The cellulase encoded by the native plasmid of Clavibacter michiganensis ssp. sepedonicus plays a role in virulence and contains an expansin-like domain

Physiological and Molecular Plant Pathology (2000) 57, 221±233 doi:10.1006/pmpp.2000.0301, available online at http://www.idealibrary.com on

The cellulase encoded by the native plasmid of Clavibacter michiganensis ssp. sepedonicus plays a role in virulence and contains an expansin-like domain M A R KO J . L A I N E 2, MI N N A H A A PA L A I N E N 1,2, TO N Y WA H L RO O S 2, KAT JA KA N KA R E 2, R I I T TA N I S S I N E N 1,2, S H A A B A N K A S S U W I 1,2 and M A RY C . M E T Z L E R 1,2* 1

Department of Biosciences, Division of General Microbiology, University of Helsinki, P.O. Box 56, Viikinkaari 9, FIN-00014 Helsinki, Finland and 2Department of Biology, Division of Plant Physiology and Molecular Biology, University of Turku, Turku, Finland (Accepted for publication October 2000 and published electronically 16 November 2000) We are examining the molecular basis of pathogenicity for Clavibacter michiganensis ssp. sepedonicus, a gram positive coryneform bacterium that causes the economically important potato ring rot disease. We present here a complete restriction map of the native plasmid pCS1, on which we localize the cellulase gene that it contains. A mutant produced by chemical mutagenesis that does not produce cellulase as well as a naturally occurring strain that does not contain the pCS1 plasmid were both shown to be markedly reduced in virulence on eggplant. Both strains became signi®cantly more virulent after the cellulase gene was introduced into the cells by transformation. The complete nucleotide sequence of the cellulase gene was determined and shown to encode a protein of 727 amino acids which would have a predicted molecular weight of 71.5 kDa. The sequence shows a leader sequence for secretion and two typical cellulase domains (a catalytic domain and a cellulose binding domain). Additionally, we identify an unexpected third domain that shows similarity to a plant protein called expansin, which is believed to c 2000 Academic Press * interact with cellulose micro®brils during plant cell expansion. Keywords: cellulase; Clavibacter michiganensis ssp. sepedonicus; expansin; Corynebacterium sepedonicum; potato ring rot.

INTRODUCTION Clavibacter michiganensis ssp. sepedonicus, a gram positive coryneform bacterium, is the causal agent of potato ring rot. The pathogen is highly biotrophic and tends to cause latent infections, in which plants carrying large numbers of bacteria can remain symptom-free. For this reason, the disease has been dicult to control, and many countries have instituted a zero tolerance policy for potato seed certi®cation programs. The bacteria do not tolerate high temperatures, and, probably for this reason, disease control has been most dicult in northern potato growing areas of Europe and North America. Clavibacter species are relatively slow growing and the molecular methods available for studying them are limited. Thus, although the bacteria can be stably transformed with recombinant plasmids [25], methods for marker exchange and transposon mutagenesis have been dicult to develop, as is also true for some other * To whom all correspondence should be addressed. E-mail: mary.metzler@helsinki.®

0885-5765/00/110221+13 $35.00/00

gram positive bacteria. This adds to the diculty of molecular studies for this genus. Nonetheless, we have been studying the molecular basis of pathogenicity for C. michiganensis ssp. sepedonicus. In our previous studies, we found that, for individual bacterial isolates, the ability to induce a hypersensitive response (HR) on the non-host plant tobacco was correlated with virulence on host plants [36]. Strains that were unable to induce an HR were always non-virulent. Extracellular enzymes such as pectinolytic enzymes and cellulases are often important factors for virulence in plant pathogens. These enzymes degrade polysaccharides in the plant cell wall, presumably enhancing the bacteria's ability to spread within the plant and simultaneously releasing nutrients to use as a source of energy. For gram negative plant pathogens, cellulase (b-1,4-glucanase) has been shown to be important for virulence for several species, including Erwinia carotovora pv. carotovora [51] and Ralstonia solanacearum [38] but has only a minor role in symptom development in Xanthomonas campestris pv. campestris [17] and Erwinia chrysanthemi [4]. For gram c 2000 Academic Press *

