugar beet arabinan were obtained from Megazyme, Inc. (Sydney, Australia). SPECTRA/POR molecularporous membrane tubing (width, 45 mm; 3,500-molecular-weight cutoff) was obtained from Spectrum Medical Industries, Inc. (Los Angeles, Calif.). Lenzing beechwood xylan and the three corn cob arabinoxylan fractions were gifts from Robert B. Hespell (Agricultural Research Service, U. S. Department of Agriculture, Peoria, Ill.). The other chemicals were reagent grade and were obtained from commercial sources. All H₂O used was double distilled (Millipore Corp.).

	RESULTS

Top Abstract Introduction Materials & Methods Results Discussion References

Enzyme activities in crude extracts. Triton extracts of C. xylanolytica contained a variety of enzymatic activities important for hydrolysis of xylans and other saccharides, including ARAF (but not -L-arabinopyranosidase), -D-xylosidase, -D- and -D-glucosidases, -D- and -D-galactosidases, and acetylesterase (Fig. 1). Likewise, native PAGE of Triton extracts revealed an array of electrophoretically separable proteins, including approximately 15 proteins with ENDOX activity distributed in the top (slowest migrating), middle, and bottom (fastest migrating) zones of the gel and detectable by zymograms with oat spelt xylan (Fig. 2A and C, lane 1). By contrast, analogous zymograms with MU-AF as the substrate revealed that ARAF activity was associated with only a single band, and this band was referred to as ArfI (Fig. 2B, lane 1). No apparent -D-glucuronidase or -D-cellobiosidase activity was observed when preparations were assayed by using 4-methylumbelliferyl-linked substrates in microtiter plate wells (Fig. 1).

View larger version (41K): [in this window] [in a new window]   FIG. 1.   Microtiter plate assay comparing the activity of purified ArfI (1st row) to activities present in a crude Triton extract of cells (3rd row) with the following 4-methylumbelliferyl derivatives: column 1, -D-galactoside; column 2, -D-galactoside; column 3, -L-arabinopyranoside; column 4, -L-arabinofuranoside; column 5, -D-glucoside; column 6, -D-glucoside; column 7, -D-glucuronide; column 8, -D-cellobioside; column 9, acetate; column 10, -D-xyloside. Each well containing purified ArfI contained 48 ng of protein (equivalent to 10.6 U of ARAF activity). Each well containing Triton extract contained 4.2 µg of protein (equivalent to 0.011 U of ARAF activity). The control (2nd row) contained test substrate, but lacked enzyme. Incubation was for 12 h at RT.

View larger version (37K): [in this window] [in a new window]   FIG. 2.   Zymograms and protein staining of a native PAGE gel for ArfI after each purification step. (A) Oat spelt xylan zymogram (stained with Congo red) of ENDOX activity. (B) MU-AF zymogram showing ARAF activity as UV-fluorescent bands. (C) Native PAGE (silver-stained protein) gel. Lane 1 contained Triton extract, and lanes 2 through 7 contained the fractions obtained after purification steps 1 through 6 listed in Table 1, respectively; lanes 1 through 7 contained 105, 123, 49, 9.1, 1.1, 0.9, and 1.2 µg of protein, respectively (B and C), and the gel for panel A was loaded with three times as much protein per lane. The arrow in panel A indicates a small zone of Congo red nonbinding induced by the presence of ArfI (see text). Note that panels B and C are magnified more than panel A.

Purification and physicochemical properties of ArfI. ArfI was purified 85-fold to homogeneity from Triton extracts by using anion-exchange and hydrophobic interaction column chromatography (Table 1; Fig. 2B and C and 3). With these procedures, 23.6% of the original activity in Triton extracts was recovered. A major step in the purification procedure was chromatography on hydroxylapatite, which alone yielded an 8-fold increase in purity. Although a slight decrease in specific activity was seen after Macro Prep 50Q chromatography (presumably because of some inactivation of ArfI), this step eliminated a significant amount of contaminating protein (compare lanes 5 and 6 of Fig. 2C and 3).

