Isolation of genes encoding β-D-xylanase, β-D-xylosidase and α-L-arabinofuranosidase activities from the rumen bacterium Prevotella ruminicola B 1 4

FEMS Microbiology

Letters 125 (1995) 135-142

Isolation of genes encoding 13-D-xylanase, D-D-xylosidase and cu-L-arabinofuranosidase activities from the rumen bacterium Prevotella ruminicola B ,4 Ales Gasparic,

Romana, Marinsek-Logar a, Jennifer Martin b, R. John Wallace b, Franc V. Nekrep a, Harry J. Flint b**

a Zootechnical Department, Biotechnical Faculty, University of Ljubljana, Groblje 3, 61230 Donuale, Slovenia b Rowett Research Institute, Greenburn Road, Bucksburn, Aberdeen AB2 9SB, UK Received 2 September

1994; revised 5 November

1994; accepted 9 November

1994

Abstract Prevotella ruminicola B,4 is a strictly anaerobic, Gram-negative, polysaccharide-degrading rumen bacterium. Xylanase activity in this strain was found to be inducible, the specific activity of cells grown on xylan being increased at least 20-fold by comparison with cells grown on glucose. Ten bacteriophage clones expressing xylanase activity were isolated from a A EMBLS genomic DNA library of P. ruminicola B,4. These clones were shown to represent four distinct chromosomal regions, based on restriction enzyme analysis and DNA hybridisation. Three groups of clones encoded activity against oat spelt xylan but not carboxymethylcellulose (CMC). In one of these groups, represented by clone 5, activities against pNP-arabinofuranoside and pNP-xyloside were found to be encoded separately from endoxylanase activity. The fourth region encoded activity against CM cellulose and lichenan, in addition to xylan, and contains an endoglucanase/xylanase gene isolated previously. Keywords:

Prevotella

ruminicola;

Xylanase; Carboxymethyl cellulase; Xylosidase; Arabinofuranosidase;

Rumen bacteria;

Gene isolation

1. Introduction

ruminicola

Prevotella

strictly account bacteria covers several on 16s

ruminicola represents a group of anaerobic, Gram-negative bacteria that can for a large proportion of the total culturable in the lumen [l]. This species at present a genetically diverse group of strains and subgroups have recently been defined based rRNA sequencing and other criteria [2]. P.

* Corresponding 224) 716 687

author. Tel.: (+ 44-224) 716 651; Fax: ( + 44-

037%1097/95/$09.50 0 1995 Federation SSDI 0378-1097(94)00488-9

of European

Microbiological

B,4 belongs to a distinct subgroup having a relatively low G + C content in its DNA by comparison with the group represented by the type strain 23 and should probably be regarded as a separate species [2]. Although they are not cellulolytic, P. ruminicola strains related to 23 and B,4 produce abundant xylanase and endoglucanase activity [3] and are thought to contribute to the degradation and utilisation of plant polysaccharides in the rumen [4]. Genes encoding xylanase and endoglucanase activities have been isolated previously from several strains of P. ruminicola [5-81, but the encoded enzymes have so far Societies. All rights reserved

136

A. Gasparic et al. /FEMS

Microbiology

been found to be related to endoglucanases of family A, and no specific family F or G xylanases have been reported [9]. In the work reported here we show that P. ruminicola B,4 possesses at least three genes encoding relatively specific xylanases, one of which is adjacent to a region encoding &xylosidase/cr-Larabinofuranosidase activity, in addition to a gene encoding an endoglucanase that has been isolated previously [S].

2. Materials and methods

2.1. Bacterial strains, plasmids and bacteriophage Preuotella ruminicola B,4 was originally obtained from J.B. Russell (Cornell University, USA). Recombinant A EMBL3 phage were grown on E. coli P2392 (a P2 lysogen of LE392) or, following purification, on E. coli Y1090. E. coli DH5a and E. coli HBlOl were used as the host strains for subclones made in plasmid pUC18. 2.2. Media and culture conditions P. ruminicola B,4 was grown anaerobically at 38°C in M2 medium [lo] under 100% CO, [3]. For induction studies, M2 medium was modified to contain oat spelt xylan (0.6%) or glucose (0.6%) as sole source of energy. Recombinant phages were plated on BBL medium (10 g 1-l BBL trypticase, 5 g 1-l NaCl, 10 mM MgSO,, with 6 g 1-l agarose (top layer) or 15 g 1-i agar (bottom layer)). For screening, oat spelt xylan was added to a final concentration of 0.2% to the top layer. E. coli cells carrying pUC plasmids were grown in LB medium containing 60 pg of ampicillin per ml. 2.3. Enzyme assays Polysaccharide substrates were obtained from Sigma; oat spelt xylan was found to contain 80% xylose and 14% arabinose, by weight, while the corresponding figures for birchwood xylan were 95% xylose and 1.3% arabinose. Sonicated extracts or phage lysates of E. coli cells were assayed for polysaccharidase and glycosidase activities as described previously [l&12]. For induction studies P. ruminicola cells were grown in 700 ml of M2