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positive plant pathogens, cellulase has been shown to play a role in the development of wilt symptoms on tomato infected with C. michiganensis ssp. michiganensis [31]. Previously, C. michiganensis ssp. sepedonicus has been shown to carry a native plasmid, pCS1, of unknown function [6, 33] and several partial descriptions of this plasmid have been published [34, 35]. The related species C. michiganensis ssp. michiganensis (the causal agent of bacterial canker of tomato) contains two native plasmids, and one of these encodes the previously mentioned cellulase that is important in virulence for that species [31]. Production of an extracellular cellulase by C. michiganensis ssp. sepedonicus has been previously demonstrated [2, 16]. This was shown to be an endoglucanase with a temperature optimum of 378C and a pH optimum of 6.0±7.0, depending upon substrate [2]. However, the relevance of the cellulase to bacterial pathogenicity has not been shown. We have previously cloned the cellulase gene from C. michiganensis ssp. sepedonicus encoded by the native plasmid [25]. In this paper, we show the location and sequence of the cellulase gene together with a restriction map of the entire 51 kb plasmid. We present evidence that cellulase activity is one factor contributing to the virulence of C. michiganensis ssp. sepedonicus, and discuss the ®nding that, based on the nucleotide sequence, this cellulase possesses an unexpected third domain, which resembles a plant expansin.

MATERIALS AND METHODS

Bacterial strains and growth conditions Bacterial strains used are listed in Table 1. C. michiganensis ssp. sepedonicus strains were grown in YGM broth [8] with

1.8 % bacto-agar (Difco) added for growth on solid medium, or in DM medium [32] with 1.7 % corn meal agar for growth on solid medium. Cultures were grown at 268C.

Chemical mutagenesis One ml of bacterial culture at an OD660 of 0.7±2.0 was centrifuged for 1±2 min at 1200 g and the supernatant discarded. The cells were washed twice with 0.1 M Nacitrate, pH 5.5 and resuspended in 1 ml of the same bu€er. Ten ml of ethyl methanesulfonate (EMS) was added and the suspension was incubated at room temperature for 1.5±6.5 h. EMS was removed by washing cells with 0.1 M Na-phosphate bu€er, pH 7.0, the cells were resuspended in 5 ml DM and grown 24 h with shaking. Cell suspensions were diluted and spread onto DM plates and grown until colonies were visible. These were replica plated onto DM and indicator plates, and colonies that grew but did not give halos when stained for enzyme activity were tested to determine if they grew to wild-type levels in liquid YGM. The use of this complex media was necessary for these tests as even wild-type C. michiganensis ssp. sepedonicus does not grow at all in minimal liquid medium and grows only very poorly on minimal agar medium.

Agarose plate assays for cellulase activity To test for cellulase production, bacterial colonies were plated onto 1.5 % agar M9 plates [40] containing 1 % carboxymethylcellulose (sodium salt, medium viscosity, Sigma) and 1 g l ÿ1 yeast extract. After growing for an average of 3±4 days, colonies were washed o€ the plate, which was then stained with 0.1 % Congo red [31]. Colonies producing cellulase gave a clear halo.

T A B L E 1. Plasmids and bacterial isolates used in this study Isolate or plasmid

Relevant genotype

C. michiganensis ssp. sepedonicus isolates Cs2, Cs7, Virulent, HR positive, cellulase positive R14, 3M P45 Very low virulence, HR positive, cellulase negative 19/Cs2 Very low virulence, HR positive, cellulase negative Plasmids pCS1 pSPE22 pSPE29 pCK3/II pCK255 pHN216 pHN216:C8

Source or reference [36] S. De Boer This study

Native plasmid of C. michiganensis ssp. sepedonicus [33±35] and this study pBluescript containing the 22 kb SpeI fragment from pCS1 This study pBluescript containing the 29 kb SpeI fragment from pCS1 This study pBluescript containing a 510 bp Sau3A±SacI fragment of C. michiganensis ssp. sepedonicus This study cellulase encoding the leader peptide plus about 1/3 of the catalytic domain pBluescript containing a 522 bp SacI±Sau3A fragment of C. michiganensis ssp. sepedonicus This study cellulase encoding most of Dom3 Shuttle vector for C. michiganensis, Neor, Gnr [25] pHN216 carrying C. michiganensis ssp. sepedonicus cellulase gene [25]

Cellulase in pathogenicity Bacterial transformation Bacteria were grown, prepared and electroporated as previously described [25] using a ®eld strength of 18 kV cm ÿ1 in 0.1 cm cuvettes. After electroporation, bacteria were plated onto 85 mm YGM plates containing 13.3 mg ml ÿ1 of neomycin.

