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Applied and Environmental Microbiology, June 2008, p. 3823-3830, Vol. 74, No. 12
0099-2240/08/$08.00+0     doi:10.1128/AEM.02701-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Chitin Hydrolysis by Listeria spp., Including L. monocytogenes

J. J. Leisner,^* M. H. Larsen, R. L. Jørgensen, L. Brøndsted, L. E. Thomsen, and H. Ingmer

Department of Veterinary Pathobiology, Faculty of Life Sciences, University of Copenhagen, Copenhagen, Denmark

Received 30 November 2007/ Accepted 2 April 2008

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Listeria spp., including the food-borne pathogen Listeria monocytogenes, are ubiquitous microorganisms in the environment and thus are difficult to exclude from food processing plants. The factors that contribute to their multiplication and survival in nature are not well understood, but the ability to catabolize various carbohydrates is likely to be very important. One major source of carbon and nitrogen in nature is chitin, an insoluble linear β-1,4-linked polymer of N-acetylglucosamine (GlcNAc). Chitin is found in cell walls of fungi and certain algae, in the cuticles of arthropods, and in shells and radulae of molluscs. In the present study, we demonstrated that L. monocytogenes and other Listeria spp. are able to hydrolyze -chitin. The chitinolytic activity is repressed by the presence of glucose in the medium, suggesting that chitinolytic activity is subjected to catabolite repression. Activity is also regulated by temperature and is higher at 30°C than at 37°C. In L. monocytogenes EGD, chitin hydrolysis depends on genes encoding two chitinases, lmo0105 (chiB) and lmo1883 (chiA), but not on a gene encoding a putative chitin binding protein (lmo2467). The chiB and chiA genes are phylogenetically related to various well-characterized chitinases. The potential biological implications of chitinolytic activity of Listeria are discussed.

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Listeria spp., including Listeria monocytogenes, are ubiquitous microorganisms in the environment, e.g., in soil, vegetation, sewage, fresh water, and low-salinity water (30), and thus are difficult to exclude from food processing plants. L. monocytogenes subtypes differ in their associations with various environments (26, 31), which may be related to differences in their abilities to survive in nature, though so far these mechanisms are not well understood (30). However, with respect to energy-yielding substrates, the ability to catabolize various carbohydrates must play an important role. As an example, the predominance of the xylose-fermenting Listeria ivanovii over other listerial species in a sawmill has been interpreted in terms of the availability of xylose, a wood breakdown product (5). In the present study, we have focused on the ability of Listeria spp. to hydrolyze chitin, the second most common carbohydrate in the environment and a major source of carbon and nitrogen (3, 10).

Chitin is an insoluble linear β-1,4-linked polymer of N-acetylglucosamine (GlcNAc), and it is a component in, e.g., cell walls of fungi and certain algae, in the cuticles of arthropods, in the peritrophic membranes of annelids and some arthropods, and in the shells and radulae of molluscs (3, 10). Chitinolytic bacteria are ubiquitous in aqueous environments, and their ability to degrade chitin is important for their interactions with zooplankton (22). For terrestrial bacteria, chitin hydrolysis may be important if arthropods are host organisms (8). This activity may also play a role as an antagonistic tool toward fungi, as shown for various bacteria, including Firmicutes (42). In addition, it is possible that chitin, present in sediments and soil, serves as a convenient substrate for saprophytic bacteria as a result of arthropod decomposition (10). In the present report, we show for the first time that various Listeria spp. are chitinolytic, and we identify in L. monocytogenes EGD two genes, lmo0105 and lmo1883, now designated chiB and chiA, that are required for chitinolytic activity.

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Examination of chitinase activities.
Listeria cultures were inoculated on a basic chitin medium (BCM) agar lawn containing per liter 10.0 g of tryptone, 5.0 g of yeast extract, 10.0 g of NaCl, 15.0 g of agar, and 2.5 g of -chitin (from crab shells; Sigma C9752) with phosphate buffer, pH 6.9; cultures were incubated under aerobic or under microaerophilic conditions at 10, 30, or 37°C and scored for hydrolytic ability (clearing zones) for up to 22 days. The effect of added glucose on the growth of a selection of species and strains during incubation at 30 and 37°C was tested by the use of BCM buffered with 0.5% glucose and tryptone soy agar (TSA; Oxoid CMO131) supplemented with 2.5 g liter^–1 chitin and with or without 0.4% glucose. Tests were performed as at least three independent replicas.

