INTRODUCTION

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Lipases (triacylglycerol acylhydrolases, EC 3.1.1.3) are hydrolytic enzymes that catalyze the hydrolysis and synthesis of a variety of acylglycerols at the interface of lipid and water (14, 18). Of bacterial extracellular lipases (4), those from the Pseudomonas species have been extensively studied for their industrially applicable properties. Pseudomonas lipases were formerly classified into three classes according to amino acid sequence homology (18). Recently, Pseudomonas lipases were reclassified into six subfamilies among family I of bacterial lipases (4), since a large number of lipases were also isolated from other genera, and some Pseudomonas species have been reclassified as Burkholderia spp. (10, 31, 35). Subfamily I.1 includes the lipases from Pseudomonas aeruginosa (7, 34) and Pseudomonas fragi (3), and subfamily I.2 includes those from Burkholderia cepacia and Burkholderia glumae (11, 20). In the crystal structures of the lipases from B. glumae, B. cepacia, and P. aeruginosa, the calcium-binding site and position of the disulfide bond as well as the catalytic triad are well conserved (23, 28, 29). Most of the lipase genes of subfamilies I.1 and I.2 are clustered with a secondary gene that is located immediately downstream of the lipase genes (1, 12, 15, 16). The protein products of the secondary genes are specifically required for forming active lipases and have been named the lipase modulator, activator, helper protein, and lipase-specific foldase (Lif). Here, the proposed general name Lif (18) was used for the known lipase-specific helper proteins. Lif is directly involved in the folding and secretion of lipase via the two-step type II secretion pathway (33).

In this study, we have cloned, sequenced, and characterized the lipase and lipase modulator genes from Pseudomonas sp. strain KFCC 10818, which produces industrially valuable enzymes, including an alkaline protease, -amylase, and lipase (this study) (19, 22, 24). When the lipase gene was isolated from Pseudomonas sp. strain KFCC 10818, a secondary gene was also identified immediately downstream of the lipase structural gene. The secondary gene was required for the formation of active lipase, as is true for known Pseudomonas Lifs. However, the deduced amino acid sequence of the secondary protein exhibited very low homology to the known Lifs. The secondary protein was named the lipase modulator since its function has not yet been characterized in detail. In a further study, we have investigated the functional interaction between the lipase and its modulator in lipase folding. Unexpectedly, a lipase variant that does not need the modulator for its correct folding was found. The changed sequences of the variant were identified, and the activity of the lipase variant as expressed without a modulator was assayed in a crude extract.

	MATERIALS AND METHODS

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Bacterial strains and growth conditions. Pseudomonas sp. strain KFCC 10818 was used as a source of a lipase gene, lipK, and a lipase modulator gene, limK. In order to isolate genomic DNA, Pseudomonas sp. strain KFCC 10818 was grown to the mid-logarithmic phase in Luria-Bertani medium at 30°C. Escherichia coli JM83 and DH5 were used for the transformation of recombinant plasmids. E. coli JM109 and XL1-Blue were used as hosts for expression of the lipK and limK genes and DNA sequencing, respectively. Ampicillin (100 µg/ml), tetracycline (13 µg/ml), and/or chloramphenicol (30 µg/ml) was added to the growth medium when necessary.

Cloning of the lipase and lipase modulator genes. To construct a genomic DNA library, genomic DNA from Pseudomonas sp. strain KFCC 10818 was isolated by the procedure described by Marmur (27) with a slight modification. Genomic DNA was partially digested with Sau3AI. DNA fragments were fractionated with a sucrose density gradient (10 to 40%, wt/vol) by ultracentrifugation at 25,000 rpm for 24 h at 20°C in a Beckman SW-28 rotor. Fractionated DNAs were ligated into the pUC19 vector at the BamHI site. Screening of the lipase gene(s) from the genomic library was carried out in two steps as previously described (2, 8, 21, 25, 26). First, colonies with halo-forming activity on tributyrin agar plates were isolated. Since both esterases and lipases can hydrolyze tributyrin, the colonies with halo-forming activity were further tested for lipase activity on the agar plates containing olive oil and rhodamine B. A recombinant plasmid carrying a lipase gene was isolated from a lipase-positive colony and sequenced. Nucleotide sequences were determined by the dideoxynucleotide chain termination method (32) with a Sequenase version 2.0 DNA sequencing kit (U.S. Biochemicals).

