Cloning and sequencing of the deacetylase gene from Vibrio alginolyticus H-8

JOURNALOFBIOSCIENCE ANDBIOENGINEEIUNG Vol. 90, No. 5, 561-563. 2000

Cloning and Sequencing of the Deacetylase Gene from Vibrio alginolyticus H-8 KAZUO OHISHI,‘* KOHJI MURASE,2 TOSHIYA OHTA,3 AND HIDE0 ETOH2 United Graduate School of Agricultural Sciences, Gifu University (Shizuoka University), 422-8529, LFaculty of Agriculture, Shizuoka University, 836 Ohya, Shizuoka 422-8529,2 and Numazu Industrial Research Institute of Shizuoka Prefecture, 3981-I Ohoka, Numazu, Shizuoka 410-0022,3 Japan

Received8 May 2OOOIAccepted 8 August 2000 A gene encoding deacetylase DA1 that is specific for N, N’-diacetylchitobiose was cloned using the shot-gun method with pUC118 and sequenced. The open reading frame encoded a protein of 427 amino acids including the signal peptide. The molecular mass of the mature enzyme estimated from the amino acid sequence data was 44.7 kDa, which is approximately similar to that, estimated by SDS-PAGE (48.0 kDa), of the purified enzyme reported previously. The N-terminal amino acid sequence deduced from the cloned deacetylase gene showed partial sequence homology with the Nod B protein from Rhizobium sp. (37% identity) and chitin deacetylase from Mucor rouxii (28%). It contained a domain, which showed homology with a chitin-binding domain of chitinase A from Bacillus circulans (39%).

[Key words: deacetylase, Vibrio alginolyticus, cloning] We previously have reported that Vibrio alginolyticus H-8 secretes deacetylase DA1 and DA2 extracellularly. Deacetylase DA1 selectively hydrolyzes the 2-acetamide group at the reducing end of N, N’-diacetylchitobiose (G~cNAc)~ and afforded iV-monoacetylchitobiose (1). The N-terminal amino acid sequence of deacetylase DA1 showed 47.5% homology with that of the Azorhizobium caulinodans Nod B protein (2). Recently, genes encoding the deacetylases from Mucor ruxii (3), Colletotrichum lindemuthianum (4), Vibrio cholerae Non-01 (5), Azorhizobium caulinodans (2), and Rhizobium sp. (6) that catalyze the hydrolysis of N-acetamide groups of poly (GlcNAc) or GlcNAc were cloned and sequenced. However, there exists little information on a specific deacetylase for (GIcNAc)~ (1). Therefore, the cloning and sequencing of the gene encoding deacetylase DA1 was carried out to investigate the functions and structure of deacetylase in V. alginolyticus H-8. Furthermore, the distribution of the deacetylase in chitinolytic marine bacteria and other bacteria was determined. A TLC method for detecting deacetylase activity to screen for clones expressing the deacetylase gene following shot-gun cloning was established. White colonies of the DNA library (7) were incubated in sodium phosphate buffer containing 10% LB broth and 1% (GIcNAc)~, and subjected to TLC analysis (isopropanol : ammonium solution =2 : 1). The reaction product (N-monoacetylchitobiose) was detected by spraying plates with a 10 mM o-phthalaldehyde and 100 mM 2-mercaptoethanol solution (8, 9), which both react with primary amines, and visualized using a UV illuminator. Three thousand clones from E. coli transformants of the genomic library were screened. The results of one TLC run in which several colonies were analyzed are shown in Fig. 1. A total of 4 colonies showed deacetylase activity, which was confirmed with the same TLC method using the reaction products of the cell extract and (G~cNAc)~. One transformant out of the 4 positive ones was further analyzed. TLC using a fluorescent reagent for screening col-