Identification and phylogenetic analyses of genes encoding putative chitinases and chitin-binding proteins in Listeria spp.
We used an authoritative online resource describing the classification of known glycosyl hydrolases and carbohydrate binding proteins to identify chitinases and chitin-binding proteins in published genome sequences of Listeria spp. (www.cazy.org/geno/acc_geno.html). The chitinase genes lmo0105 (AL591973) and lmo1883 (AL591981) and a gene encoding a chitin-binding protein, lmo2467 (AL591983), from the genome sequence of the L. monocytogenes EGD-e strain (9) were employed as probes to search for similar sequences by using the microbial resource BLAST at the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov/). A group of up to 14 to 16 sequences most similar to the EGD-e genes was selected for further phylogenetic analyses in addition to sequences representative of other bacterial or eukaryotic groups and sequences representing bacteria with well-characterized chitinolytic systems, including Bacillus circulans WL-12 (38, 39, 40), Vibrio cholerae O1 biovar El Tor strain N16961 (12, 19), and Vibrio harveyi BB7 (34). Protein domain predictions, including determination of the glycosyl hydrolase family 18 (Glyco_18) domain, were obtained from NCBI as well as from http://smart.embl-heidelberg.de/. Signal peptide predictions were performed using SignalP, version 3.0, from the Center for Biological Sequence Analysis, BioCentrum-DTU, Technical University of Denmark (www.cbs.dtu.dk/services/SignalP/).

Glyco_18 domains of sequences selected from BLAST searches were aligned to the Glyco_18 domains of the proteins encoded by lmo0105 (amino acids [aa] 38 to 436) and lmo1883 (aa 44 to 327). Alternatively the entire sequences were aligned to the entire sequences of lmo0105, lmo1883, and lmo2467 products. Phylogenetic trees were constructed by the neighbor-joining method using the Clustal X (version 1.81) (36) and MEGA, version 4 (35), software packages with default settings.

The presence of lmo0105- and lmo1883-related sequences in L. monocytogenes F2365 was examined by PCR and using the following primers: lmo0105-fo, 5'-TTACGGTGATTGGTCGA-3'; lmo0105-re, 5'-CCACGCCTTGTTTATCCA-3'; lmo1883-fo, 5'-CTTTTGGCAATAGGTGGA-3'; and lmo1883-re, 5'-CATCCACATTGGCTGGTA-3'.

Construction of deletion mutants.
To examine the effect of the genes annotated as catalyzing chitin degradation (lmo0105 and lmo1883) or adherence (lmo2467) on the chitinolytic phenotype, mutants of L. monocytogenes EGD with deletions of one of each gene were constructed in addition to a double mutant lacking both presumed chitinase genes. Mutants with in-frame deletions in the coding regions of lmo0105, lmo1883, and lmo2467 were constructed by gene splicing using the overlap extension method (11). Primers were designed to amplify two fragments, one comprising the 5' end of the gene and upstream sequences (primers lmoxxxxa [i.e., lmo0105a, lmo1883a, or lmo2467a] and lmoxxxxb) and the other comprising the 3' end of the gene and downstream sequences (primers lmoxxxxc and lmoxxxxd, where xxxx refers to the numbers in the locus tag) (Table 1). The fragments were joined to create PCR fragments containing 2,226-bp, 1,011-bp, and 1,392-bp in-frame deletions of the lmo0105, lmo1883, and lmo2467 genes, respectively. These PCR products were cloned into pCR2.1-TOPO (Invitrogen) and thereafter cloned into the BamHI and XbaI restriction endonuclease sites of the temperature-sensitive shuttle vector pAUL-A (4). Subsequent electroporation and double crossover were performed as described previously (18). The lmo0105 lmo1883 double mutant was constructed in the lmo0105 deletion strain by electroporation and double crossover with pAUL-A with the lmo1883 deletion PCR fragment. The mutated alleles were sequenced to verify the presence of in-frame deletions of 2,226 bp, 1,011 bp, and 1,392 bp in the coding regions of the lmo0105, lmo1883, and lmo2467 genes, respectively, of the mutants.