DNA isolation To isolate pCS1 DNA, strain Cs7 was grown as lawns on DM plates and the bacterial cells were scraped o€ and suspended in 10 mg ml ÿ1 lysozyme ( four plates per 5 ml of lysozyme solution). This suspension was incubated at 378C for 90 min. Ten ml of lysis solution (0.2 M NaOH, 1 % SDS) was added, and the solution was shaken and then incubated on ice for 10 min. 7.5 ml of 3 M potassium acetate, pH 4.8, was added and the solution vortexed and placed on ice for 5 min. Debris was pelleted by centrifugation, and the supernatant was extracted with equal volumes of bu€er-equilibrated phenol and chloroform : isoamyl alcohol (24 : 1). The phases were separated by centrifugation and DNA was precipitated from the aqueous phase by adding 25 ml of ethanol. After incubation at room temperature for 10 min, DNA was pelleted by centrifugation. The pellet was resuspended in 8 ml of distilled water, and plasmid was puri®ed using a CsCl gradient by standard methods [40]. To isolate genomic DNA from C. michiganensis ssp. sepedonicus strains, two loops of cells were harvested from lawns growing on YGM plates, and DNA was isolated from them using the Phytopure plant DNA extraction kit (Amersham, Inc.) modi®ed as follows. The cells were resuspended in 1 ml ice-cold TEN bu€er, centrifuged and washed twice with the same bu€er, and then resuspended in 200 ml TEN bu€er to which was added 7.5 ml of 20 mg ml ÿ1 lysozyme, 6 ml of 1 mg ml ÿ1 RNase A and 7.5 ml 0.5 M EDTA. The solution was incubated at 378C for 4 h, after which 600 ml Phytopure Reagent 1 and 200 ml Phytopure Reagent 2 were added. The rest of the puri®cation followed the manufacturer's protocol.

RNA isolation Strain Cs7 was grown in 40 ml of liquid YGM for 1±3 days at 268C. The cells were harvested by centrifugation and washed with 20 ml of RNA bu€er (20 mM Tris, pH 7.5, 200 mM NaCl, 5 mM EDTA). Cells were collected again by centrifugation at 48C. The pellet was resuspended in 2 ml of RNA bu€er, and 100±200 mg of glass beads (150±212 mm, Sigma) was added. The tube was vortexed 3  2 min, then 2 ml of hot (1008C) RNA bu€er and 4 ml of hot phenol (658C) were added and the tube was vortexed for 5 min. After centrifugation, the aqueous phase was re-extracted twice with an equal

223

volume of phenol (RT), twice with an equal volume of phenol : chloroform (1 : 1), then ®nally with an equal volume of chloroform. The aqueous phase was collected and one-®fth volume of 10 M LiCl and an equal volume of ethanol were added and mixed, and the solution was placed at ÿ208C for at least 3 h. After thawing on ice, the sample was centrifuged at 12 000 g for 15 min at 48C, and the pellet was washed with 70 % cold EtOH and evaporated to dryness under a vacuum. The pellet was resuspended in 50 ml of RNAse free water and the concentration measured spectrophotometrically.

Northern blot analysis RNAs (13 mg per lane) were separated on 1.2 % agaroseglyoxal gels and transferred to Hybond nylon membrane (Amersham) by blotting overnight. The membrane was washed in 6  SSC, dried and the RNA was ®xed under u.v. light and de-glyoxylated by heating at 808C for 2 h. Fragments to be used as probes were labeled with digoxigin and hybridizing bands detected using the DIG detection system (Boehringer Mannheim) according to the manufacturer's instructions.

Primer extension To end label oligonucleotides, 10 pmole of oligonucleotide P7 (50 -CGGTGGCGGCTAAGGCAGGC30 ), which hybridizes to the message at positions 879±859 (numbering as per Fig. 4), was labeled with g-P32 ATP using T4 polynucleotide kinase. After incubation at 378C for 1 h, the labeled oligonucleotide was puri®ed using TE midiSELECT-D G-25 spin columns (5prime-3prime, Inc) following the manufacturer's instructions. For hybridization and extension, 8 ml of total RNA (approximately 16 mg) plus 1 ml of labeled primer were mixed, heated to 908C for 10 min, and chilled on ice. Five ml of reaction mix (Pharmacia Biotech First Strand cDNA Synthesis Kit) and 1 ml of dithiothreitol were added and the sample was incubated at 378C for 1 h. The samples were then either stored at ÿ808C or run directly on sequencing gels. Sequencing reactions were done using a Sequenase version 2.0 kit, according to the manufacturer's instructions (United States Biochemical). cDNA and sequencing reaction products were run on polyacrylamide/urea gels according to standard methods [40].

DNA gels and Southern hybridization Bacterial genomic DNA was digested with PstI, run on agarose gels and blotted onto nitrocellulose membrane using standard methods [40]. Probe for Southern blots was prepared by cutting out the 22 kb fragment from pSPE22 using SpeI. This fragment was gel puri®ed and

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digested with MscI, then labeled with 32P using a Multiprime kit (Amersham) and used as a probe.