onies expressing the deacetylase gene following shot-gun cloning was very convenient and useful. The plasmid isolated from the transformant was designated pVAD. An inserted 3.0-kb fragment of pVAD was digested with various restriction enzymes and ligated into pUC118. These ligation mixtures were inserted into E. coli JM109. Subclones carrying pVAD-1 (1.8-kb KpnI-PstI fragment) or pVAD-2 (1.4-kb BamHI-PstI fragment) expressed the deacetylase activity. The nucleotide sequence of the deacetylase gene was determined by sequencing plasmids pVAD-1 and pVAD2 using the ABI PRISMTM 310 Genetic Analyzer (Perkin Elmer Applied Biosystems, USA) and the Tag DyeDeoxy terminator cycle sequencing ready reaction kit (Perkin Elmer). Nucleotide sequence data were analyzed with GENETYX-Mac (Software Kaihatsu Co., Tokyo). An open reading frame (ORF) starting at base 256 and end12345678

e

FIG. 1. Detection of N-monoacetylchitobiose by TLC to screen for clones expressing the deacetylase gene. Lane 1, (GlcNAc)z;lane 2, the reaction product of (GIcNAc)~and purified deacetylase DA1 (Nmonoacetylchitobiose); lane 3, LB medium: lanes 4,5,6,7, and 8, the reaction product of (GIcNAc)~and transformants. The arrow indicates the spots corresponding to N-monoacetylchitobiose.

* Correspondingauthor. 561

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GRRR~TCGRRRflTGRRRTTRRflTRRRCTGGCTRTTQCRRCOCTTQTRRCiTQcCQCT 300 ,,KLNKLAlRTLUSRA

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CTRTCTCRRTRTGCF,TTTGCCCRRRCTOClCRCCRRflGGRRCCRTTTRTCTQRcOTTT~330 LSQVRFRQTOTKGTIVLTFO

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GRTGGCCCRRTCRRCGCCTCRRTTGRcBTCRTTRRTOTGCTRRRlC~QRR~TRRRR OGPINRSIOUINULNQEEUK

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GCGRCGTTTTRCTllY+RTGCGTGGCRCCTRGRTGGTATTGGTORTGRRRFMRRGRCWM 480 RTFVFNRWHLOGIGOENEOR

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GCGTTRGRGGCRCTRRRRCTGGCGTTOGRTRGCGGCCflCRTCGTCQcRRRCCRcRJ3TTRT 540 RLERLKLRLOSGHIURNHSV

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GRCCRTRTGGTTCFtCRRCTGTCfTTG!W53RRTTTOQCCcRRRclWTOCQGCRGRRTGTRRT ma OHt,UHNCUEEFGPNSRRECN

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GCRRCGGGTGRTCRCCRGRTCRRCTCTTRTCRRGRTCcQGcClRCQRlGccTcGRTGTTT660 ATGOHQINSVQOPRVORS~F

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720 BCRGRRRRCTTQTCRGTRCTRGRRRRRTRTRTTOCCGlWcRTTRcGRGCTRCCCRRflCTRT RENLSULEKYLPNITSVPNV

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780 RARQCGRRTGRGTTTQCTCGTTTGCCGTRTRccRRTOGTTGQCGCQTCRCTRRRG8cTTC KRNEFRRLPVTNGURUTKOF

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840 RARGCRQRCGQCTTRTOTGCCRCGTccGRTGRlCTTRRRCCTTQ3GRGCCTGGCTRTTCR KROGLCRTSOOLKPUEPGVS

841

900

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960 GCGRRTRRTGGCTRTcRRRCTCRCWTTOGGRTDTOGRTT~QQCCCCTGRRRRCTW3GGT RNNGVQTHQUDUDURPENWG

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RTTGCGRTGCCRQCRRRCffiCCTT~RGW0cTQRRCCRTTCCTTQGQTRTGTCQRTTCC IO20 IRt4PRNSLTEREPFLQVUDS

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GCGCTRRRTRCTTGTGCGCCTRCGRCRRTTRflCccTRTCRRCTCCRRRGcRCRRG~TTC1000 RLNTCRPTTINPINSKRQEF

1081

C~TGTGOlR~TTTGCTGCTO(ITRRROTTRTTQTGCTRRCTCRCGRRTTCCTOTTT PCGTPLHRDKUIULTHEFLF

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CRMFICCTGCCGRRGCTCGCRRGTTTRTC 1200 GRRGRCGGTRRRCGTBGCRTGGGTQCRRCT EDGKRGNGRTQNLPKLRKFI