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TABLE 1. Primer sequences used to obtain internal deletions in the lmo0105, lmo1883, and lmo2467 genes by double crossover recombinationa

Growth of L. monocytogenes EGD and mutant strains.
Aliquots of 30 ml of BCM without chitin but with 0.2% glucose or 0.2% GlcNAc added and brain heart infusion broth were used as media to compare growth responses of cultures inoculated at an initial optical density of 0.1 and incubated as either static or shaken cultures at 30°C. Growth was measured by the optical density at 600 nm.

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Screening of chitinase activities among Listeria spp.
We investigated the presence of chitinase activity among 28 strains of all six Listeria spp. by using a plate assay based on chitin-containing agar plates. We found chitinolytic activity for the three genome-sequenced strains Listeria innocua Clip 11262, L. monocytogenes EGD, and Listeria welshimeri LMG 11389^T; delayed activity for strains of L. ivanovii and Listeria seeligeri; and no activity for Listeria grayi (Table 2; see also Fig. 4). Indeed, the ability to hydrolyze chitin is common for a wide range of L. monocytogenes strains, with NCTC 7973 and F2365 as the lone exceptions (Table 2). Homologues of lmo0105 and lmo1883 are found in the genome of L. monocytogenes F2365, and the presence of these genes in our isolate of strain F2365 was shown by PCR (data not shown).

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TABLE 2. Chitinolytic activity of Listeria strains

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FIG. 4. Glucose and temperature influence the chitinase activity of Listeria spp. Bacterial suspensions of L. innocua Clip 11262 (1), L. monocytogenes Scott A (2), L. seeligeri LMG 11386 (3), L. monocytogenes EGD (4), L. welshimeri LMG 11389T (5), and L. monocytogenes 4446 (6) were spotted onto TSA plates containing 2.5 g liter–1 chitin without glucose (A and C) or supplemented with 0.4% glucose (B) and incubated for 3 days at 30°C (A and B) or at 37°C (C).

lmo0105 and lmo1883 encode L. monocytogenes EGD chitinases.
The majority of bacterial chitinases belong to Glyco-18 family, and two members of this family (lmo0105 and lmo1883 in L. monocytogenes EGD) are found in all listed genome-sequenced isolates of L. innocua (Clip 11262), L. monocytogenes (e.g., 4b H7858, HPB2262, FSL N1-017, 10403S, 1/2a F6854, FSL N3-165, F6900, J0161, J2818, EGD-e, and F2365), and L. welshimeri (LMG 11389^T). In addition, all sequenced isolates carry one gene annotated as a carbohydrate-binding family 33 module (e.g., lmo2467 in L. monocytogenes EGD-e) that is possibly involved in chitin binding. In all cases these genes are not organized in an operon but are located far apart on the genome. The lmo0105 gene encodes a 756-aa protein with a 24-aa signal peptide and includes a 398-aa N-terminal Glyco_18 domain and a 41-aa family 5 carbohydrate-binding module. The lmo1883 gene encodes a 352-aa protein with a 283-aa Glyco_18 domain and a 29-aa signal peptide but contains no carbohydrate-binding module. The lmo2467 gene encodes a 476-aa protein with a 29-aa signal peptide that includes four domains, three of which are characterized as chitin or chitin/cellulose binding domains; the fourth encodes a sequence homologous to fibronectin type III domains.

In order to study the contribution of the individual genes lmo0105, lmo1883, and lmo2467 to the chitinolytic activity of L. monocytogenes, we inactivated these genes by introducing in-frame deletions and compared the chitinolytic activities of the resulting mutants with those of the wild type. Interestingly, the chitinolytic activity was completely abolished in the strain lacking lmo0105 (lmo0105), and it was partially reduced in the lmo1883 strain. In contrast, deletion of lmo2467 had no effect on the ability to hydrolyze chitin (Fig. 1). As expected, no chitinolytic activity was observed in a mutant strain in which both lmo0105 and lmo1883 had been deleted. The differences in chitinolytic activities revealed by EGD wild-type and mutant strains were not due to differences in growth rates as all strains showed similar growth in brain heart infusion broth and in BCM without chitin but supplemented with 0.2% glucose or 0.2% GlcNAc (results not shown). Thus, both lmo0105 and lmo1883 are necessary for the wild-type chitinolytic phenotype, and therefore we designate lmo0105 chiB and lmo1883 chiA.