Eggplant virulence assays and tests of hypersensitive response in tobacco C. michiganensis ssp. sepedonicus strains were tested for virulence by inoculating them as previously described [36] into 28 day old eggplant. For each individual plant, disease symptoms were scored after 18 days as percentage of the leaf surface area showing wilting symptoms, such that, for example, a plant with four leaves in which half of each of two leaves were completely wilted would be scored as having 25 % symptoms. For tests of statistical signi®cance, pairwise anova single factor comparisons were done in Microsoft Excel. All virulence tests of mutants and transformed strains were done at least three times with at least nine plants per treatment and gave qualitatively similar results. Strains were tested for the ability to induce a hypersensitive response in Samsun tobacco as described previously [36].

DNA sequencing Restriction fragments of the C. michiganensis ssp. sepedonicus cellulase gene were subcloned into pBluescript KS and sequenced from universal and reverse primers using an automated sequencing machine (ALF) at the sequencing facility of the Institute of Biotechnology, University of Helsinki. Areas of ambiguity arising from the high GC content of the gene were resolved by producing speci®c oligonucleotide primers and repeating the sequencing reactions, numerous times if necessary, at high temperatures. The nucleotide sequence for the cellulase from C. michiganensis ssp. sepedonicus has been deposited in GenBank under accession number AY007311.

been presented. To facilitate studies of possible pathogenicity factors carried on the plasmid, we generated a complete restriction map. We found that pCS1 could be cut with the restriction enzyme SpeI to generate two large fragments, of 22 and 29 kb. We cloned these fragments into pBluescript KS to generate plasmids pSPE22 and pSPE29. These plasmids were digested with various restriction enzymes, and we used these and previously published data [34] to generate a complete restriction map of pCS1 (Fig. 1). The gene encoding the cellulase (celA), which we had previously cloned [25] and which encodes a cellulase from C. michiganensis ssp. sepedonicus, was localized on this map based on hybridization and restriction fragment patterns. Having a complete restriction map also allowed us to localize on the plasmid the repetitive element, IS1121 [26] that is present in many copies on the bacterial genome [33] and in two copies on pCS1 [35]. Comparison of our map with the restriction map given in a previous study for a small region containing the IS [35] shows that it is present as two separate inverted repeats, both located around the two ClaI sites on the 29 kb SpeI fragment (Fig. 1). Probing the chromosome of C. michiganensis ssp. sepedonicus with pSPE29 on a Southern blot gave a large number of bands of varying intensity, corresponding to the many copies of the insertion sequence present on the chromosome. The pattern was similar to that seen when probing the chromosome with the entire pCS1 plasmid. When pSPE22 was used as a probe, only a limited number of bands of approximately equal intensity were seen; these corresponded to copies of the plasmid in the genomic DNA preparations. This demonstrated that only the 29 kb SpeI fragment of pCS1 contains repetitive chromosomal elements (data not shown).

Virulence of cellulase negative isolates restored to cellulase expression

DNA sequence analysis Primary sequence data was analyzed using the Wisconsin package, version 8 (September 1994), Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, 53711, U.S.A. Database searches were performed using the Blast facility at the National Center for Biotechnology Information of the National Library of Medicine (http:// www.ncbi.nlm.nih.gov/BLAST).

RESULTS

Mapping of native plasmid pCS1 and localization of cellulase gene Although several descriptions of pCS1 have been published, no restriction map of the plasmid has ever

Mutants that no longer produced cellulase were made from wild-type virulent strains by chemical mutagenesis. One of the non-cellulase producing mutants, 19/Cs2, was chosen for further study because it was HR positive on tobacco (data not shown), and it grew to wild type levels on a reduced nutrient media (YGM without casamino acids). Mutant 19/Cs2 was inoculated into eggplant and disease symptoms scored after 18 days. It showed greatly reduced virulence in comparison to the parent strain, Cs2 (Fig. 2). The plasmid pHN216:C8, which is a C. michiganensis ssp. sepedonicus cloning vector carrying the celA gene [25] was transformed into mutant 19/Cs2. This restored cellulase expression to the mutant (data not shown). In eggplant, virulence was greatly enhanced in the transformant carrying pHN216:C8, but not in a transformant carrying vector alone (Fig. 2).