1201

CRGCTRGCCRRACROGCTGQTTRTQTCTTCGRTRCCRTGGRTRRCT~R~CC~RTT~ QLRKQRGVUFOTMDNVTPNW

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C~GTTQGTRRCRRCTFGCGCCGOtMltTRCOTTCT~R~T~GTRCGQTRTRT~fl QUGNNVSflGOVULHLGTUVa

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G~)GTRRCFIRGCCRTR~GCAACAAORTTGGGCTCCRTC~CPRCRTCTRGCTTQTGQ 1380 RUTSHTRQQOWAPSPTSSLU

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1440 RCBRRTGCGGRTCCFJGCTRC~~~~~QGXTCRQRRTGTTT~RTRCRRFICRRGGCGRTOTG TNROPRTNWTPNUSVKPQOU 1441

1500 QTQRCTTRTCRRCGTTTQCGTTRCCTRQTTRRTGTRcCQCRCQTRTCTCRRGcRORCTGQ UTVQGLRVLUNUPHUSQROU

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RCTCCGRRCTCRCRDRRCRtCTTG~C~RGCTCTRTRRGCTGTTCCCTRCTRT~TCRT TPNSQNTLFTRL*

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FIG. 2. Nucleotide sequence of the deacetylase gene and deduced amino acid sequence. Putative promoter elements are underlined. The SD sequence is doubly underlined. The inverted repeat sequence is indicated by arrows in opposite directions.

ing at base 1536 was found (Fig. 2). This sequence of 1281 nucleotides encoded 427 amino acid residues. A putative Shine-Dalgarno (SD) sequence, AGGA, was found upstream of the start codon (ATG). A putative promoter region was detected upstream of the SD sequence. The -35 region was TTACTA and the - 10 region was TTAATTT. In the region downstream of the terminal codon, an inverted repeat sequence composed of an 8-bp stem and a loop of 5 bases was found. The N-terminal amino acid sequence of the protein encoded by the deacetylase gene exhibited typical features of a signal sequence. The N-terminal amino acid sequence of

the deacetylase DAl, QTDTKGTIYLTFDDGPINASI, was corrected from QTDTKGTIYLTFFNGPINASI reported previously (1). This corrected sequence coincided with the amino acid sequence starting from Gln-23 of the deduced amino acid sequence of deacetylase DA1 . Thus, the N-terminal 22-amino acid sequence is the signal peptide and the cleavage site is between Ala-22 and Gln-23. These results indicate that the cloned gene encodes deacetylase DAl. The molecular mass of the mature protein calculated from the deduced amino acid sequence (44,742 Da) was lower than that (48 kDa) estimated by SDS-PAGE reported previously (1). This discrepancy might be due to the electrostatic charge and the conformation of the enzyme in SDS-polyacrylamide gels. The amino acid sequence deduced from the deacetylase DA1 gene was compared with the sequence of other proteins in the SWISS-PROT protein data bank. Although the entire deduced amino acid sequence of deacetylase DA1 did not show significant homology with any protein, the N-terminal amino acid sequence showed some homology with the Bacillus stearothermophilus fumarase (35%) (lo), Rhizobium sp. Nod B protein (37%) (6), Clostridium thermocellum xylanase U (26% unpublished, accession no. AF047761-2), and Mucor rouxii chitin deacetylase (28%) (3) (Fig. 3A). Thus, this Nterminal domain, whose function is unclear, and which is present in various types of enzymes, seems to be a common feature of deacetylases such as Nod B and chitin deacetylase that hydrolyze the N-acetamide groups of GlcNAc residues. Nod B deacetylates the non-reducing GlcNAc residue of chitooligosaccharides (11) and participates in the synthesis of Nod factor which causes leguminous plants to form root nodules. Chitin deacetylase hydrolyzes the N-acetamide groups of GlcNAc residues in chitin to produce chitosan, which is a cell wall component of fungi. However, this N-terminal domain did not show homology with that of N-acetylglucosamine-6-phosphate deacetylase (5). Interestingly, the C-terminal amino acid sequence showed some homology with the C-terminal amino acid sequences of the Serratia marcescens chitinase (37%) (12), Bacillus circulans chitinase A (39%) (14), and N-terminal amino acid sequence of Aeromonas sp. chitinase (36%) (13) (Fig. 3B). However, the C-terminal amino acid sequence of chitinase B which was one of six chitinases isolated from V. alginolyticus H-8 (7) showed a low level of homology with that of deacetylase DA1 (25% data not shown). The homologous regions in the above chitinases are chitin-binding domains, but deacetylase DA1 did not exhibit chitin-binding ability (data not shown). Therefore, the function of the C-terminal domain in deacetylase DA1 is unknown. Plasmid pVAD2, which expressed deacetylase DA1 which was missing 40 amino acids from the C-terminal sequence, still showed deacetylase activity. This suggested that the remaining 55 amino acids of the C-terminal domain were sufficient for normal catalytic function in this enzyme. No sequence homology with other proteins was found for the region between the C and N termini. Since a deacetylase specific for (GlcNAch has only been found in V. alginolyticus H-8, the distribution of deacetylases in other bacteria was determined. v. alginolyticus H-8, V. alginolyticus IFO15630, Vibrio parahaemolyticus IF012711, Vibrio harveyi IF015634, AIteromonas sp. O-7, Serratia marcescens 2170, S. marcescens QMB1466, Shewanella sp. EY410, and Pseu-

