Cloning and characterization of the lipase and lipase activator protein from Vibrio vulnificus CKM-1

Biochimica et Biophysica Acta 1678 (2004) 7 – 13 www.bba-direct.com

Cloning and characterization of the lipase and lipase activator $ protein from Vibrio vulnificus CKM-1 Jer Horng Su a,b, Ming Chung Chang c, Yeong Sheng Lee d, I Cheng Tseng b, Yin Ching Chuang e,* a

Department of Biotechnology, Chia-Nan University of Pharmacy and Science, Tainan, Taiwan b Department of Biology, National Cheng Kung University, Tainan, Taiwan c Department of Biochemistry, Medical College, National Cheng Kung University, Tainan, Taiwan d Center for Disease Control, Department of Health, Taipei, Taiwan e Departments of Medical Research and Internal Medicine, Chi Mei Medical Center, 901, Chung Hwa Road Yung Kang, Tainan, Taiwan Received 17 October 2003; received in revised form 5 January 2004; accepted 21 January 2004

Abstract The gene (lipA) encoding the extracellular lipase and its downstream gene (lipB) from Vibrio vulnificus CKM-1 were cloned and sequenced. Nucleotide sequence analysis and alignments of amino acid sequences suggest that LipA is a member of bacterial lipase family I.1 and that LipB is a lipase activator of LipA. The active LipA was produced in recombinant Escherichia coli cells only in the presence of the lipB. In the hydrolysis of p-nitrophenyl esters and triacylglycerols, using the reactivated LipA, the optimum chain lengths for the acyl moiety on the substrate were C14 for ester hydrolysis and C10 to C12 for triacylglycerol hydrolysis. D 2004 Elsevier B.V. All rights reserved. Keywords: Vibrio vulnificus; Extracellular lipase; Lipase activator protein

1. Introduction Vibrio vulnificus is a halophilic and gram-negative bacterium which causes both serious wound infections and fatal septicemia in humans [1,2]. This estuarine/marine bacterium occurs frequently in seafoods. Its ingestion through raw oysters also results in a ca. 60% mortality in persons who are vulnerable to this bacterium [3]. Although the exact mechanism for the pathogenesis of V. vulnificus is not presently understood, several potential virulence factors such as siderophores, capsule polysaccharide, and type IV pili have been described [4– 6]. In addition, V. vulnificus is also known to secrete a number of degrative enzymes such as lecithinase, lipase, protease, DNase, hemolysin and elastase [7]. Correlations between extracellular proteins and virulences of V. vulnificus strains have been reported

$ The DNA sequence reported in this paper has been deposited to the GeneBank under accession no. AF436892. * Corresponding author. Tel.: +886-6-281-2811x2612; fax: +886-6251-7849. E-mail address: [email protected] (Y.C. Chuang).

0167-4781/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.bbaexp.2004.01.003

[8], although the potential roles of some extracellular proteins in the pathogenicity of V. vulnificus infections are still being debated [9,10]. Lipase was chosen for this study based on the potentially pathological role of V. vulnificus extracellular proteins. It has been suggested that lipase may provide bacterial nutrients [11] and constitute virulence factors either by enhancing the movements of human granulocytes [12] or affecting several immune system functions through longchain unsaturated fatty acids generated by lipolytic activities [13]. An extracellular lipase (LipA) of Pseudomonas aeruginosa has been reported as an important virulence factor which acts in synergy with bacterial phospholipase C to degrade phospholipids from lung surfactants, thus promoting the invasion by this pathogen [14,15]. In addition, a number of potential industrial applications for microbial lipase have been developed [16]. This consequently leads to the expected increase in the usage of the enzymes in the industry. In this study, we first described the cloning and expression of an extracellular lipase gene, lipA, from a clinically isolated V. vulnificus CKM-1. A nucleotide sequence downstream of lipA was then identified as being another gene,

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lipB. Next, productions of active LipA in recombinant Escherichia coli cells were tested in both the presence and absence of lipB. We showed that LipA is a member of the lipase family I.1 and the presence of its activator protein LipB was essential for obtaining active lipase. We further demonstrated that the optimal chain fatty acid substrates preferred by LipA from V. vulnificus are found to be different from those preferred by LipA from Pseudomonas.