Cellulase in pathogenicity

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51300,Nhe I 51200,Pvu II Spe I,1 50300,Pvu II Bgl II,200 49500,Nhe I Sna BI,400 49100,Nde I Nde I,2500 49000,Apa I Bgl II,4700 46800,Pvu II Nhe I,5300 45200,Apa I Apa I,5800 45100,Cla I Pvu II,6500 44900,Nhe I 43700,Apa I 42700,Bgl II 41300,Nhe I 41200,Nhe I 41000,Cla I 40900,Apa I

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F I G . 1. Restriction map of pCS1. The position and direction of the celA gene and the two copies of IS1121 are marked. HindIII, EcoRI and BclI do not cut the plasmid. The enzymes BamHI, KpnI, MluI, MscI, NcoI, NotI, PstI, SacI, StuI and XhoI all cut at least three times but the sites were not localized on this map. The NdeI site at 37.9 is refractory to complete digestion.

F I G . 2. Virulence of C. michiganensis ssp. sepedonicus strains on eggplant. Thirteen eggplants per treatment were inoculated and symptoms evaluated as described in materials and methods. Plants were inoculated as follows: mock, bu€er alone; Cs2/19, cellulase non-producing mutant; Cs2/19 ‡ vector, mutant Cs2/19 transformed with pHN216 vector alone; Cs2/19 ‡ celA, mutant Cs2/19 transformed with pHN216 : C8; Cs2, wild type strain Cs2; P45, wild type strain P45; P45 ‡ vector, P45 transformed with pHN216 vector alone; P45 ‡ celA, P45 transformed with pHN216 : C8. All bars identi®ed with the same lower case letter do not signi®cantly di€er from each other (P 5 0.05).

Because of the uncertain nature of the mutation in mutant 19/Cs2, we examined a strain that has been reported to lack the native plasmid pCS1 [34]. This strain, P45, induces an HR on tobacco but has a low level of residual virulence. We found no production of extracellular cellulase based on agar plate assays (data

not shown). Using the 22 kb SpeI fragment from pCS1, which contains the cellulase gene, as a probe, we found that P45 did not contain any hybridizing DNA (Fig. 3), whereas all other tested strains did. This demonstrates that the cellulase encoded on pCS1 is lacking in isolate P45. This isolate was transformed to express cellulase

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2

3

4

5

6

8.0 5.0 3.0 1.5

F I G . 3. Southern blot hybridization of C. michiganensis ssp. sepedonicus genomic DNAs probed with the 22 kb SpeI (cellulasecontaining) fragment from pCS1. Lane 1, PstI-digested pSPE22 plasmid DNA; lanes 2±6, PstI digested genomic DNAs from C. michiganensis ssp. sepedonicus strains Cs7 (lane 2); Cs2 (lane 3); R14 (lane 4); P45 (lane 5); 3M (lane 6).

using the plasmid pHN216:C8 and restoration of cellulase expression was con®rmed on cellulase indicator plates (data not shown). This transformed strain, as well as P45 transformed with vector alone, were then tested on eggplant and only the transformant carrying the cellulase gene had signi®cantly gained virulence (Fig. 2).

The nucleotide sequence of celA and analysis of transcript size and promoter region DNA (3.05 kb) containing the cellulase gene was sequenced. This revealed an open reading frame of 2181 bp starting from codon ATG768 and terminating at TGA2951 , encoding a deduced protein of 727 amino acids that we refer to as CelA (Fig. 4). The length of the celA message was determined by Northern blot analysis. One transcript of 2.4 kb was found using either a 510 bp fragment from the N-terminal end ( pCK3/II; data not shown) or a 522 bp fragment from the C-terminal end ( pCK255; data not shown) of the gene to probe total RNA isolated from strain Cs7. The transcription start site of the celA message was determined by primer extension analysis. The start of transcription was located 204 nucleotides upstream from the start codon of the celA open reading frame, corresponding to nucleotide C at position 566 (data not shown).

Analysis of the deduced amino acid sequence of the celA gene The cellulase open reading frame (Fig. 4) begins with a 45 amino acid signal peptide for secretion. This shows typical features of a signal peptide [54], including a cationic N-terminus, a hydrophobic central region and an anionic C-terminus with a typical signal peptidase site (Ala-Val-Ala) just before the presumptive start of the catalytic domain. After removal of the signal peptide, the