Letters I25 (1995) 135-142

medium for 60 h at 38°C and samples (10 ml> taken anaerobically. Cells were centrifuged (20 min, 3000 X g, 4”C), washed twice with 50 mM Na-phosphate buffer pH 6.5, frozen at -2O’C and disrupted by osmotic shock before assaying polysaccharidase activities. Methods for detecting xylanase activity following SDS-PAGE were described previously [3]. 2.4. Library construction and subcloning Ligations, transformations and restriction enzyme analysis all followed standard procedures [13]. Chromosomal DNA was extracted from P. ruminicola B,4 as described previously [2,3] except that the CsCl step was omitted and RNA was removed by treating with RNase A. A genomic library was constructed by ligating 9-23-kb fragments derived from a Sau3A partial digest of P. ruminicola DNA with A EMBL3 BamHI-digested arms, followed by packaging in vitro with Gigapack II gold packaging extract (Stratagene). Bacteriophage DNA was isolated by the Diagen (DIAGEN GmbH, Dusseldorf, Germany) large-scale preparation method. Subclones were obtained by digesting bacteriophage DNA completely with Sal1 and partially with EcoRI, followed by ligation with SalI/EcoRI or EcoRI cut pUC18 and transformation of E. coli HBlOl. In the case of phage 6, attempts were also made to subclone fragments from EcoRl/HindIII partial digests. 2.5. Southern hybridization DNA from A EMBL3 clones was completely digested with SalI/EcoRI, separated on a 0.4% agarose gel and transferred to Genescreen membrane (NEN, Dreiech, Germany). Probes were prepared from EcoRI/SalI-digested phage DNA by the oligo priming method using kits and radiochemicals ([ 32P]dCTP) from Amersham UK. Hybridisation and washing of filters as described previously [ll], at 65°C.

3. Results 3.1. Isolation of xylanase genes from P. ruminicola B,4 Ten xylanase-positive plaques were obtained from approximately 3000 plaques screened, from a ge-

A. Gasparic et al. /FEMS

Microbiology Letters 125 (1995) 135-142

137

Table 1 Four non-overlapping DNA

chromosomal

Clone number

fragments

encoding

xylanase

Xylanase activity a (nmol min-’ (ml lysate)-

3 5 6 8

activity,

‘)

osx

BWX

0.23 2.7 0.55 0.57

0.70 2.8 0.8 0.18

isolated from a A EMBL3 library of Preuofella ruminicola B,4

Approx. insert size (kb)

Internal EcoRI restriction fragments

22 17 32 14

0.8, 1.2, 4.8, 10.1 0.9, 1.9, 3.7, 7 11.4, 5, 7.2 0.9, 1.1, 1.6, 1.75, 2.3, 6

b

a OSX, oat spelt xylan; BWX, birch wood xylan. Activities were determined from concentrated phage lysates. CM-cellulase and lichenase activities were also detectable in lysates of phage 8, while pNP-xylosidase and pNP-arabinofuranosidase activities were detectable in phage 5 (see Table 2). These activities were not detected for phages 3 or 6 (not shown). b These sum to give the estimated insert size when the sizes of the terminal EcoRl/SaiI fragments (not shown) are included.

nomic library of P. ruminicola B,4 constructed in the A vector EMBL3. These xylanase-positive phages were classified into four groups following analysis of

restriction digests with EcoRI and Sal1 and one representative of each group was chosen for further investigation (Table 1). In order to confirm that four Actlvlty XYL

pNPX pNPA

1 Kb t

,

E

3/24

B

HP

E

I

ma

E

P

x’

N’

I

I

I

I

PPH

P

Ev*

X’