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FIG. 1. Hydrolysis of chitin by Listeria monocytogenes EGD and lmo0105, lmo1883, lmo2467, and lmo0105 lmo1883 mutants. Ten microliters of bacterial suspensions was inoculated on TSA supplemented with 2.5 g liter–1 chitin and incubated for 4 days at 30°C. Arrows point to the clearing zones. The image is representative of three independent experiments.

Phylogenetic analyses of ChiB, ChiA, and putative chitin-binding proteins in Listeria spp.
The gene products of chiB (lmo0105), chiA (lmo1883), and lmo2467 of L. monocytogenes EGD-e are closely related to homologous proteins in other Listeria species and strains of L. monocytogenes (Fig. 2 and 3; also results not shown), but their phylogenetic relationships to similar proteins in other bacterial genera are very different. The sequence of the putative catalytic region embedded in the Glyco_18 domain (aa 38 to 436) of the protein encoded by chiB (lmo0105) is similar to group III, as outlined by Svitil and Kirchman (34), that includes firmicute chitinases. In agreement with this result, the ChiB Glyco_18 domain is related to a well-characterized chitinase produced by Clostridium paraputrificium (25) and a hypothetical protein encoded by the related Eubacterium ventriosum; the ChiB Glyco_18 domain has less similarity to a large group of gene products annotated as chitinases in other Clostridium spp., albeit without strong bootstrap support (Fig. 2). More distantly related Glyco_18 domain sequences annotated as chitinases were also found for various Bacillus spp. including the well-characterized chitinase A-1 produced by B. circulans WL-12 (38, 39) and various Vibrio spp. and Shewanella spp. (Fig. 2). A phylogenetic analysis of the entire L. monocytogenes ChiB (lmo0105 gene product) gave essentially the same result (results not shown).

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FIG. 2. Alignment of the amino acid sequence of the L. monocytogenes lmo0105 protein (aa 38 to 436) Glyco_18 domain (in bold) with representative sequences of Glyco_18 domains in other species and strains. Sequences marked with an asterisk are referenced in the text.

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FIG. 3. Alignment of amino acid sequence of the L. monocytogenes lmo1883 protein (aa 44 to 327) Glyco_18 domain (in bold) with representative sequences of Glyco_18 domains in other species and strains. Sequences marked with an asterisk are referenced in the text.

In contrast to the sequence of ChiB, the sequence of the putative catalytic region encoded by chiA (lmo1883) is similar to group IV as outlined by Svitil and Kirchman (34). The Glyco_18 domain (aa 44 to 327) of L. monocytogenes ChiA comprises most of the sequence of this protein and is related to the Glyco_18 domain of the Enterococcus faecalis V583 EF0361 gene product. More distantly related Glyco_18 domain gene products are encoded by Serratia marcescens, Lactococcus lactis, Pseudomonas spp., Vibrio spp., including the well-characterized ChiA chitinase produced by V. harveyi (34), and bacteria of other genera (Fig. 3).

The entire lmo2467 sequence is related to genes annotated as chitin-binding proteins encoded by various lactic acid bacteria, including Enterococcus faecalis V583, Enterococcus faecium DO, Lactobacillus sakei subsp. sakei 23K, and Lactobacillus plantarum WCFS1, and more remotely related to genes encoded by a variety of other bacteria (results not shown).

Effects of temperature and glucose on chitin hydrolysis by Listeria.
With the aim of determining how environmental conditions affect the chitinolytic activity of Listeria spp., we examined chitinase activity at various temperatures and in the presence of glucose. We observed chitin hydrolysis at 10°C, 30°C, and 37°C (Table 2), but the activity was in general diminished at 37°C and was very low at this temperature for the L. monocytogenes 4239 and LO28 strains as well as for L. ivanovii Div-B1 and absent for L. seeligeri LMG 11386 (Table 2 and Fig. 4; also data not shown). At 10°C, the activity level was also diminished, but at this temperature, growth was also reduced (results not shown). The effect of glucose on chitinolytic activity was examined for six strains: L. innocua Clip 11262; L. monocytogenes strains 4446, Scott A, and EGD; L. seeligeri LMG 11386; and L. welshimeri LMG 11389^T. The addition of 0.4% glucose to TSA chitin agar plates reduced the clearing zones of all examined strains (Fig. 4). The addition of 0.5% glucose to BCM gave similar results (results not shown).