View larger version (84K): [in this window] [in a new window]   FIG. 3.   SDS-PAGE (silver-stained) gel of fractions obtained during purification of ArfI. Lane 1 contained Triton extract, and lanes 2 through 7 contained the fractions obtained after purification steps 1 through 6 listed in Table 1, respectively; lanes 1 through 7 contained 10.5, 9.8, 4.9, 3.6, 2.3, 1.7, and 2.4 µg of protein, respectively. Lane Std contained the following molecular weight markers: rabbit muscle phosphorylase B (molecular weight, 97,400), bovine serum albumin (66,200), hen egg white ovalbumin (45,000), bovine carbonic anhydrase (31,000), soybean trypsin inhibitor (21,500), and hen egg white lysozyme (14,400).

View larger version (45K): [in this window] [in a new window]   FIG. 4.   IEF gel of purified ArfI (1 µg of protein per lane). (A) Gel stained with crocein scarlet. (B) Gel overlaid with an MU-AF zymogram gel. Lane 1, purified ArfI; lane 2, purified ArfI plus pI standards (-lactoglobulin B [pI 5.1], bovine carbonic anhydrase [pI 6.0], and human carbonic anhydrase [pI 6.5]).

Enzymatic activity and stability of ArfI. With pNP-AF as the substrate, ArfI had optimum activity at pH 5.8 (at 45°C) and 45 to 50°C and exhibited a K_m of 0.504 ± 0.034 mM and a V_max of 319 ± 6.6 µmol · min¹ · mg of protein¹. ArfI was highly specific for the -L-arabinofuranoside linkage, as no hydrolytic activity was seen with the following p-nitrophenyl derivatives: -L- and -L-arabinopyranosides, -D- and -D-glucopyranosides, -D- and -D-galactopyranosides, -L-rhamnopyranoside, -L-fucopyranoside, -D-lactopyranoside, -D-xylopyranoside, -D-cellobioside, -D-mannopyranoside, -D-glucuronide, and acetate. This specificity was also observed when 4-methylumbelliferyl derivatives of many of these same compounds were tested (Fig. 1).

View larger version (80K): [in this window] [in a new window]   FIG. 5.   Thin-layer chromatogram of various arabinoxylans and an arabinan before (controls) and after exposure to purified ArfI. Lanes 1 and 19, xylose, xylobiose, xylotriose, xylotetraose, and xylopentaose xylooligosaccharide standards; lanes 2 and 18, arabinose standard; lane 3, rye arabinoxylan plus ArfI; lane 4, rye arabinoxylan control; lane 5, wheat arabinoxylan plus ArfI; lane 6, wheat arabinoxylan control; lane 7, sugar beet arabinan plus ArfI; lane 8, sugar beet arabinan control; lane 9, arabinose and xylose standards; lane 10, CCXA plus ArfI; lane 11, CCXA control; lane 12, CCXB plus ArfI; lane 13, CCXB control; lane 14, CCX plus ArfI; lane 15, CCX control; lane 16, oat spelt arabinoxylan plus ArfI; lane 17, oat spelt arabinoxylan control. A, arabinose; X1, xylose; X2, xylobiose; X3, xylotriose; X4, xylotetraose; X5, xylopentaose.

Interaction of ArfI with ENDOXs. The documented ability of many ARAFs to interact synergistically with ENDOXs (3, 11, 21, 23, 28, 34, 46, 50) prompted us to explore this phenomenon with the analogous enzymes of C. xylanolytica. ENDOX components from three main zones separable by native PAGE of Triton extracts (Fig. 2A) were partially purified and designated ENDOX I, ENDOX II, and ENDOX III (Fig. 6). The ENDOX II pool consisted of at least four ENDOX enzymes, including a small but significant amount of a component(s) present in ENDOX III. In contrast, the ENDOX I and ENDOX III pools each contained only one or perhaps two electrophoretically resolvable ENDOX components. Each ENDOX pool, however, contained far less non-ENDOX, Coomassie blue-stainable material than crude Triton extracts (Fig. 6, lane 4). Size-exclusion FPLC of each ENDOX pool yielded a single activity peak that eluted at a position corresponding to 130 kDa for ENDOX I, 63 kDa for ENDOX II, and 43 kDa for ENDOX III.

View larger version (79K): [in this window] [in a new window]   FIG. 6.   Oat spelt xylan zymogram (A) and Coomassie blue protein staining (B) of a native PAGE gel of partially purified ENDOX pools. Lane 1, ENDOX I; lane 2, ENDOX II; lane 3, ENDOX III; lane 4, Triton extract. Lanes 1 through 4 contained 0.24, 1.03, 1.30, and 110.9 µg of protein, respectively.