III

I

I

I

_

_

+

+

E

_ +

L2L3

_

E \I/

e/l9

+ a

_

-

_

a

K’ B ”

”

PPH

P

K’

Fig. 1. Three non-overlapping DNA fragments isolated from P. ruminicola B,4 specifying xylanase activity. Clones 3/24, 5/4a and 8/19 were obtained as subclones derived from phages 3, 5 and 8, respectively, in pUC18. Restriction endonuclease cleavage sites are indicated as follows: E, EcoRI; H, Hi&III; B, BglII; K, E@nI; EV, EcoRV; P, PstI; X, XbaI. Sites marked with an asterisk were not mapped for all three clones: KpnI sites were not mapped for 3/24 and 5/4a, while EcoRV and XbaI sites were not mapped for 8/19. The small arrows indicate the direction of ZacZ transcription. Further subclones from 5/4a are also shown; L2/L3 and Ll/L4 refer to fragments cloned in opposite orientations in pUCl8 and pUC19 (Ll and L2 into pUC18, L3 and L4 in pUC19). Activities shown on the right hand side refer to the results of qualitative assays for pNP-arabinofuranoside (pNPA), pNP-xylosidase (pNPX) and oat spelt xylanase (XYL) activities (see also Table 2).

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Microbiology Letters 125 (1995) 135-142

non-overlapping chromosomal regions had been isolated, 32P-labelled probes were prepared from DNA of phages 3, 5 and 8 and hybridised against EcoRIdigested chromosomal DNA from P. ruminicola B,4, and against SaZI/EcoRI-digested DNA from phages 3, 5, 6 and 8. Hybridisation to insert-derived fragments was specific, establishing that each phage carried non-homologous, non-overlapping regions of the P. ruminicola chromosome. Consistent with this, different bands hybridised with each probe in EcoRI digests of chromosomal DNA (results not shown).

3.2. Evidence that endoxylanase and @-(1,4)-xylosidase / cx-L-arabinofuranosidase activities are separately specified by clone 5 A 6.8-kb EcoRI fragment from phage 5, subcloned in pUC18, was found to specify activity against oat spelt xylan, birchwood xylan, pNP-xyloside (pNPX) and pNP-arabinofuranoside (pNPA) (Fig. 1, Table 2). All of these activities were highly temperature-labile under standard assay conditions and 50-90% of activity was lost after 10 min incuba-

hours of growth

hours of growth

Fig. 2. Activities against oat spelt xylan and CMC-cellulose found in Preootella rwninicola B,4 cells growing in M2 medium containing either glucose (A) or oat spelt xylan (B) as the sole energy source. Total cell protein is also shown. Inocula were from cells grown on M2 medium with glucose.

A. Gasparic et al. / FEMS Microbiology Letters 125 (1995) 135-142 Table 2 Enzyme activities detected in E. coli cells carrying xylanase genes from Preuotella ruminicola B,4 subcloned in pUC18/19 Activity (nmol min-’

Oat spelt xylan Birchwood xylan Lichenan Barley B-glucan CM-cellulose pNF’-xyloside pNP-arabinofuranoside

(mg protein)-‘)

3/24

S/19

5/4a

L30

L3

1.3 1.0 < 0.5 0.6 < 0.5 < 0.2 < 0.2

2.1 1.2 274.6 1812.2 1.8 < 0.2 < 0.2

26.6 72.0 0.6 < 0.5 < 0.5 0.4 1.9

3.8 8.1 ND a ND ND < 0.2 < 0.2

4.8 146.0 ND ND ND 8.0 15.5

a ND, not determined.

tion at 50°C (results not shown). A subcloned XbaI fragment (4.1 kb, clone L30) specified activity against birchwood xylan and oat spelt xylan, but not against pNPX or pNPA. Meanwhile an overlapping 3.1-kb subclone (L3) retained all four activities, but the activity against oat spelt xylan was only 3% of that against birchwood xylan, compared with 47% and 37% in the clones L30 and 5/4a, respectively (Table 2). These results suggest that subclone L30 encodes an endoxylanase while subclone L3 encodes a distinct catalytic domain, enzyme, or enzymes, capable of hydrolysing pNPX and pNPA, and of hydrolysing birchwood xylan preferentially over oat spelt xylan. Similar activities were found when the 3.1-kb fragment from L3 was cloned in the opposite orientation in pUC19, suggesting that expression was from a promoter present within the cloned fragment (results not shown).