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This study demonstrates that many species of Listeria possess chitinolytic activity and that the chitinase encoded by lmo0105 in L. monocytogenes EGD is necessary for chitinolytic activity, whereas the chitinase encoded by lmo1883 is required for maximum activity. Consequently, lmo0105 was designated chiB, and lmo1883 was designated chiA.

The chitinolytic ability was shown to be common for a wide range of Listeria species and strains. Generally, Listeria spp. hydrolyzed chitin at 10, 30, and 37°C although the activity was reduced at 10 and 37°C. Furthermore, the addition of glucose to chitin agar plates reduced the overall size of the clearing zones, suggesting that chitinase activity is subjected to catabolite repression mediated by easily fermentable carbohydrates and regulated by temperature.

Only L. grayi LMG16491 and two L. monocytogenes strains (NCTC 7973 and F2365) could not hydrolyze chitin (Table 2). While L. monocytogenes NCTC 7973 is anomalous regarding the effect of carbohydrates on virulence gene expression (24), L. monocytogenes F2365 is known to carry several mutations (27). When we examined the genome sequence for L. monocytogenes F2365 in more detail, we found that ChiB in this strain harbors two conserved amino acid substitutions and one nonconserved amino acid substitution, while the F2365 ChiA exhibited four nonconserved substitutions relative to the L. monocytogenes EGD-e strain. None of the substitutions is located in the conserved sequences of the putative catalytic regions of these proteins. The F2365 homologue of the lmo2467 gene product shows a larger number of substitutions, including 14 conserved and 19 nonconserved; however, this protein was shown in this study not to be necessary for chitinase activity by the EGD strain. The promoter regions of F2365 chiB and chiA do not show any divergence in their sequences from EGD-e. PCR using primer sequences for lmo0105 and lmo1883 revealed the presence of fragments with the predicted sizes, showing that sequences encoding chiB and chiA, indeed, appear to be present in the L. monocytogenes F2365 strain included in this study. Taken together, these results indicate that the lack of chitinolytic activity of the F2365 strain is not due to the mutations in coding and upstream sequences of the two chitinase genes but is probably a result of changes in regulatory genes located elsewhere in the genome. In support of this notion, a specific ATP-dependent HPr kinase/phosphorylase which is stimulated by intermediates of the glycolytic pathway represses expression of both lmo0105 and lmo1883 (23), and a number of genes, including ccpA, sigB, and mogR, affect the expression of lmo1883 (15, 23, 32).

Using bioinformatics tools, we investigated the putative functional domains of ChiB and ChiA. The group III ChiB (Lmo0105) chitinase of L. monocytogenes EGD-e contains both a catalytic Glyco_18 domain and a chitin-binding family 5 module. The central role of the ChiB chitinase for enzymatic activity toward native chitin found in our study resembles the results obtained for the related C. paraputrificium ChiB and B. circulans WL-12 A-1 chitinases (Fig. 2) (25, 38). Both of these enzymes contain a carbohydrate domain that is important for activity toward native chitin but not processed chitin, such as colloidal chitin. It is intriguing that the group IV ChiA (Lmo1883) chitinase contains only a catalytic Glyco_18 domain and not a chitin-binding domain, which may explain the lack of chitinolytic activity of the mutant strain that expresses only ChiA and not the ChiB chitinase (Fig. 1). Consistent with the group III C. paraputrifium and B. circulans chitinases, the group IV chitinase A (Fig. 3) produced by V. harveyi BB7 does not hydrolyze colloidal chitin as well if the putative chitin-binding domain is deleted (34). It is well known that chitinolytic systems in, e.g., Alteromonas sp. strain O-7, B. circulans WL-12, Vibrio furnissii, and V. cholerae all produce multiple chitinases which differ in substrate specificities and subsequently have different roles in the chitin catabolic cascade (12, 16, 19, 28, 38). Additional studies, including the determination of the activity of purified enzymes, are necessary to further investigate the catalytic specificities encoded by chiB (lmo0105) and chiA (lmo1883). Another interesting question to explore is whether ChiA is cell associated because this may offer an alternative explanation of the lack of chitinolytic activity of the mutant that expresses only this enzyme and not ChiB.