Construction of expression plasmids. The fragment containing both lipK and limK was amplified by PCR using the M13/pUC reverse sequencing primer (5'-AGCGGATAACAATTTCACACAGGA-3') and the Mod-3 primer (5'-GGCATGCATCTTATA CTATCTTATTGACC-3') from pLIP172, which carries a 2.7-kb fragment containing lipK and limK in the pUC19 vector. In all PCR amplifications for subcloning, Pfu DNA polymerase (Stratagene) was used. The PCR product was digested with EcoRI and NsiI and ligated to the EcoRI and PstI sites of the E. coli expression vector pKK223-3 (Amersham Pharmacia Biotech) containing the strong tac promoter and ampicillin resistance gene, resulting in pKLM11 (lipK plus limK). To express the lipK and limK genes separately, the lipK gene was amplified by PCR with the M13/pUC reverse sequencing primer and Lip-II (5'-GGACAAGCTTACAGTCCAAGTTGTTG-3'). The amplified fragment was inserted into pKK223-3 at the EcoRI and HindIII sites to make pKL11 (lipK). The limK gene was amplified by PCR using primers Mod-1 (5'-CGAGAATTCATGATGCGTTATAAACCCA-3') and Mod-3. The amplified fragment was digested with EcoRI and NsiI and inserted into the EcoRI and PstI site of pACYC-tac, yielding pACTM12 (limK). Plasmid pACYC-tac was constructed in this study by inserting the tac promoter, ribosomal binding site, and rrnB transcription terminator from pKK223-3 into PvuII- and NcoI-digested pACYC184 (New England Biolabs). Plasmid pACYC184 is a cloning vector compatible with vectors such as pKK223-3 and pUC19 and has tetracycline and chloramphenicol resistance genes. Plasmids pLIP171H and pKLM11H have also been constructed by inserting a 1.4-kb fragment with lipK and truncated limK into pUC19 and pKK223-3, respectively.

Mutagenesis of lipK and limK by PCR. The high spontaneous error rate (6) of Taq DNA polymerase was utilized to obtain various random mutations. To isolate limK mutants that cannot support the formation of active lipase, the limK gene was amplified from pLIP172 by PCR using primers Mod-1 and Mod-3 under standard conditions. Wild-type limK of pACTM12 was replaced with the amplified products. The resulting plasmids were introduced into E. coli JM109 cells harboring pKL11. The lipase activities of the transformants were tested on tributyrin agar plates supplemented with both ampicillin and tetracycline. From the colonies lacking halo-forming activity, plasmids that contain limK mutant were isolated and put together. Then, lipK mutants that could suppress the putative loss-of-function mutation of limK were screened. The lipK gene was amplified from pLIP172 with the M13/pUC reverse sequencing primer and Lip-II primer under standard conditions. The amplified product was digested with ClaI and HindIII and inserted into the ClaI and HindIII sites of pKL11. The resulting plasmids were introduced into E. coli JM109 cells harboring plasmids containing the limK mutant. The transformants were tested for lipase activity on tributyrin agar plates. From the colonies forming halos on tributyrin agar plates, plasmids derived from pKL11 were isolated and sequenced to identify mutations. Primers Lip438 (5'-CCATTCTAGCCCTAACC-3') and Lip744 (5'-CACTGGCTTTTAATCGTC-3') were used for DNA sequencing.

Lipase activity assay. Tributyrin agar plates (Luria-Bertani medium, 0.5% tributyrin [Sigma], and 1.5% agar [Difco]) were used to detect lipase activity (26). Lipase activity forms halos around colonies. Rhodamine B-olive oil agar plates (nutrient broth, 1% olive oil [Sigma], 0.001% rhodamine B [Sigma], and 1.5% agar [Difco]) were also used for the cloning of true lipase (8, 25), since tributyrin is hydrolyzed by esterases as well as lipases. The interaction of rhodamine B with fatty acids released during hydrolysis of triglycerides causes intense fluorescence around colonies upon UV irradiation. For the liquid assay, p-nitrophenyl ester was used as a substrate (5). The lipase assay in liquid was slightly modified by replacement of p-nitrophenyl butyrate with p-nitrophenyl palmitate. The assay measured the increase in absorbance at 410 nm due to the hydrolytic release of p-nitrophenol. One unit of lipase activity was defined as the activity releasing 1 µmol of p-nitrophenol per min at 25°C.

Nucleotide sequence accession number. The lipK and limK DNA sequences have been deposited in the GenBank, EMBL, and DDBJ databases under accession no. AF125523.