Cellular distribution of ArfI and ENDOX activities. The relatively high molecular weight of most arabinoxylans (1, 32) requires that they undergo hydrolysis to fragments small enough to pass through the cell membrane before further dissimilation can take place by cytoplasmic enzymes. Inasmuch as most of the enzymes of the xylanase system of C. xylanolytica were cell associated, it seemed likely that some or all of them, including ArfI, might be on or in the outer membrane or periplasm of this gram-negative bacterium, where they would have access to the native substrate or to large fragments released from it. This notion was supported by the ready extractability of the xylanase system from whole cells with a low concentration (0.2%) of the detergent Triton X-100, a treatment which caused no obvious disruption of cells, as assessed previously by phase-contrast and electron microscopy (13). However, Triton extracts were recently found to exhibit GPD activity (a typically cytoplasmic enzyme), and 0.8% agarose gel electrophoresis of Triton extracts revealed the presence of ethidium bromide-stainable material that migrated to a position typical of that of RNA (data not shown). These observations suggested that Triton X-100 did in fact disrupt the cytoplasmic membrane enough to liberate cytoplasmic components. Therefore, a cell fractionation procedure was used to determine more precisely the cellular location of ArfI and other enzymes of the xylanase system.

View larger version (69K): [in this window] [in a new window]   FIG. 7.   Specificity of anti-ArfI antiserum. (A) MU-AF zymogram of native PAGE gel. (B) Native PAGE (silver-stained) gel. (C) Western blot with anti-ArfI antiserum. Lane 1, purified ArfI; lane 2, soluble cell extract (obtained by sonication and centrifugation); lane 3, Triton extract of particulate cell material (obtained by sonication and centrifugation); lane 4, Triton extract of untreated whole cells; lane 5, concentrated cell-free growth medium. Lanes 1 through 5 contained 0.6, 75.0, 72.5, 60.0, and 72.0 µg of protein, respectively.

View larger version (146K): [in this window] [in a new window]   FIG. 8.   Oat spelt xylan zymogram of a native PAGE gel showing the distribution of ENDOX components in cells and culture fluids of C. xylanolytica. Lane 1, concentrated cell-free growth medium; lane 2, soluble cell extract; lane 3, soluble cell extract plus Triton X-100; lane 4, Triton extract of particulate cell material; lane 5, osmotic shock fluid (= cell periplasmic fraction); lane 6, soluble (cytoplasmic) fraction of spheroplasts; lane 7, Triton extract of particulate fraction of spheroplasts; lane 8, Triton extract of untreated whole cells. Lanes 1 through 8 contained 0.014, 3.7, 3.7, 2.8, 0.094, 5.1, 1.8, and 4.4 mg of protein, respectively.

ArfI-like proteins in other strains of C. xylanolytica. Polyclonal antiserum produced against purified ArfI was specific for ArfI in C. xylanolytica XM3, as judged by a Western immunoblot analysis of native PAGE gels containing proteins from various cell fractions (Fig. 7C). Like zymograms, Western immunoblots failed to demonstrate that ArfI was present in cell-free spent culture fluids (Fig. 7A and C, lane 5). This anti-ArfI antiserum was then used to determine whether ArfI-like proteins were present in soluble cell-free extracts of other freshwater and marine strains of C. xylanolytica isolated previously (12). Zymograms of native PAGE gels revealed that most of the strains possessed ARAF activity, which appeared as a single band migrating to a position that was similar (but not necessarily identical) to the position of ArfI (Fig. 9A). However, three marine strains (OP2E, OP2F, and PR2L) (Fig. 9A, lanes 11 through 13, respectively) did not appear to express any ARAF activity, and the ARAF activity bands for two other marine strains (EPA and EPB) (Fig. 9A, lanes 9 and 10, respectively) were faint.