2. Materials and methods 2.1. Bacterial strains, plasmids and cultivation condition The V. vulnificus CKM-1 used in this study was isolated from the blood culture of a patient at the National Cheng Kung University Hospital and identified by the Culture Development Center, Food Industry Research and Development Institute, Hsinchu, Taiwan, Republic of China. E. coli XL1B (Stratagene) and E. coli BL21 (DE3) [17] were used as transformation or protein expression hosts. V. vulnificus CKM-1 and E. coli were grown in Luria-Bertani (LB) medium [17] at 28 and 37 jC, respectively. Plasmid pUC19 [17] was used in the subcloning experiments. Plasmid pET21b (Novagen, Madison, WI) was used in protein overexpression. For screening lipolytic clones, tributyrin plates containing 0.4% caso agar (Merck), 0.1% gum arabic, 1.0% (v/v) tributyrin, 50 Ag ampicillin ml 1 and 1.5% agar were used. Whenever necessary, the medium was supplemented with isopropyl-h-D-thiogalactopyranoside (IPTG) to a final concentration of 1 mM. 2.2. Construction and screening of the genomic library All DNA manipulations were carried out as described by Sambrook et al. [17]. The genomic library containing V. vulnificus CKM-1 genomic DNA in the vector pBR322 [17] was prepared as described previously [18]. The gene bank was used to transform E. coli XL1B and transformants were screened on tributyrin plates. After 48 h of incubation at 37 jC, the colonies that had clear zones around them were isolated.

1.0 min, 52 jC for 1.0 min, and 72 jC for 1.0 min. A final extension step of 10 min at 72 jC was also included. Taq polymerase was used. The resulting PCR product was digested with HindIII/KpnI and NdeI/XhoI, respectively, and then inserted into the vectors pUC19 and pET21b, respectively, and was digested with the same enzymes. 2.4. Expression of lipA and lipB in E. coli For bacterial expression, the pUAB and pUA were constructed by oligonucleotide-directed mutagenesis with pUAB1 as the template using the Altered Sites II Mutagenesis System (Promega). The pUAB contains lipA –lipB and the pUA contains lipA. DNA oligonucleotide primers (Table 1) were designed to allow the PCR amplification of the entire lipA-lipB and lipA from plasmid pUAB1. The pUAB and pUA coding sequences were confirmed by sequence analysis and then subcloned into the HindIII and KpnI restriction sites of expression vector pUC19, respectively. E. coli XL1B was likewise transformed with the plasmid pUAB and pUA, respectively. The transformants were induced with IPTG and tested on tributyrin plates. The lipase activity was assayed spectrophotometrically using p-nitrophenyl myristate as substrate. The reaction was carried out at 37 jC in 20 mM phosphate buffer (pH 7.6) containing 0.2% Triton X-100, 5 mM p-nitrophenyl myristate, and enzyme as described previously [19]. In some cases, lipase activity was also quantitated by a titrimetric assay using triacylglycerol emulsions as substrate [19]. One unit of lipase activity was defined as the amount of enzyme that liberated 1 Amol p-nitrophenol per minute from pNPP and fatty acids from triacylglycerol emulsion, respectively. Protein concentrations were determined using a protein

Table 1 Oligonucleotide primers used for plasmid construction

2.3. Sequencing, DNA analysis and PCR The DNA was sequenced by automated DNA sequencing (ABI 377; Applied Biosystems, Inc., Foster City, CA). Sequence analysis was done with PC/GENE software package (Intelligenetics). The nucleotide sequence and its deduced amino acid sequence were analyzed using the BLAST E-mail network server at the National Centre for Biotechnology and Information. PCR was performed with a model PE 2400 automated thermocycler with MicroAm tubes (Perkin-Elmer Cetus, Norwalk, CT). Primers were synthesized by MDBio Inc. The PCR conditions were as follows: 30 cycles at 94 jC for

Shading indicates substituted nucleotides, and restriction enzyme sites are underlined.