calculated molecular weight of the protein would be 71.5 kDa. Analysis of the remainder of the sequence shows an overall domain structure with three discreet and presumably functionaly distinct regions of the mature protein. After the signal peptide, there is a catalytic domain of 344 amino acids. It shows clear identity with a number of other bacterial cellulases, the highest identity being 51 % over the entire domain with the endo-b-1,4glucanase of Bacillus polymyxa [3]. This is followed by a region of 33 amino acids that contains six prolines and three serines. Since interdomain linkers are typically rich in proline, serine and threonine [15], the amino acid composition suggests that this may be an interdomain linker. The next domain is a sequence of 84 amino acids encoding what appears to be a family IIA cellulose binding domain (CBD; Fig. 5) based on its clear similarity (26±35 %) with other members of this family [48]. It is followed by a linker region of 36 amino acids. The last 18 amino acids in this linker consist of a hexapeptide, ``TPPSQA'', repeated three times. Finally, the C-terminus of the open reading frame encodes a novel domain of 202 amino acids which we refer to as Domain 3 (Dom3; Fig. 6). It shows signi®cant sequence identity (36 % over 174 aa) with a reading frame of unknown function, called YoaJ, from Bacillus subtilis. Without a known function, little can be concluded from this similarity. However, in addition, a lower level of sequence identity can be seen with a family of plant proteins called expansins. Although the overall level of identity is not high (maximum of 30 % over 120 aa), it is seen with many (at least 15) members of the expansin family and suggests that Dom3 has the same overall fold as expansin.

DISCUSSION A complete description of the native plasmid pCS1 was a necessary prerequisite for studying pathogenicity factors that it encodes, including the cellulase. We also determined the location of the two copies of the insertion sequence IS1121, and this was helpful for resolving the status of strain P45, a naturally occurring strain that has been reported to be plasmid free [34]. Having not been able to cure any of our strains of the plasmid (unpublished data), we anticipated that P45 would be very useful for examining functions of plasmid-encoded factors. However, strain P45 had previously been reported to be cellulase positive [2], although we saw no evidence of cellulase production. Southern blot analysis using the 29 kb SpeI fragment of pCS1 as a probe to examine the chromosome of P45 gives a pattern similar to that seen with other strains that clearly carry pCS1 (data not shown). In contrast, using the 22 kb SpeI fragment of pCS1 as a probe, we obtained no hybridization to strain P45 genomic DNA, demonstrating that

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F I G . 4. Cont. Complete deduced amino acid sequence of C. michiganensis ssp. sepedonicus cellulase. The nucleotide sequence is shown above, with the amino acid sequence, along with italicized numbering, shown below. The domains and linkers are delineated with lines underneath the amino acid sequence. The conserved TG in the promoter region is underlined, and the four direct repeats located around start of transcription are shown with arrows underneath. The start of transcription is shown with an arrowhead above the nucleotide sequence. Residues involved in the active site of the catalytic domain are shown in bold.

this strain does not carry the cellulase gene and lacks a large portion of pCS1. This suggests that hybridization of P45 genomic DNA to pCS1 is due to the repetitive element IS1121, of which there are two copies on the 29 kb SpeI fragment of pCS1, and con®rmed that P45 does not contain the cellulase gene. Previous reports that P45 expresses cellulase [2] may be due to di€erences in substrates used or use of a mixed culture containing P45 plus another, cellulase producing strain. For mutant Cs2/19 there was the possibility that other, unwanted mutations had been introduced during the treatment with mutagen. Thus strain P45 was useful for con®rming that cellulase was a virulence factor in C. michiganensis ssp. sepedonicus. It should be noted that P45, as well as the mutant Cs2/19, are both HR positive on tobacco. We have found previously that wild-type virulent strains of C. michiganensis ssp. sepedonicus give an HR in tobacco, and wild-type non-virulent strains do not; the only exception being P45, which has very low virulence on eggplant but gives an HR on tobacco. The molecular basis of the hypersensitive response in C. michiganensis ssp. sepedonicus is not understood at this time ( for example, no hrp genes have been isolated); however, the clear correlation between virulence and HR induction suggested that the ability to induce HR on non-hosts, although not sucient by itself, might be a pre-requesite for full virulence. Thus we felt that the ability to induce an HR on tobacco was important for strain P45 if we hoped to enhance its virulence through expression of the cellulase gene. The low level of virulence

of both cellulase non-producing mutants and the clear increase in virulence seen when either is transformed to express cellulase is consistent with a model in which the cellulase accounts for a portion, but not all, of C. michiganensis ssp. sepedonicus virulence. Determining the nucleotide sequence of the region containing the cellulase gene from pCS1 allowed us to examine the upstream regions of the gene for regulatory sequences. In contrast to Clavibacter species, for which only a few genes have been isolated and sequenced, many genes from the related genus Streptomyces have been characterized, providing a basis for comparison. The C. michiganensis ssp. sepedonicus cellulase gene, as in most Streptomyces promoters [47], does not contain ÿ10 or ÿ35 regions with signi®cant homology to the Escherichia coli s70 promoter consensus sequence. A conserved gram positive dinucleotide motif (TG) between the ÿ10 and ÿ35 regions has previously been described in Clavibacter [10, 19]. Several tandem direct repeats are seen around the transcription start site. Regulatory regions, such as direct and inverted repeats, located at the transcription start site in Streptomyces promoters that do not contain typical s70 promoter consensus sequences, have been previously described [22]. Direct repeats have been suggested to play a role in gene regulation for both gram negative and gram positive bacteria [5, 9, 50] and have been observed in previously described Clavibacter promoters [10, 19]. Initial sequencing results suggested that the fragment carrying the cellulase could also contain another gene,

