Biochimica et Biophysica Act a, 1131 (1992) 253-260 © 1992 Elsevier Science Publishers B.V. All rights reserved 0167-4781/92/$05.00

253

BBAEXP 92394

Cloning, nucleotide sequence and expression in Escherichia coli of a lipase gene from Bacillus subtilis 168 V6ronique Dartois, Alain Baulard, Karin Schanck and Charles Colson UnitJ de G~n~tique, Unicersit~Catholique de Loucain, Loucain-la-Neuce (Belgium)

(Received 23 January 1992)

Key words: Lipase; Nucleotide sequence; (B. subtilis) The gene coding for an extracellular lipase of Bacillus subtilis 168 was cloned and found to be expressed in Escherichia coli. Enzyme activity measurements showed no fatty acid chain length preference. A set of Tn5 insertions which inactivate the gene were localized and used to initiate its sequencing. The nucleotide sequence was determined on two independent clones expressed in E. coli. In one of these clones, the sequence revealed a frameshift, due to the presence of an additional adenine in the N-terminal region, which caused the interruption of the open reading frame, probably allowing translation to initiate at a second ATG codon. The sequence of the wild-type lip gene from B. subtilis was confirmed on the chromosomal fragment amplified by polymerase chain reaction (PCR). When compared to other lipases sequenced to date, the enzyme described here lacks the conserved pentapeptide GIy-X-Ser-X-Gly supposed to be essential for catalysis. However, alignments of several microbial lipase sequences suggest that the pentapeptide Ala-X-Ser-X-Gly present in the lipase of B. subtilis may function as the catalytic site. Homologies were found in the N-terminal protein region with lipases from different Pseudomonas species. The predicted Mr and isoelectric point for the mature protein are 19348 and 9.7 respectively.

Introduction

Lipases (triacylglycerol acylhydrolases, EC 3.1.1.3) are a class of enzymes which hydrolyse ester bonds of triacylglycerols to yield free fatty acids, diacylglycerol, monoacylglycerol and sometimes glycerol. Lipases are often difficult to assay and the interpretation of their kinetics of hydrolysis is not straightforward. This is mostly due to the fact that diacylglycerols and monoacylglycerols released during the reaction can be also subjected to enzymatic hydrolysis and that lipases act at oil/water interfaces. This unique feature separates lipases from closely related hydrolytic enzymes as esterases, although lipases also hydrolyse water-soluble substrates. Lipases are widely distributed throughout animals, plants and microorganisms. In particular, the genes for several microbial lipases have been cloned and D N A sequences are available for the lipase genes from Staphylococcus aureus [1], Staphylococcus hyicus [2],

Correspondence to: C. Colson, Unit~ de G~n6tique, Universit6 Catholique de Louvain, Place Croix du Sud 4, Louvain-la-Neuve, Belgium. The sequence data reported in this paper have been submitted to the EMBL/Genbank Data Libraries under the accession number M74010.

Moraxella TA144 [3-5], Bacillus stearothermophilus [6], various Pseudomonas [7-12], Geotrichum candidum [13] and Mucor miehei [14]. Comparison of all the known lipase sequences, from mammalian as well as microbial sources, reveals the presence of a conserved pentapeptide Gly-XI-Ser-X2-GIy which is reported to contain the nucleophilic serine residue essential for catalysis, as shown in recent work [15,16]. We observed that B. subtilis strain 168 exhibits an extracellular lipolytic activity. No report on the genetics of lipase synthesis in B. subtilis has appeared yet. It should be noted, however, that a biochemical characterization of lipolytic activity in B. subtilis 168 has been previously published [17]. B. subtilis is, among Grampositive bacteria, the model organism in which a number of genetic tools have been developped [18] and in which extensive genetic analysis is available. In addition, the genetics of other extracellular hydrolytic enzymes, e.g., proteinases and amylase has been investigated thoroughly [19-21]. Thus, we have undertaken the genetic study of its lipolytic activity. We report in this paper the molecular cloning, expression in E. coli and nucleotide sequence of the lipase gene of B. subtilis. Comparison of the amino acid sequence of the protein with those of other lipases revealed that the lipase of B. subtilis is the first, among all lipases already sequenced, in which the conserved sequence Gly-XI-Ser-X2-Gly is not present.

254

Materials and Methods

Bacterial strains, plasmids and phages The bacterial strains, plasmids and phages used in this study are listed in Table I.