139

patterns obtained for the subclone 8/19 (Fig. 1) and for the parental phage clones correspond with a region isolated from the same strain by Matsushita et al. [8]. The gene described previously was reported to encode an enzyme of 82-88 kDa having CM-cellulase and xylanase activities [8]. 3.4. Xylanases produced by P. ruminicola B,4 In P. ruminicola B,4, xylanase activity was found to be increased between 20- and 40-fold after 12 h growth with xylan as energy source, compared to growth on glucose (Fig. 2). CM-cellulase activity was also higher in xylan grown cells, but by a smaller factor between 2- and 7-fold. It was shown previously that the major bands of CM-cellulase activity detected by SDS-PAGE in xylan-grown cells were between 82 and 88 kDa, in agreement with Matsushita et al. [8], whereas the major xylanase was around 63-66 kDa [3]. In the present work an additional band of around 26-29 kDa was also detected in xylan-grown cells (results not shown). Thus the inducible xylanase activity of P. ruminicolu B,4 cannot be attributed mainly to an endoglucanase/ xylanase, and is assumed to be due to the products of the other genes isolated here. Consistent with this, xylanase activity bands of 63-66 kDa were detectable in extracts of clone 3/24, and bands of 26-29 kDa in extracts of clone 5/4a (results not shown).

4. Discussion 3.3. Activities encoded by phage clones 3, 6 and 8 Subclones expressing xylanase activity detectable by the formation of clear zones in oat spelt xylan overlays were obtained from phage 3; these carried a 4.7-kb EcoRI fragment (Fig. 1). No xylanase-positive subclones were obtained from phage 6 (see Materials and methods). Xylanase was the only significant activity detected in lysates of phage clones 3 and 6, or in plasmid subclones derived from phage 3 (Tables 1, 2). In the case of phage 8, however, a subclone carrying a 6-kb EcoRI fragment expressed activity against CM-cellulose and the g&3-1,4)glucans lichenan and barley l3-glucan, in addition to xylanase activity (Table 2). The restriction digest

This work shows that at least four chromosomal regions specify xylanase activity in P. ruminicola B,4. One of these regions was isolated previously [8] and is known to encode an endoglucanase that may also be responsible for the activities detected here against g-(1,3-1,4) glucans and against xylan. Our evidence shows that another region, represented by clone 5, not only specifies endoxylanase activity but also encodes pNP-xylosidase and pNP-arabinofuranosidase activities which are due to other catalytic domains or enzymes. The ability of the L3 subclone to degrade birchwood xylan in preference to oat spelt xylan may be explained by differences in the state of substitution of the main chain, since the

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birch wood xylan used is almost free of arabinose substituents, whereas the oat spelt xylan contains approximately 14% arabinose. It is possible that the pNPX, birchwood xylanase, and perhaps also the pNPA, activities are due to a single enzyme that can hydrolyse substituted xylo-oligosaccharides or unsubstituted xylans, but not substituted xylans. Single gene products specifying pNPA and pNPX activities have been reported from Butyrivibrio fibrisolvens and Clostridium stercorarium [14,15] but xylanase activity was not reported to be associated with these enzymes. Close linkage of xylanase and g-(1,4)xylosidase genes has been observed in several species previously including the related Bacteroides ovatus [16,17].

Since CM-cellulase activity was induced by a lower factor than total xylanase activity by growth of P. ruminicola B,4 on xylan, a broad specificity endoglucanase/xylanase cannot account for the induced xylanase activity in this strain, and the more specific xylanase genes we have identified here are likely to contribute significantly to the induced xylanase activity. Similar conclusions were reached previously for the rumen cellulolytic bacterium Ruminococcus flauefaciens

17 1121.

Xylan is a complex and variable heteropolymeric substrate and a range of debranching enzymes as well as multiple xylanases with differing specificities is likely to be required for its efficient degradation and utilisation by microorganisms. Multiple endoxylanase genes have now been found in several microorganisms, including the cellulolytic rumen species Ruminococcus flavefaciens [ 111. Comparison at a molecular level of the xylanolytic system of a non-cellulolytic rumen species such as P. ruminicola with that of cellulolytic rumen species should provide important insights into the roles of individual gene products in plant cell wall breakdown.