The most closely related homologues of the Glyco_18 domain of the listerial ChiB gene product were distributed within Clostridiales, including C. paraputrificium, E. ventrosum, and other Clostridium spp., which mostly are soil bacteria (Fig. 2). More distantly related sequences were found in the bacterial genera Bacillus, Shewanella, and Vibrio that include mainly soil and marine bacteria in addition to the genera Myxococcus and Saccharopolyspora. For marine bacteria, chitinolytic activity may facilitate attachment to zooplankton (22), whereas such activity may support a saprophytic lifestyle of soil-associated Firmicutes or contribute to antagonistic activities toward fungi, as suggested for Clostridium thermocellum ATCC 27405 endochitinase Chi18A (Fig. 2) (42).

Closely related homologues to the Glyco_18 domain of the chiA (lmo1883) gene product were comprised of Listeria spp. and the related firmicute E. faecalis (Fig. 3). Chitinolytic activity of E. faecalis has been suggested to support survival in aqueous environments by promoting attachment to and survival on zooplankton (33).

The putative catalytic regions of the Glyco_18 domains of L. monocytogenes ChiB and ChiA belong to different groups according to Svitil and Kirchman (34), and phylogenetic analyses show them to be very distantly related (Fig. 2 and 3), suggesting that they do not have a monophyletic origin. This result is supported by the fact that the genes are distantly localized on the genome and, moreover, also located far away from the lmo2467 gene that encodes a putative chitin-binding protein. This situation is in contrast to that found in other bacterial chitinolytic systems in which many of the genes involved are organized in operons, including V. cholerae N16961 (12, 19, 22) and V. furnissii (19).

The products of L. monocytogenes ChiB and ChiA are very distantly related to similar V. cholerae O1 biovar El Tor strain N16961 genes, VCA0027 and VC1952 (Fig. 2 and 3), that are components of the best-understood bacterial chitinolytic system (12, 22). Taken together, the genetic information on lmo0105 and lmo1883 does not present a clear indication of the biological roles of their enzymatic activities. One possibility is that these enzymes may enable Listeria to attach to and persist in chitin-containing invertebrates and thereby add to their virulence, as exemplified by Vibrio infection of the crab Cancer pagurus (37). A note of caution toward this hypothesis is offered, however, by the scarcity of reports on the presence of Listeria in arthropods even though several other Firmicutes, including Bacillus, Clostridium, Enterococcus, and Lactococcus, are frequently isolated from this habitat (1, 2, 7, 13). L. monocytogenes, including the EGD strain, has been shown to exert virulence toward Drosophila melanogaster (14, 20). This effect cannot, however, be attributed to chitinolytic activity as infection was established by injection of bacterial cells into the thorax, thereby bypassing the first natural step of infection, including entry of the pathogen over the chitin-containing cuticle. As an alternative, the chitinolytic activity may instead support a saprophytic lifestyle in soil and sediments. Indeed, L. monocytogenes may survive for considerable periods of time in such environments (6, 41).

At present, we do not know how the chitinolytic activity of L. monocytogenes affects the biology of the organism and impacts food safety. However, this phenotype may prove to be of considerable importance as it has been shown that chitin enhances the survival of L. monocytogenes (29) and that this species shows increased resistance to biocides while attached to chitin (21). It will be of considerable interest to further explore the role of chitinolytic activity for growth and survival of L. monocytogenes in the environment and for its transmission into food manufacturing plants.

 
Christel Galschiøt Buerholt, Jan Pedersen, and Vi Phuong Thi Nguyen are thanked for excellent technical assistance. Henrik Christensen, Department of Veterinary Pathobiology, University of Copenhagen, is thanked for valuable input to the phylogenetic analyses of chitinase genes.

 
* Corresponding author. Mailing address: Department of Veterinary Pathobiology, Faculty of Life Sciences, University of Copenhagen, Grønnegårdsvej 15, 1870 Frederiksberg C, Denmark. Phone: 45 3533 2768. Fax: 45 3533 2757. E-mail: jjl{at}life.ku.dk

Published ahead of print on 18 April 2008.

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Applied and Environmental Microbiology, June 2008, p. 3823-3830, Vol. 74, No. 12
0099-2240/08/$08.00+0     doi:10.1128/AEM.02701-07
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