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B VADE333:PN~V~KTSffiDYYLHLGTV~~AV~~~--QQDIAP5P~SSf~~ADPATNWTQNVSYKQGDWWPGLR~LVNTPIJVSQ~NSQNTtFTAL 427 SMCP386 : SLTDSGLTPATQYSYFOAA~S~~~LPSSieLAV-~ATDGTP-PDE~~HS~~~D~-SPTCIQAll~~N~~D~$--476 APCP 29:-ArrqEeTmTM;TWTYNGHnUqSLVT~~~YVG~S~T~~S~P~~KDL~~T~GT~PT~TPTSTPWTVKPTTPVTS~~------------------- 106 BCCA616 : ---------DTSIITF~KA~AA(;IYYSAASN14VS~-~AAE-~--~~~YS~~VN~~~T~GQL~-~N~~LQ~~8LA~~S~~~--696 FIG. 3. Alignment of the N-terminal and the C-terminal deduced amino acid sequences of deacetylase and other related enzymes. (A) The N-terminal sequence of the I/. alginolyficus H-8 deacetylase (VADE) is aligned with that of the B. stearofhermophilus fumarase (BSFM), Rhizobium sp. Nod B (RSNO), C. thermocellum xylanase U (CTXV), and 44. rouxii chitin deacetylase (MRCD). (B) The C-terminal sequence of the V. alginolyticus H-8 deacetylase (VADE) is aligned with that of the S. marcescens chitinase (SMCP), Aeromonas sp. chitinase (APCP), and B. circulans chitinase A (BCCA). Similar residues are shaded.

stutzeri IZ-208 were cultured in LB medium seawater and chitin. Streptomyces griseus HUT6037, Streptomyces lividans 66, and Bacillus circulans WL-12 were cultured in LB medium containing chitin. Then, the deacetylase activities of these culture broths were assayed using the TLC method described above, of the bacteria tested V. afginolyticus H 8, V. alginolyticus IF015630, and V. parahaemolyticus IF012711 showed deacetylase activity. It is presumed that these bacteria secrete deacetylase extracellularly and uptake N-monoacetylchitobiose into cells to be utilized as a carbon and nitrogen source. However, the reason why (GlcNAc)* is converted to N-monoacetylchitobiose by deacetylase has not been elucidated. To elucidate the role of the deacetylase in V. alginolyticus H-8, the construction of a deacetylase DA1 gene disruptant by homologous recombination is now in progress. domonas containing

We are very grateful to Prof. Takeshi Watanabe of Niigata University, Prof. Hiroshi Tsujibo of Osaka University of Pharmaceutical Sciences, and the Marine Biotechnology Institute for providing bacterial strains and to Dr. Ken Tokuyasu of the National Food Research Institute for useful advice.

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