Multigene families of Cellulomonas flavigena encoding endo-β-1,4-glucanases (CM-cellulases)

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Gene, 74 (1988) 549-553 Elsevier GEN 028 18

Multigene families of Cellulomonus fluvigena encoding endo-/?-1,4_glucanases (CM-cellulases) (Gene cloning; endocellulases; lulosome; plasmids)

multiple genes; hybridisation;

activity gels; enzyme characterisation;

cel-

M.W. Akhtar -‘, M. Duffy I, B.C.A. Dowds*, M.C. Sheehan b and D.J. McConnell’ Departments of a Genetics and bBiochemistry, Trinity College, Dublin (Ireland) Tel. (01) 772941 and ‘Institute of Chemistry, University of the Punjab, Lahore, (Pakistan) Tel. : Lahore 54349 Received 21 January 1988 Accepted 30 June 1988 Received by publisher 5 October 1988

SUMMARY

Multiple genes coding for endo+- 1,4-glucanases (CM-cellulases) have been isolated from a newly discovered highly cellulolytic strain of Celhhmonasj7avigena. Clones of C. jlavigena DNA were isolated in Escherichia cofi and screened for gene expression on CM-cellulose plates staining with congo red. Six clones produced CMcellulase activity as detected in liquid assays, and on activity gels. They fell into three groups within which the sequences cross-hybridised. There were small differences in the pH and temperature optima of the enzymes encoded by representatives of the three groups of clones.

INTRODUCTION

The enzymatic breakdown of cellulose to glucose requires the activity of endo+l&lucanase (EC 3.2.1.14)and exo+1,4glucanase(EC 3.2.1.91) Correspondenceto: Dr. B.C.A. Dowds at her present address: Dept. of Biology, St. Patrick’s College, Maynooth, Co. Kildare, Ireland. Tel. (01)285222. Abbreviations: Ap, ampicillin; bp, base pair(s); CM cellulose, carboxymethylcellulose; EDTA, ethylenediaminetetraacetic acid; IPTG, isopropyl-/I-D-thiogalactopyranoside; kb, loo0 bp; SDS, sodium dodecyl sulfate; SSC, 0.15 M Nail 0.015 M Na, . citrate, pH 7.6; XGal, 5-bromo4chloro-3-indolyI-/3-ogalactopyranoside; [ 1,designates plasmid carrier state.

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which act synergistically to break down cellulose to cellobiose, and j?-1,4-glucosidase (EC 3.2.1.21) which converts cellobiose to ducose. Many bacterial, yeast and fungal species are known to produce one or more of these enzymes. However in spite of extensive studies, the biochemistry and molecular biology of these enzymes are not well understood (Coughlan, 1985). Molecular cloning is now being used to facilitate analysis of the structure and function of these enzymes, their biosynthesis and excretion, their evolution, and to enhance their production for testing in experimental cehulolytic systems. A potent cellulolytic species of CMdomonasj7avigena has been isolated from a soil sample in Pakistan. Division)

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At least four endoglucanases could be separated chromatographically. It also produces significant extracellular exoglucanase activity. This paper describes the cloning and expression of several putative C.flavigena endoglucanase genes as judged by the clones’ ability to direct degradation of CM-cellulose (i.e. CM-cellulase genes). The data show that there are multiple genes which belong to three different gene families.

EXPERIMENTAL AND DISCUSSION

(a) Cloning of CelZulomonasjZuvigena CM-cellulase genes Recombinant plasmid DNA was constructed by ligating 2-lo-kb Sau3AI fragments of a partial digest of chromosomal DNA with linearised and dephosphorylated pUC18 DNA using standard methods (Maniatis et al., 1982). Escherichia coli K-12 Tgl (lac pro supE44 thi hsdR [F’ traD36 proAB + 1acP ZAM 151) was transformed with the ligation mixture and plated on agar containing Ap, XGal and IPTG. About 70% of the colonies were white showing a high yield of recombinants. 4000 white colonies, equivalent to several complete genomic banks, were