View larger version (55K): [in this window] [in a new window]   FIG. 9.   ArfI-like proteins and ARAF activity of various freshwater isolates (strains XM3, SL1, MA3, and EW1) and marine isolates (strains EPFW, EPA, EPB, OP2E, OP2F, and PR2L) of C. xylanolytica after growth in freshwater medium (FW medium) or marine medium (MM medium). (A) MU-AF zymogram of native PAGE gel showing ARAF activity (viewed by UV light). (B) Western immunoblot of a native PAGE gel with polyclonal anti-ArfI antiserum. Lane 1, purified ArfI; lane 2, strain XM3 (FW medium); lane 3, strain XM3 (MM medium); lane 4, strain MA3 (FW medium); lane 5, strain EW1 (FW medium); lane 6, strain SL1 (FW medium); lane 7, strain EPFW (FW medium); lane 8, strain EPFW (MM medium); lane 9, strain EPA (MM medium); lane 10, strain EPB (MM medium); lane 11, strain OP2E (MM medium); lane 12, strain OP2F (MM medium); lane 13, strain PR2L (MM medium). Lanes 1 through 13 contained 0.6, 91.0, 142.0, 138.0, 121.0, 166.0, 146.0, 68.0, 226.0, 201.0, 85.0, 130.0, and 59.0 µg of protein, respectively. Lanes 2 through 13 contained soluble cell extract produced after sonification and centrifugation. Note that panel B is magnified more than panel A.

	DISCUSSION

Top Abstract Introduction Materials & Methods Results Discussion References

A cytoplasmic arabinofuranosidase (ArfI) from C. xylanolytica was purified 85-fold by column chromatography and was judged to be a single protein species on the basis of SDS-PAGE, native PAGE, and IEF analyses, all of which yielded a single protein band (the latter two preparations retained ARAF activity in zymograms), as well as on the basis of FPLC (during which ArfI eluted as a single, sharp symmetrical peak) and on the basis of a Western immunoblot analysis with anti-ArfI antiserum (which revealed only one cross-reactive protein, ArfI, in cell extracts of strain XM3). We obtained no evidence which suggested that ArfI existed in multiple forms, as has been observed with some ARAFs and ENDOXs produced by other organisms (20, 30, 44, 46).

ArfI accounted for virtually all of the ARAF activity in cultures of C. xylanolytica XM3, and it appeared to be the only ARAF produced by this bacterium under our growth conditions. Likewise, single ARAFs were produced by certain other freshwater and marine strains of C. xylanolytica isolated from geographically distant sites, and most of these cross-reacted with anti-ArfI antiserum, suggesting that they are structurally similar to ArfI. A recent study (44) suggested that some ARAFs may go undetected because of their inability to cleave pNP-AF, the substrate commonly used to assay for ARAF activity. However, MU-AF was also used as a substrate in the present study, and again the only MU-AF-hydrolyzing activity observed was that attributable to ArfI and present as a single electrophoretically separable band throughout the purification procedure (Fig. 2B). The trace amount of extracellular ARAF activity occasionally observed in cell-free culture fluids of xylan- and xylose-grown cells (13) was probably the result of lysis of some cells in the population, since the protein responsible for such activity has properties similar to those of ArfI during column chromatography and IEF. However, although ArfI was the only ARAF detected in the present study, it may not be the only ARAF that cells are capable of producing. Indeed, in another paper (19) Kim et al. describe the cloning and sequencing of the following two different ARAF-encoding genes from C. xylanolytica: arfI, which encodes ArfI; and arfII, which encodes an ARAF (ArfII) expressed by Escherichia coli, but which has not yet been observed in cells or culture fluids of C. xylanolytica. In any case, the single protein (ArfI) accounting for ARAF activity was in marked contrast to the multiple ENDOX activities which segregated into three major zones (slow, moderate, and fast migrating) during native PAGE and which were associated with ca. 15 individual bands. Such a multiplicity of endoxylanases is not uncommon in xylanolytic systems (8, 49).

Some of the properties of ArfI from C. xylanolytica (see above) were typical of the properties of ARAFs from various other organisms; these properties include subunit molecular mass (which is usually 30 to 95 kDa), an acidic pH optimum (typically between pH 2.5 and 6.9), and the ability to release arabinose from pNP-AF, MU-AF, arabinoxylans, and arabinan (but otherwise the substrate specificity is narrow). However, the apparent native molecular mass of 160 to 210 kDa, which was consistent with the molecular mass of a trimer or tetramer composed of 56-kDa subunits, was higher than that of many other ARAFs. Among purified ARAFs, ArfI bears perhaps the closest resemblance to the analogous enzyme from Butyrivibrio fibrisolvens GS113 in terms of physical parameters (similar pIs, identical subunits [56 and 31 kDa, respectively], and location [both cytoplasmic] [see below]), catalytic properties (similar K_m values and temperature and pH optima), and narrow substrate specificity (i.e., they are specific for the furanoside configuration, with no activity on arabinogalactan [16]).