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F I G . 5. Sequence alignment of cellulose binding domains from C. michiganensis ssp. sepedonicus cellulase and the eight most similar CBDs of family IIa. Residues that are conserved in all nine sequences are marked in bold, as well as the incompletely conserved cysteines at the N and C termini of most of the sequences. The CBDs are from the following proteins: Srochei, EglS from Streptomyces rochei, accession number X7953 [37]; Slivid, CelB from S. lividens, accession number U04629 [52]; C®miD, CenD from Cellulomonas ®mi, accession number B47093 [30]; C®miB, CbhB from C. ®mi, accession number S59077 [43]; Tfusca, cellulase E2 from Thermomonospora fusca, accession number M73321 (unpublished); Mbispora, CelA from Microbispora bispora, accession number P26414 [55]; Mcellulo, McenA from Micromonospora cellulolyticum, accession number S76408 [27]; P¯uor, CelB from Pseudomonas ¯uorescens, accession number X52615 [13]; Cmsep, celA from C. michiganensis ssp. sepedonicus, accession number AY007311.

especially considering that the reported molecular weight for the enzyme was 28 kDa [2]. For this reason, we did Northern blot hybridizations using as probes regions both from the 50 end of the fragment, which clearly carried the leader peptide and part of the catalytic domain, plus a fragment from the 30 region, corresponding to Dom3. Both probes gave the same size hybridizing band (2.4 kb) supporting the conclusion that we were dealing with a single transcript. Further sequence analysis con®rmed that we had only one open reading frame, and suggested that the reported molecular weight for the protein was incorrect. Examining the sequence of the open reading frame encoded by the cellulase gene from C. michiganensis ssp. sepedonicus revealed that it was very similar (84 % identity) to the cellulase from C. michiganensis ssp. michiganensis [23]. Both proteins have a multi-domain structure containing three domains separated by linker regions. Cellulases often contain two or more domains, one responsible for the catalytic activity (the catalytic domain) and the other(s) for enhancing binding to cellulose (the cellulose binding domain, or CBD [28]). In general, the CBD strongly enhances cellulase activity. A number of cellulases also contain other domains, and in many cases, the function of these other domains is unknown. The large number of sequenced cellulases has allowed the domains to be placed in families based on amino acid similarity. Based on its similarity to other endoglucanases, the CelA catalytic domain can be placed in cellulase family A [21], also known as glycosyl hydrolase family 5 [20]. It

contains the seven invariant amino acids previously identi®ed for this family [21] and con®rmed as being located in the active site of the enzyme based on the Xray crystallographic structure of endoglucanase A from Clostridum cellulolyticum [11]. The strongest similarity is to the endo-b-1,4-glucanase of B. polymyxa [3], with 51 % identity over the entire domain. Other similar sequences are the E1 endo-b-1,4-glucanase from Acidothermus cellulolyticus (accession number U33212) with 50 % identity over the entire domain, the EngXCA endo-b1,4-glucanase of Xanthomonas campestris pv. campestris with 44 % identity [18], and the CelB endo-b-1,4-glucanase from Caldocellum saccharolyticum with 33 % identity [41]. Lower levels of identity can be found to protein sequences for similar enzymes from numerous gram negative and gram positive bacteria. After the ®rst presumed linker is a family IIA (CBD) based on its clear similarity to other CBDs from this family [48]. Sequence identity to the most closely related members of this CBD family is in the range of 26±35 % (Fig. 5). It lacks the typical C and N terminal cysteines, which normally form a disul®de bridge to connect the termini of this domain [14]. Several other CBDs of this family also lack one or both of these terminal cysteines [48]. The three conserved tryptophans are particularly important, as they interact with the glycosyl rings of the cellulose chain [49]. The most unusual feature of CelA is its third unique domain, Dom3, which is most similar to a-expansins.