TABLE I Specific activity of CM-celhtlase in lysates of E. coli pWM Plasmid B

Specific activity b

pWM79 pWM95 pWM114 pWM147 pWM191 pWM196

0.105 0.155 0.165 0.113 0.104 0.088

a Plasmids are described in EXPERIMENTAL AND DISCUSSION, section a. b Measured as p moles/liter of reducing sugar produced per mg of lysate protein. Midlog phase cells were treated with lysozyme (2 mg per ml of cell suspension in 10 mM Tris, pH 8) for 1 h on ice. The suspension was sonicated at 20 MHz for 3 min in an ice bath, centrifuged at 20000 x g for 1 h and the supernatant used for enzyme assays. CM-cellulase was assayed by the method of Miller et al. (1960) and protein content was estimated using a Biorad Protein Assay Kit.

screened for CM-cellulase activity. Eight were found to be positive, giving clear haloes on CM-celluloseagar after staining with congo red (see Cantwell and McConnell, 1983, for staining technique). Plasmid DNA was prepared from the positive clones and named pWM79, pWM95, pWM114, pWM118, pWM147, pWM191, pWM194 and pWM196. In each case the plasmid DNA was capable of transforming E. coli Tgl at a high frequency to CM-cellulase positive. Each plasmid was analysed with various restriction enzymes. Double digests with EcoRI + Sal1 removed the vector as a single band but cleaved the inserts into multiple bands (data not shown). pWM118 and pWM194 showed identical digest patterns to pWM95 and pWM191, respectively, and were not studied further. A variety of restriction digests were performed for each clone. There were no EcoRI or Hind111 sites in any of the clones. There were several Sal1 sites in each clone; Sac1 and XhoI sites were frequent in pWM79, 147 and 196. The sizes of the inserts in pWM79, pWM95, pWM114, pWM147, pWM191 andpWM196were11.7,2.7,5.3,4.3,3.9and7.6kb, respectively, as measured from different digests including EcoRI + Hind111 digests in which the inserts are excised as a single band. In general the digests indicated that the clones differed markedly from each other (data not shown). Cell extracts from each of the six clones were assayed for CM-cellulase. AI1 showed significant activities (Table I). There is less than a factor of 2 in the differences between the clones. pWMl14 showed the maximum (0.165 PM reducing sugar produced per mg protein), closely followed by pWM95 which has the smallest insert of 2.7 kb. CM-cellulase activity was also detected in activity gels (data not shown). Apart from pWM95 which showed two bands, all the others showed one band at about the same position in the gel. pWM95 specified the same band as the other clones as well as a second band of slower mobility. These could represent the products of two different genes or a single protein subject to post-translational modification. (b) Homology among the inserts and chromosomal DNA 32P-labelled probes from each plasmid were hybridised (Dowds et al., 1988) to EcoRI + Hind111

Fig. 1. Southern blots ofdigests ofplasmid pWM79,95,114,147,191 and 196 DNA, each cut with Hind111 + EcoRI with the exception of pWM95 which was cut with Hind111 + SacI, and of C.Javigena and E. coli DNA cut with EcoRI + HindIII. The samples on the gel are pUCl8, pWM79,95,114,147,191 and 196, C.flavigena chromosomal DNA, and E. coli chromosomal DNA (lanes l-9, respectively). Seven blots (panels A-G) were probed with pUCl8, pWM79,95, 114, 147, 191 and 196, respectively. Digests of recombinant plasmid DNA (0.05 pg) and chromosomal DNA (5 pg) were electrophoresed on seven replica 0.7% agarose gels. After electrophoresis the gels were soaked in 0.25 N HCl for 15 min before denaturing, neutrabsing and blotting as described in Maniatis et al. (1982). DNA (1 pg) from each of the six recombinant clones and from pUCl8 was labelled with alpha-32P ATP by nick-translation as described in Maniatis et al. (1982). Hybridisation was performed as described in Dowds et al. (1988) except that the temperature was changed to 42°C.