	RESULTS AND DISCUSSION

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Cloning of the lipase and lipase modulator genes of Pseudomonas sp. strain KFCC 10818. The genomic DNA library of Pseudomonas sp. strain KFCC 10818 was constructed and screened to isolate the lipase gene. Out of approximately 10,000 E. coli transformants screened on tributyrin agar plates, 27 halo-forming colonies were detected and further analyzed on rhodamine B-olive oil agar plates to distinguish between lipase and esterase activity. Out of 27 colonies, 1 colony exhibited true lipase activity. Finally, a plasmid named pLIP172, which has a 2.7-kb fragment carrying a lipase gene and a secondary gene, was obtained. The restriction maps of pLIP172 and its derivatives used in this study are represented in Fig. 1. The open reading frame of the lipase gene named lipK begins from a GTG initiation codon and putatively encodes a protein of 311 amino acids. The deduced amino acid sequence of the lipase LipK revealed high homology (36 to 53%) to the subfamily I.1 and I.2 Pseudomonas lipases (11, 20, 34). A secondary gene was located immediately downstream of lipK, similar to what occurs in subfamily I.1 and I.2 Pseudomonas lipase operons. The secondary gene was designated lipase modulator gene limK. The limK gene can encode a hydrophilic protein of 279 amino acids, which is 60 to 74 residues smaller than the previously known Pseudomonas Lifs. An analysis using the TMpred program (European Molecular Biology Network) indicated that the LimK protein is possibly a membrane-associated protein, with amino acids 6 to 25 forming a transmembrane helix. However, the deduced amino acid sequence of LimK shares only 17 to 19% homology with the Lifs, in comparison to the significant homology (28 to 58%) among the known Lifs. These results suggest that Pseudomonas sp. strain KFCC 10818 is distantly related to subfamily I.1 and I.2 Pseudomonas species.

View larger version (13K): [in this window] [in a new window]   FIG. 1.   Restriction maps of the plasmids used in this study. The thin arrows indicate the direction of transcription. Plac and Ptac represent the lac and tac promoters, respectively. EV, EcoRV; C, ClaI; Nc, NcoI; Hc, HincII; St, StuI; P, PstI; H, HindIII; E, EcoRI; aa, amino acid.

Effect of the limK gene on lipase expression. The Lifs are known to function as a chaperone for correct folding and efficient secretion of lipase. Therefore, we investigated whether LimK is required for the formation of active LipK. First, a deletion analysis of the limK gene was performed. Plasmid pLIP171H, which contains the lipK gene and a truncated limK gene, was introduced into E. coli JM109 cells and the phenotype was compared to that of E. coli JM109 harboring pLIP172. While E. coli JM109 harboring pLIP172 expressed an active lipase, E. coli JM109 harboring pLIP171H did not show lipase activity, which implies that the limK gene is necessary for the expression of active lipase (Fig. 2A).

View larger version (47K): [in this window] [in a new window]   FIG. 2.   Expression of the lipK and limK genes in E. coli JM109. (A) Effect of limK on the expression of lipK in cis. The following plasmids were introduced into E. coli JM109 cells: pUC19 (colony row 1), pLIP171H (lipK and truncated limK) (colony row 2), and pLIP172 (lipK and limK) (colony row 3). (B) Effect of limK on the expression of lipK in trans. The following plasmids were introduced into E. coli JM109 cells: pKLM11 (lipK and limK) and pACYC-tac (colony 1), pKK223-3 and pACYC-tac (colony 2), pKL11 (lipK) and pACYC-tac (colony 3), pKLM11H (lipK and truncated limK) and pACYC-tac (colony 4), pKL11 (lipK) and pACTM12 (limK) (colony 5), and pKLM11H and pACTM12 (colony 6). The transformants were grown on a tributyrin agar plate containing 1% tributyrin for 48 h at 37°C.

Identification of a lipase variant: formation of active enzyme without LimK. To further investigate the functional interactions of the LipK and LimK proteins, we have attempted to isolate loss-of-function mutations of limK and secondary mutations in lipK that suppress them. As a first step, random mutations were introduced into the limK gene by PCR as described in Materials and Methods. Out of 350 transformants, 27 colonies did not show lipase activity on tributyrin agar plates, indicating they were putative limK mutants that cannot support the formation of active lipase. From the colonies, plasmids containing mutant limK genes were isolated and put together. Next, lipK mutants that could suppress the putative loss-of-function mutation of limK were screened. The plasmids carrying randomly mutated lipK genes were introduced into E. coli JM109 harboring the plasmids containing mutant limK genes. Out of approximately 80,000 transformants, 26 colonies formed halos on tributyrin agar plates. From the colonies, plasmids derived from pKL11 were isolated and sequenced to identify the mutations. All lipK genes from the 26 clones had an identical CA mutation at codon 112, which converted a Pro residue to a Gln residue, and some had an additional silent TC mutation at codon 204.

View larger version (26K): [in this window] [in a new window]   FIG. 3.   Comparison of the lipase activities of LipK and its mutant LipKP112Q. (A) Plasmids were introduced into E. coli JM109 cells. E. coli transformants were grown on a tributyrin agar plate containing 1% tributyrin for 72 h at 37°C. Row 1, pKK223-3 and pACYC-tac; row 2, pKL11 (wild-type lipK) and pACYC-tac; row 3, pKL-S1 (encoding LipKP112Q) and pACYC-tac; row 4, pKL-S1 and pACTM12 (limK); row 5, pKLM11 (wild-type lipK and limK) and pACYC-tac. (B) E. coli JM109 transformants were induced with 1 mM IPTG (isopropyl--D-thiogalactopyranoside). The cells were harvested at 3 h after induction. The lipase activities of the crude extracts were measured.