One curious property of the ArfI of C. xylanolytica was its ability to suppress Congo red binding to oat spelt arabinoxylan zymogram gels (Fig. 2A). However, we are reluctant to attribute true endoxylanase activity (hence bifunctionality) to this enzyme because (i) little or no reducing sugar was liberated (above the amount expected from release of arabinose residues alone) when ArfI acted on oat spelt arabinoxylan, (ii) neither a significant amount of reducing sugar nor spots on TLC plates corresponding to xylooligomers were liberated by ArfI from essentially arabinose-free Lenzing beechwood xylan (data not shown), and (iii) no apparent xylooligomers accompanied the release of arabinose from rye or wheat arabinoxylan (Fig. 5). Nevertheless, it is conceivable that a small amount of xylan-depolymerizing activity might be associated with ArfI and that this activity might be akin to that seen with certain ENDOXs of Clostridium thermocellum (which liberate too little reducing sugar to be detected by conventional colorimetric assays, but can effect enough depolymerization of the substrate to be detected in Congo red-stained zymograms [26]) or the ARAF of Streptomyces lividans (which hydrolyzes the xylan backbone after prolonged incubation [46]). Such activity might also be responsible for the small amount of saccharide trailing the arabinose spot after TLC of ArfI digests of corn cob and oat spelt arabinoxylans (Fig. 5). However, the ArfI of C. xylanolytica bears little resemblance to the arabinose-releasing ENDOXs that release arabinose side groups before attacking the xylan backbone (30). Such debranching ENDOXs do not act on MU-AF and pNP-AF, whereas the ArfI of C. xylanolytica had significant activity on these substrates.

An osmotic shock procedure, originally developed during the isolation of inner and outer membrane components from several Cytophaga species (10), was part of a cell fractionation study that implied that the location of ArfI is almost entirely cytoplasmic. In contrast, this same study revealed a broad cellular distribution of ENDOX components, including at least one component whose location was primarily periplasmic (Fig. 8, lane 5), and it also revealed several ENDOX components that appear to be secreted from cells and comprise the small, but significant, fraction of total ENDOX activity that is not cell associated (Fig. 8, lane 1). However, we regard this assessment of the cellular location of ENDOX components as a first approximation. Other workers have shown that the distribution of xylanase components can be affected by the amount and type of growth substrate, the culture conditions employed, and the growth phase of cells at the time of harvest (24, 25, 31, 37).

In light of the largely (if not entirely) cytoplasmic nature of ArfI, it seems reasonable to assume that the major role of this enzyme in C. xylanolytica is to remove -L-arabinofuranosyl residues from xylooligosaccharide fragments that are small enough to pass through (or be actively transported through) the cytoplasmic membrane and whose presence might otherwise impede the action of cytoplasmic ENDOXs and/or -xylosidases. Such xylooligosaccharide fragments are undoubtedly liberated from arabinoxylans by ENDOX components external to the cell membrane. This interpretation is consistent with the ability of ArfI to release arabinose from various arabinoxylans (Fig. 5) and to interact synergistically with various ENDOX components (Table 2), including some components present in the cytoplasmic fraction of cells (Fig. 8, lanes 2 and 6). Such synergy is not unusual and has been reported previously for the xylanolytic enzyme systems of other organisms (3, 30). Although one might assume that xylooligosaccharides capable of passing through the cytoplasmic membrane are restricted to short oligomers, preliminary evidence that uptake of large oligosaccharides occurs has been obtained for Bacteroides thetaiotaomicron (39), a member of the same phylogenetic group as C. xylanolytica (i.e., the Bacteroides group in the Flexibacter-Cytophaga-Bacteroides phylum [27]).

Given the ability of C. xylanolytica to compete so well for the degradation of insoluble xylan particles in anoxic enrichment cultures, further studies on the nature and cellular location of specific ENDOXs and other debranching enzymes, as well as on the mechanisms and limits of saccharide uptake by cells, should undoubtedly refine our concept of arabinoxylan degradation by cells. Hence, we consider this study only one step in dissecting the complex xylanase system of this fascinating gliding bacterium.