J.H. Su et al. / Biochimica et Biophysica Acta 1678 (2004) 7–13

assay kit (Bio-Rad) with bovine serum albumin as a standard. Substrate specificity was measured using p-nitrophenyl esters of different chain lengths (Sigma), which were emulsified completely by sonication in the presence of 0.5% Triton X-100. The substrates used include: pNP acetate (2), pNP butyrate (4), pNP caproate (6), pNP caprylate (8), pNP caprate (10), pNP laurate (12), pNP myristate (14), pNP palmitate (16), and pNP stearate (18), where the numbers in parentheses denote the number of carbon atoms in the alkyl chain. LipA and LipB were separated by 0.1% sodium dodecyl sulfate (SDS)-15% polyacrylamide gel electrophoresis (PAGE) [20]. pEmA, pEB and pEtB were constructed by PCR with pUAB1 as a template and two oligonucleotides (Table 1) introducing an NdeI site at the 5V end and a XhoI site at the 3V end of the lipA and lipB, respectively. The pEmA, pEB and pEtB coding sequences were confirmed by sequence analysis and were subcloned into the NdeI and XhoI restriction sites of the overexpression vector pET21b, respectively. The modified lipA (mlipA) was constructed by removing the signal sequence of lipA and fusing with the His-tag-encoding sequence at the 3V end. The full lipB was constructed and fused with the His-tag-encoding sequence at the 3V end. The truncated lipB (tlipB) was constructed by removing the first 63 nucleotide sequence at the 5V end of lipB and fusing with the His-tag-encoding sequence at the 3V end. mLipA, LipB and tLipB, all with a His-tag sequence fused at the C-terminal end, have been overproduced in E. coli BL21 (pEmA), E. coli BL21 (pEB) and E. coli BL21 (pEtB). mLipA, LipB and tLipB can be purified in a onestep procedure by affinity chromatography on Ni2 +-nitriloacetate resin, respectively [21]. Western blots were carried out according to standard protocol [17]. Either polyclonal rabbit anti-mLipA or anti-tLipB was used as the primary antibodies at a dilution of 1:4000.

3. Results and discussion 3.1. Cloning of the lipase gene To clone the gene encoding the extracellular lipase of V. vulnificus CKM-1, a genomic library of this organism was constructed in E. coli XL1B. Transformed E. coli cells were screened on tributyrin-containing medium to select tributyrin-degrading clones. Among the approximately 2000 transformants, one colony forming a clear zone was detected. The plasmid from this clone was isolated and subjected to restriction endonuclease mapping. The results of subcloning indicated that a 3.2-kb KpnI –KpnI fragment was sufficient for the lipase production in E. coli XL1B. This fragment was subcloned into pUC19, yielding plasmid pUAB1 and pUAB2. The DNA fragment inserted in pUAB2 was oriented in a direction opposite to that of pUAB1. Since lipase was expressed from both pUAB1 and pUAB2, an active promoter on the cloned fragment