restriction digests of C. jlavigena and E. coli chromosomal DNA, and to digests of each plasmid DNA, which had been electrophoresed and blotted onto nitrocellulose. For live of the plasmids the restriction digest chosen (EcoRI + Hind111 digest) gives two bands, one the vector and one the insert. For the sixth plasmid, pWM95, the SacRI + Hind111 digest gives three bands, one the vector and two smaller ones from the insert (there is some uncut DNA in the pWM95 digests which hybridises with all probes including the pUCl8 probe). As shown in Fig. 1, pUC18 and the six recombinant plasmids all hybridised to the pUC18 vector band and to a band in E. coli DNA, which is probably from the lac hybridised to the operon. The recombinants C. jlavigena DNA, but quite poorly, and the signal was only seen on a long exposure of the auto-

radiogram. In fact, hybridisation of these clones was peculiar in another way - when the inserts were cleaved into multiple bands, e.g. with Sal1 and Southern-blotted, some of the bands did not hybridise to a nick-translated probe of the same plasmid! Blots B, E and G show that the insert from each of the recombinants pWM79, pWM147 and pWM196 hybridised to each other (lanes 2, 5 and 7), but not to E. coli DNA (lane 9) or to the other inserts. The insert DNA of pWM114 and pWM191 likewise hybridised (blots D and F) to each other (lanes 4 and 6). The pWM95 insert DNA hybridised (blot C) to itself (lane 3), but not to any other plasmid. All recombinants hybridised to Cellulomonas DNA but the weak signal was only seen on a long exposure of the autoradiograph (data not shown). From the hybridisation results it is apparent that

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the six clones represent at least three different Cellulomonas CM-cellulase genes. There may be more than three. pWM95, for example, gives rise to two activity bands, so this clone might contain two genes. The restriction digests of pWM79, pWM147 and pWM196 are sufficiently different from each other to suggest that they are from ditTerent chromosomal loci. Since they are homologous in Southern hybridisations they may code for enzymes which are closely related. Likewise pWM114 and pWM191 differ sufficiently from each other to suggest that they may be clones of different but homologous genes. (c) Characteristics of the enzymes encoded by the recombinant plasmids Extracts from pWM95, pWM147 and pWM191, representative of the three homology groups gave linear time courses for 60 min on carboxymethylcellulose with very similar activities (approximately 10 pg glucose released per min per ml). Crude cellulose (DL/S/4’, ICI) and microcrystalline cellulose from straw (PTS 13, ICI) were digested by extracts of pWM147 and pWM191 at about the same rate as carboxymethylcellulose, with the pWM95 extract being about 50% as active as on CM-cellulose. Temperature and pH profiles using CM-cellulose as substrate are shown in Fig. 2a,b. Extracts of Tgl pWM191 and Tgl pWM147 have very similar temperature profiles with optima at 50°C. The Tgl pWM95 extract gave a slightly different profile with an optimum at 55 ‘C. Each extract has a broad but distinctive pH profile.

(d) Conclusions Biochemical data show at least four different secreted CM-cellulases which can be separated by chromatography (Sami et al., 1988). These chromatographic variants could be due to post-translational modification of a single polypeptide, or to multiple genes, or both factors could contribute to the multiplicity of biochemical forms. It is known that fragments of endoglucanases retain enzymatic activity (M.W.A., unpublished observations; MacKay et al., 1986; Fukumori et al., 1987). However, it is now clear from the cloning that some of the variation in C. flavigena cellulases is due to the presence of at least three genes. Most bacterial species or strains contain only one CM-cellulase gene. There are three exceptions. Two endo+ 1,4glucanase clones, pEC2 and pEC3 were isolated from CeZZuZomonasfimi(Gikes et al., 1984) and Fukumori et al. (1986) obtained two CM-cellulase clones, pNK1 and pNK2 with strong homology to each other from Bacillus sp. strain N-4. More strikingly, Aubert and coworkers have identified seven CM-cellulase genes (ccl) in Clostridium thermocellum (see JolilT et al., 1986a,b for full references). Four, coding for CM-cellulases A, B, C and D, have been studied intensively. It is interesting that they show little sequence homology to each other. Except for short regions at the 3’ ends which were noted after sequencing, DNA from each of the four clones does not cross-hybridise. The Cellulomonasfravigena strain which we have studied is similar to C. thermocellum in two respects. It has multiple

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Temperature% PH Fig. 2. Temperature (panel A) and pH (panel B) profiles of the CM-cellulase activity conferred on E. coli by plasmids pWM95 (O), 147 (+) and 191 (A). Samples were prepared and assayed as described in the footnote to Table I. pH optima were determined using as buffer systems: citric acid. Na,HPO, (pH 4-7.5), Tris-HCl (pH 8-9) and glycine-NaOH (pH 10).