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F I G . 6. Sequence alignment of expansin-like domain from C. michiganensis ssp. sepedonicus, the open reading frame of unknown function from B. subtilis yoaJ, accession number Z99114 [24], and eight similar plant expansins. Amino acids that are conserved in at least nine of the sequences are marked with bold, with valine and alanine being considered equivalent to each other, and the aromatic residues tryptophane, phenylalanine and tyrosine being considered equivalent. The expansins are from the following organisms: B. napus, accession number AJ000885 [12]; tomato, accession number U82123 [39]; cotton, accession number G2244736 [44]; cucumber, accession number U30382 [42]; loblolly pine, accession number U64893 (Hutchison et al., unpublished); Rice, accession number U30477 [42]; A. thaliana 1, accession number U30476 [42]; A. thaliana 2, accession number U30481 [42].

Although sequence identity is low, expectation values from Blast for comparisons of Dom3 to expansins start at 2  10 ÿ4. This indicates signi®cant levels of identity [1] that suggest a common evolutionary origin. Expansins are plant proteins located in the cell wall that are involved in plant cell expansion. Isolated plant cell walls can be stretched up to 50 % of their original length. However, treatments that inactivate cell wall proteins make the cell walls brittle, causing them to break when stretched. Puri®ed expansin can be added back to such cell wall fragments, and this restores elasticity [29]. Current models propose that expansin acts as a kind of molecular grease, allowing the cellulose micro®brils to slip past each other [7]. A catalytic function has not been determined for expansins, although they contain a His-

Phe-Asp motif as well as conserved cysteine residues, as do members of the family 45 group of glycosyl hydrolases (also known as family K cellulases). The similarity of Dom3 to a-expansins begins near the middle of the expansin sequence, starting at the His-Phe-Asp motif (His-Leu-Asp in Dom3) and continues nearly to the end of both proteins. Overall, in spite of the low identity between Dom3 and expansins, certain structural similarities can be observed. In expansins, there is a cysteine-rich region at the Nterminal end of the mature protein, a basic region, and ®nally a region containing several conserved tryptophans [7]. The cysteine-rich region is believed to contain a number of disul®de bridges, although the signi®cance of this is not known. In comparison, Dom3 also contains

Cellulase in pathogenicity cysteine in the N-terminus; although the location of the cysteines within the sequence is not conserved, all four cysteines in the protein occur within the ®rst 70 amino acids of the domain (the total domain length is 202). The central, basic region of expansins is enriched in lysine and arginine; for example, there are nine arginine and lysine residues within the 80 central residues in the cucumber expansin (Fig. 6). Similarly, for Dom3, the central 80 amino acid residues include eight arginine and lysine residues; only one of which, however, is strictly conserved. The third region is also similar between the two proteins, with two of the three tryptophans conserved. These may be involved in binding to cellulose, as is the case for the conserved tryptophan residues in CBD family II. No expansin-like domain had been reported associated with a cellulase prior to the determination of the amino acid sequences for CelA from C. michiganensis ssp. michiganensis [23] and C. michiganensis ssp. sepedonicus (this report). Interestingly, however, the recent determination of the genome sequence for the causal agent of citrus variegated chlorosis, Xylella fastidiosa [45], reveals that it contains an open reading frame with identity to both the catalytic domain (35 % identity) and Dom3 (28 % identity) of CelA from Clavibacter. The Xylella reading frame is not, however, big enough to encode a CBD as well, but rather appears to contain only two domains. Additionally, a small, single-domain cellulase has recently been described from Mytilus edulis (blue mussel) that has a low level of identity to expansin [53]. It does not, however, show signi®cant identity with Dom3 from CelA. The presence of an expansin-like domain makes biological sense, as it might enable the cellulase to break down the plant cell wall more easily by improving access to the (insoluble) substrate. The presence of a domain in this bacterial cellulase that is related to a plant gene may not be as surprising as it seems, as the genome sequencing project for Chlamydia has revealed a number of genes that appear to have been acquired from plants [46]. Cellulases are generally associated with necrotrophic pathogens where the bacteria cause disease through destruction of host tissue. In such organisms, many di€erent hydrolytic enzymes with di€erent speci®cities and roles are found. However, massive and rapid breakdown of host tissues is not part of the life cycle of the highly biotrophic pathogen C. michiganensis ssp. sepedonicus, and the role of the single cellulase in pathogenicity is not clear. Future investigations will focus on both the overall role of the cellulase in pathogenicity, and the speci®c functions of the various domains of cellulase. The authors would like to thank Professor Rudolf Eichenlaub for his gift of the cellulase gene from Clavibacter michiganensis ssp. michiganensis, and Dr Solke De Boer for assistance with testing virulence of chemical mutants.

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We thank Adrian Goldman for critically reading the manuscript. This research was supported by the Finnish Academy.

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