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must have transcribed the lipase gene. Southern hybridization studies confirmed that the gene responsible for lipase activity must have originated from V. vulnificus CKM-1 (data not shown). 3.2. Features of the sequence The nucleotide sequence of the 3.2-kb DNA fragment was determined for both strands. The sequence analysis revealed the presence of two ORFs with the same orientation: ORF1, located at positions 434 to 1375, and ORF2, located at positions 1395 to 2234. The 10 and 35 regions of the putative promoter sequence show good homology with the E. coli consensus sequences [22] (position 367 through 372 and 391 through 396, respectively) with an optimal spacing of 16 nucleotides. Furthermore, an A + T-rich region, which had been shown to be involved in transcription regulation in E. coli [23], was found immediately upstream from the consensus sequences. The possible ribosomal binding sites (Shine – Dalgarno sequences) [24], AGGA and GGAA, were found 8 and 4 upstream from the presumptive start codon of the ORF1 and ORF2, respectively. At the point of 26 bp downstream from the stop codon of ORF2, there was a stable hairpin structure (free energy of 12.6 kcal mol 1). This structure is expected to make it an efficient transcriptional terminator. These data indicated that the ORF1 and ORF2 may be transcriptionally linked in a single operon. 3.3. Comparison of the deduced amino acid sequences of ORF1 and ORF2 with other proteins It was deduced that ORF1 is the lipase structural gene lipA. Analysis of the putative amino acid sequence of LipA revealed that the first 19 amino acids have the characteristics of a bacterial signal peptide for the initiation of translocation across the cytoplasmic membrane. The predicted cleavage site was between alanine 19 and glycine 20, as determined by the PSIGNAL program (PC/Gene). The deduced amino acid sequence of LipA contains a sequence, Val-Asn-LeuIle-Gly-His-Ser-His-Gly-Gly (positions 104 to 113), which matches almost completely with the consensus sequences and is known to be conserved in the active center of lipases (Fig. 1A). A search of the available protein sequence databases revealed that LipA has high homology to some lipases, especially to the family I.1 proteobacterial lipases [11,25– 27]. Fig. 1A shows that the LipA has 75% (with 84% similarity), 58% (with 71% similarity), and 51% (with 62% similarity) amino acid sequence identity to the family I.1 lipases from V. cholerae O1, P. aeruginosa PAO1, Acinetobacter calcoaceticus RAG-1, respectively, using the multiple sequence analysis program [28]. The predicted molecular mass of the mature form of LipA is 31.0 kDa, which corresponds well with those of proteins belonging to family I.1 lipase with molecular mass ranging from 30 to 32 kDa [27]. In addition, some functionally important residues

10 J.H. Su et al. / Biochimica et Biophysica Acta 1678 (2004) 7–13 Fig. 1. Optimal alignments of the deduced amino acid sequence of the V. vulnificus CKM-1 lipase (A) and lipase activator protein (B) with those of other bacterial lipases and lipase activator protein, respectively. V. vulnificus CKM-1 (VvLipA), this study, AF436892; V. cholerae O1 (VcLipA), P15493; P. aeruginosa PAO1 (PaLipA), P26876; A. calcoaceticus RAG-1 (AcLipA), AAD29441; V. vulnificus CKM-1 (VvLipB), this study, AF436892; V. cholerae (VcLipB), CAA68635; P. aeruginosa (PaLipB), Q01725; A. calcoaceticus BD413 (AcLipB), Q43961. Boxes in black indicate positions at which the amino acids are identical in all four proteins. Boxes in gray indicate the location of similar residues of the four protein sequences. Closed circles indicate strictly conservation. The closed triangles indicate the Ser, Asp, and His residues, which comprise the catalytic triad. The disulfide bond between Cys and Cys of the lipase is indicated by a dashed line. Open circles indicate signal peptide residues. Open triangles indicate transmembrane hydrophobic helix. The dashes represent gaps introduced during the alignment process. Numbers refer to the amino acid located at the end of each line.