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CM-cellulase genes and some are not related to each other sufficiently to cross-hybridise. This suggests that multiple enzyme activities may be required to degrade the highly heterogeneous cellulose substrate normally encountered in the wild. It will be interesting to determine if the multiple CM-cellulases of Cellulomonas are organised in a multienzyme complex - the cellulosome - as has been shown in C. thermocellum (Lamed and Bayer, 1988). At least one Celiulomonas species has been found to make cellulosomes (Lamed and Bayer, 1988). Preliminary sequence analysis (A. Al-Tawheed, personal communication) indicates that the pWM95 clone contains two open reading frames, one of which is homologous to the ceIB gene of C. thermocellum (Comet et al., 1983). The second open reading frame displays homology to the exe-/I-1,Cglucanase (cex) and one of the endo+ 1,Cglucanase genes (ce&) of C.fimi (O’Neill et al., 1986; Wong et al., 1986).

ACKNOWLEDGEMENTS

This work was supported by grants from the United Nations Industrial Development Organisation, Imperial Chemical Industries (ICI), the National Board for Science and Technology of Ireland, and the Science Research Council of Pakistan. We thank Dr. A. McHale for activity gels and Dr. P. Sharp and Dr. C. Bailey for useful discussions.

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Coughlan, M.P.: The properties of fungal and bacterial cellulases with comment on their production and application. Biotechnol. Genetic Eng. Rev. 3 (1985) 39-109. Dowds, B.C.A., Sheehan, M.C., Bailey, C.J. and McConnell, D.: Cloning and characterization of the gene for a methanolutilizing alcohol dehydrogenase from Bacillus stearothetmophilus. Gene 68 (1988) 11-22. Fukumori, F., Sashihara, N., Kudo, T. and Horikoshi, K.: Nucleotide sequence of two cellulase genes of alkalophilic Bacillus sp. N-4 and their strong homology. J. Bacterial. 168 (1986) 479-485. Fukumori, F., Kudo, T. and Horikoshi, K.: Truncation of an alkaline cellulase from an alkalophilic Bacilfus species. FEMS Lett. 40 (1987) 311-314. Gilkes, N.R., Langsford, M.L., Kilbum, D.G., Miller Jr., R.C. and Warren, R.A.J.: Mode of action and substrate specificities of cellulases from cloned bacterial genes. J. Biol. Chem. 259 (1984) 10455-10459. Joliff, G., Beguin, P., Juy, M., Millet, J., Ryter, A., Poljak, R. and Aubert, J-P.: Isolation, crystallisation and properties of a new cellulase of Clostridium thermocellum overproduced in Escherichia coli. Biotechnology 4 (1986a) 896-900. Joliff, G., Beguin, P. and Aubert, J-P.: Nucleotide sequence ofthe cellulase gene ceZD encoding endoglucanase D of CIostridium thermocellum. Nucleic Acids Res. 14 (1986b) 8605-8613. Lamed, R. and Bayer, E.A.: The cellulosome concept: exocellular/extracellular enzyme reactor centers for efficient binding and cellulolysis. FEMS Sympos. 43 (1988) 101-l 16. MacKay, R.M., Lo, A., Willick, G., Zuker, M., Baird, S., Dove, M., Moranelli, F. and Seligy, V.: Structure of a Bacillus subtiks endo-/I-1,4glucanase gene. Nucleic Acids Res. 14 (1986) 9159-9170. Maniatis, T., Fritsch, F.F. and Sambrook, J.: Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1982. Miller, G.L., Blum, R., Glemon, W.E. and Burton, A.L.: Measurement of carboxymethylcellulase activity. Anal. Biochem. 1 (1960) 127-132. O’Neill, G., Goh, S.H., Warren, R.A.J., Kilbum, D.G. and Miller Jr., R.C.: Structure of the gene encoding the exoglucanase of Celbdomonasjbni. Gene 44 (1986) 325-330. Sami, A.J., Akhtar, M.W., Malik, N.N. and Naz, B.A.: Production of free and substrate-bound cellulases of Cellulomonas flovigena. Enzyme Microb. Technol. 10 (1988) In press. Wong, W.K.R., Gerhard, B., Guo, Z.M., Kilbum, D.G., Warren, A.R. and Miller Jr., R.C.: Characterisation and structure of an endoglucanase gene celA of Celhdomonas fvni. Gene 44 (1986) 315-324. Communicated by J.K.C. Knowles.