Acknowledgements

We would like to thank the British Council/ALE link for supporting exchange visits that enabled this work to be carried out. The work was supported also by the Scottish Office Agriculture and Fisheries Department and by Gist-brocades. We are grateful to Peter Dewey for carbohydrate analyses.

Letten 125 (19951 135-142

References [l] Van Gylswyk, N.O. (1990) Enumeration and presumptive identification of some functional groups of bacteria in the rumen of dairy cows fed grass silage-based diets. FEMS Microbial. Ecol. 73, 243-254. [2] Avgustin, G., Wright, F.W. and Flint, H.J. (1994) Genetic diversity and phylogenetic relationships among strains of Preuotella (Bacteroides) ruminicoZa from the rumen. Int. 3. Syst. Bacterial. 44, 246-255. [3] Avgustin, G., Flint, H.J. and Whitehead, T.R. (1992) Distribution of xylanase genes and enzymes among strains of Preuotella (Bacteroides) ruminicola from the rumen. FEMS Microbial. Lett. 99, 137-144. [4] Dehority, B.A. (1993) Microbial ecology of cell wall fermentation. In: Forage cell wall structure and digestibility (H.G. Jung, D.R. Buxton, R.D. Hatfield and J. Ralph, Eds.), pp 425-453. ASA-CSSA-SSAA, Madison, WI. [S] Whitehead, T.R. (1993) Analyses of the gene and amino acid sequence of the Preuotella (Bacteroides) ruminicola 23 xylanase reveal unexpected homologies with endoglucanases from other genera of bacteria. Curr. Microbial. 27, 27-33. [6] Whitehead, T.R. and Lee, D.A. (1990) Cloning and comparison of xylanase genes from ruminal and colonic Bacteroides species. Curr. Microbial. 23, 15-19. [7] Vercoe, P.E. and Gregg, K. (1992) DNA sequence and transcription of an endoghtcanase gene from Bacteroides (Preootella) ruminicola AR20. Mol. Gen. Genet. 233, 284292. [8] Matsushita, O., Russell, J.B. and Wilson, D.B. (1991) A Bacteroides ruminicola 1,4 - !3-D-endoglucanase is encoded in two reading frames. J. Bacterial. 173, 6919-6926. [9] Gilkes, N.R., Henrissat, B., Kilbum, D.G., Miller, R.C. Jr. and Warren, R.A.J. (1991) Domains in microbial B-1,4glycanases: sequence conservation, function and enzyme families. Microbial. Rev. 55, 303-315. [lo] Hobson, P.N. (1969) Rumen bacteria. Methods Microbial. 3B, 133-149. [ll] Flint, H.J., McPherson, C.A. and Bisset, J. (1989) Molecular cloning of genes from Ruminococcus flavefaciens encoding xylanase and t3(1-3,1-4) glucanase activities. Appl. Environ. Microbial. 55, 1230-1233. [12] Flint, H.J., McPherson, C.A. and Martin, J. (1991) Expression of two xylanase genes from the rumen cellulolytic bacterium Ruminococcus flauefaciens 17 cloned in pUC13. J. Gen. Microbial. 137, 123-129. [13] Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. [14] Sakka, K., Yoshikawa, K., Kojima, Y., Kanta, S., Ohmiya, K. and Shimado, K. (1993) Nucleotide sequence of the Clostridium stercorarium xylA gene encoding a bifunctional protein with B-D-xylosidase and cu-L-arabinofuranosidase activities, and properties of the translated product. Biosci. Biotechnol. Biochem. 57, 268-272. (151 Utt, E.A., Eddy, C.K., Keshav, K.F. and Ingram, L.O. (1991) Sequencing and expression of the Butyriuibrio fibrisoluens

A. Gasparic et al. /FEMS Microbiology xylB gene encoding a novel bifonctional protein with B-Dxylosidase and cY-L-arabinofuranosidase activities. Appl. Environ. Microbial. 57, 1227-1234. [16] Panbangred, W., Kondo, T., Negoro, S. Shinmyo, A. and Okada, H. (1983) Molecular cloning of the genes for xylan degradation from Bacillus pumilus and their expression in Escherichia coli. Mol. Gen. Genet. 192, 335-341.

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[17] Weaver, J., Whitehead, T.R., Cotta, M.A., Valentine, P.C. and Salyers, A.A. (1992) Genetic analysis of a locus on the Bacteroides ouarus chromosome which contains xylan utilization genes. Appl. Environ. Microbial. 58, 2764-2770.