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in family I.1 lipases are strictly conserved in LipA, such as a pentapeptide Gly-X-Ser-X-Gly (108 to 112), a single functional disulfide bond connecting residues Cys212 to Cys264, and a catalytic triad Ser110 Asp258 His280 identified by homology with P. aeruginosa lipase and its known crystal structure [29]. The serine residue is common to both the pentapeptide and the catalytic triad [27]. Based on sequence similarity and approximate molecular size, it is proposed that the LipA belongs to the family I.1 lipases. ORF2 (lipB) starts at 16 bp downstream from ORF1 and encodes a protein of 280 amino acids with a molecular mass of 30.7 kDa. (Fig. 1B). A search through the available protein sequence databases revealed that LipB has significant homology to those of lipase activator proteins. In Fig. 1B, the amino acid sequence of the LipB aligns with the three proteins of lipase activator with the highest degree of homology [25,30,31], using the multiple sequence analysis program. Like the three lipase activators, LipB also has a short hydrophobic N-terminal region that comprises of a potential transmembrane segment (amino acids 2 to 22) and a hydrophilic C-terminal region [32] (PC-Gene, programs RAOARGOS and HELIXMEN). This suggests that LipB might be a lipase activator for the expression of LipA in V. vulnificus. 3.4. In vivo activation of lipase in E. coli To investigate whether LipB is a lipase activator required for the production of active LipA lipase, plasmid pUA containing lipA and plasmid pUAB containing lipA – lipB were constructed by PCR amplification and lipase activity of E. coli containing pUA or pUAB was determined. A crude cell extract of E. coli XL1B (pUAB) had lipase activity of 65 U mg 1 ( p-nitrophenyl myristate was used as substrate) when cells were cultivated on tributyrin-LB broth and induced with IPTG. In contrast, E. coli XL1B (pUA) containing only the lipA had no lipase activity. Halo formation around the colony on tributyrin-LB agar also indicated that E. coli XL1B (pUA) had less activity than E. coli XL1B (pUAB) (data not shown). Expressions of lipA in E. coli XL1B (pUA) and those of lipA and lipB in E. coli XL1B (pUAB) were detected by Western blot, as shown in Fig. 2. The sizes of LipA and LipB were very similar to those predicted for the products of the LipA and LipB, respectively. No major polypeptides were detected in E. coli cells or E. coli cells carrying only the pUC19 vector (data not shown). Although Western blot analysis indicated that E. coli XL1B (pUA) produced as much LipA as did E. coli XL1B (pUAB), no lipase activity could be detected in E. coli XL1B (pUA). These results indicate that the production of active LipA in recombinant E. coli cells is lipB-dependent. The 31-kDa polypeptide of mature LipA could be detected immunochemically in both concentrated supernatant of culture broth and crude cell extracts of V. vulnificus CKM-1 (Fig. 2). Since LipA could be detected only in crude

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Fig. 2. (A) Western blot analysis of LipA. Lanes 1 and 2, crude cell extracts from E. coli XL1B carrying pUA and pUAB, respectively; lanes 3 and 4, crude cell extracts and concentrated supernatants of culture broth from V. vulnificus CKM-1, respectively. (B) Western blot analysis of LipB. Lanes 1 and 2, crude cell extracts from E. coli XL1B carrying pUA and pUAB, respectively; lane 3, crude cell extracts from V. vulnificus CKM-1. The culture medium was supplemented with IPTG. Samples were separated by SDS-PAGE, blotted and incubated with antisera. Lane M, molecular mass standards (Pharmacia); values in kilodaltons.

cell extracts of E. coli XL1B (pUAB) and E. coli XL1B (pUA) when cells are grown in the same culture conditions, LipA apparently is not exported to the cultured medium in E. coli XL1B. It has been reported that the Pseudomonas lipases of family I.1 are exported via a two-step mechanism [33]. Since LipA has been predicted to be synthesized as a precursor with an N-terminal signal peptide of 19 amino acids and the mature form of LipA demonstrated overall similarity to Pseudomonas family I.1 lipases, it is speculated that the LipA seems to be secreted via a two-step-like mechanism. Such mechanism requires the signal peptide for transport across the inner membrane into the periplasm and auxiliary proteins for transport across the outer membrane into the cultured medium [33]. Thus, LipA could not have been exported to the cultured medium from recombinant E. coli cells. This may be due to the absence of auxiliary protein translocation mechanism in host cells. 3.5. In vitro activation of lipase expressed in E. coli The role of LipB as a chaperon for LipA has been demonstrated by Hobson et al. [34] with the lipase from P. cepacia. Noriko et al. [35] proved that the P. aeruginosa lipase activator, LipB, acts neither on the transcription nor translation of lipase gene, lipA, but instead assists the folding of the LipA into an active structure. Furthermore, Hiroyuki et al. [36] also showed that a truncated lipase activator, D21LipB of P. aeruginosa, which lacks hydrophobic N-terminal residues, disperses homogeneously in a solution, and that the resulting reactivated and unfolded LipA is more effective than the full-length activator LipB. The objective of this study was to find out whether the same is true of V. vulnificus LipA and LipB. Overexpressions and purifications of both the modified LipA (mLipA) and truncated LipB (tLipB) were performed, and in vitro

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Table 2 Activity of the reactivated mLipA towards various p-nitrophenyl esters and triacylglycerols Acyl group substrate

Activity of the lipase against p-nitrophenyl derivatives %

Acetate Butyrate Caproate Caprylate Caprate Laurate Myristate Palmitate Stearate

a

3 27 18 64 61 83 100 58 30

Triacylglycerols %a 0 5 10 67 95 100 88 50 28

a Relative activity was expressed as the percentage of the maximum activity detected (100%). Data are means of triplicate determinations.

reactivation of mLipA lipase was determined. mLipA was incubated with tLipB at a molar ratio of 1:1 at 4 jC for 24 h as described by Noriko et al. [35]. In this procedure, the lipase activity of mLipA was 450 U mg 1. In contrast, the lipase activity of mLipA was only 110 U mg 1 with fulllength LipB in the same conditions. These results suggested that LipA from V. vulnificus CKM-1 could be produced in E. coli and truncated LipB reactivated unfolded LipA more effectively than full-length LipB. To find the optimal substrate specificity of LipA from V. vulnificus CKM-1, we tested esterase and lipase activity by using p-nitrophenyl ( pNP) derivatives and triacylglycerol, respectively, with the reactivated mLipA. The results (Table 2) indicate that lipase activities exist in the presence of substrates with chain lengths from C2 to C18. The highest esterase activity was observed with the substrate p-nitrophenyl myristate (C14) and maximum lipase activity was detected with tricaprin (C10) or trilaurin (C12). Whether the production of middle chain fatty acid may affect several immune system functions and contribute to the preferential location of V. vulnificus at deep sites of infection remains to be discovered. In addition, the lipolytic activity of reactivated mLipA could be detected by analyzing the halo formation on N-broth agar plates supplemented with an emulsion of tributyrin, triolein, olive oil (plus rhodamin B), or egg yolk. An A. calcoaceticus BD413 extracellular lipase LipA, which has been categorized as a member of the family I.1 lipases [27], has also been classified as a true lipase because it shows specificity toward water-insoluble longchain triglycerides and displays activity in olive oil or egg yolk emulsions [37]. Based on the aforementioned enzymatic properties and the deduced amino acid sequence which manifest a high homology to A. calcoaceticus LipA, LipA can also be classified as a true lipase. However, a kinetic study should be done to further investigate the role of LipA in V. vulnificus. We have demonstrated that the LipA belongs to the family I.1 lipases based on the molecular size, amino acid sequence, typical signal sequence, and that LipA requires a

LipB activator protein for production of an active lipase. The prototype enzyme of family I.1 is the 29-kDa extracellular lipase from P. aeruginosa [29]. This enzyme has been well studied mainly because it is a virulence factor of P. aeruginosa infections [38] and has many uses in biotechnological applications [39]. However, the precise functions of the LipA in V. vulnificus are still unclear. We have cloned the LipA and LipB from V. vulnificus and have begun to characterize the preliminary enzymatic properties of LipA. Further studies with genetic modifications for functional analysis may help to elucidate whether LipA is a virulence determinant in the pathogenesis of V. vulnificus.

Acknowledgements The work was supported by grants DOH91-DC-1015 and DOH91-DC-1016 from the Center for Disease Control, Department of Health, Taipei, Taiwan, Republic of China.

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