Media Bacteria were grown in L broth containing 1% tryptone, 1% yeast extract (Difco, Detroit, USA) and 0.5% NaCI. For infection with bacteriophage, E. coli was grown in L broth plus 0.2% maltose and 10 mM MgSO 4. A-infected cells were plated in 3 ml soft agar (L broth supplemented with 0.7% agar and 10 mM MgSO 4) poured on top of standard L plates. For phage infection with M13, 2 × TY medium (1.6% tryptone, 1% yeast extract, 0.5% NaC1) was used. The screening of lipase-positive colonies was a two-step procedure involving two distinct media. The first was a combination of 'Spirit Blue Agar' medium (Difco) and lipase reagent (Difco). The second [22] contained triolein (Sigma, Germany) as the substrate and a fluorescent agent, the basic dye, Rhodamine B (Sigma) [23]. Rhodamine B (1 mg/ml) was dissolved in distilled water and sterilized by filtration. L medium (1 I) was autoclaved and cooled to about 60°C. Then 31.25 ml of filter-sterilized triolein (Sigma) and 10 ml of Rho-

damine B stock solution were added with vigourous stirring and emulsified by mixing for 1 rain with a domestic mixer (Braun AG, Frankfurt, Germany). The medium was then poured into Petri dishes. Tests of commercial lipase and esterase on Spirit Blue Agar and Rhodamine B plates showed that Spirit Blue is not strictly selective for lipases but also screens for esterase activity while Rhodamine B allows the screening of true lipases only. Because of the very limited growth of E. coli on this medium, it was not used to directly screen the library for true lipase activity. Phage A would probably not develop normal plaques on Rhodamine B medium. When appropriate, antibiotics (Boehringer-Mannheim, Germany) were added at the following concentrations: tetracycline (Tc), 12.5 ~ g / m l , ampicillin (Ap), 50 ~zg/ml for pBR328-derived plasmids; 100/xg/ml for pBluescript SK-derived plasmids, kanamycin (Kin), 50/xg/ml, chloramphenicol (Cm), 30 # g / m l for plasmid-borne resistance gene; 3/xg/ml for chromosome-borne resistance gene.

Construction of the genomic library High-molecular weight genomic DNA was extracted from B. subtilis strain QBl133 and purified by CsC1EtBr gradient centrifugation [24]. The DNA was subjected to partial Sau3A digestion. DNA fragments

TABLE I

Bacterial strains, plasmids and phages used in this study Strain, plasmid or phage

Genotype or phenotype

Source or reference

metB sacA amyE arol

NCIB 11979

lacY1 galK2 rpsL20 xyl5 mtll recA13 aral4 proA2 supE44 hsdS recBC sbcB endA gal met rpsL A(lac-proXIII) recA56 arg su ° [F' lacl q L8 Pro + ] Nal r Rift F hsdR514 hsdM supE44 supF58 lacY1 galK2 gaiT22 rnetBl trpR55 metB supF hsdR NM538 (P2 cox3) recA1 endA1 gyrA96 thi hsdR17 supE44 relA1 lac [F' proAB lacIq ZAM15 Tnl0(Tcr)]

51 52 53

BaciHus subtilis168 QBlI33

Escherichia coil HB101 BJ5183 NK5830 LE392

NM538 NM539 Xll Blue CL2001 Plasmids pBR322 pBR328 pLIP1 pLIP10 pL1Pll3 pL1Pll5 pLIP2 pLIP3 Phages A467 AEMBL4 ALIPI ALIP2

NK5830(pL1P1) Ap r Tc r Apr Tc r Cm r pBR328 pBR328 pBR328 pBR328 pBR328 pBR322

Lip + Lip + Lip Lip + Est + Lip +

Cm ~ Cm ~ Tc s Cm ~ Tc ~ Cm s Tc ~

Ab221 rex::Tn5 ci857 Oam29 P a m 8 0 AsbhlA1 ° b189 int29 ninL44 c1857 trpE KH54 chiC srIA4 ° nin5 srlA5 ° AEMBL4 Lip + SpiAEMBL4 Est + Spi

54 25 25 Stratagene this study

55 56 this this this this this this

study study study study study study

28 25 this study this study

255 sized between l0 and 20 kb were ligated to phage AEMBL4 BamHI-SalI arms [25]. After ligation, the DNA was packaged in vitro using a packaging kit (Amersham). Samples of dilutions of the packaging mixture were plated on both NM538 (P2) and NM539 (P2) + E. coli strains in order to determine the ratio of recombinant phages by their Spi- phenotype [26].

DNA manipulations The methods described by Sambrook et al. [24] were used for construction and isolation of recombinant DNA. Restriction, ligation, end-filling were performed with enzymes from Boehringer-Mannheim (Germany) under conditions recommended by the supplier. DNA fragments were recovered from agarose gels using the G E N E CLEAN kit from BIO101 (La Jolla, CA). Transformation Competent cells of E. coil were prepared and transformed by usual procedures [24] and by electroporation with the Bio Rad gene pulser (Richmond, CA) under the conditions recommended by the supplier. Transformation of B. subtilis with chromosomal DNA was performed in competent cells of B. subtilis [27]. DNA concentrations from 0.5 to 1 /zg/ml of competent cells were used. Transposon Tn5 mutagenesis Stocks of phage A467 (A::Tn5)were prepared by the soft agar layer technique [27] using E. coli strain LE392. Mutagenesis of the non-suppressive E. coli strains CL2001 and CL2002 with Tn5 was performed as described by de Bruijn and Lupski [28]. Briefly, an E. coil culture in late exponential phase was infected with A467 at a multiplicity of infection of 1 to 10 and kanamycin resistant cells were selected on plates. Colonies were washed off the plates and their plasmid DNA was extracted. This DNA was used to transform E. coli strain BJ5183 to Tc r and Km r. Insertions in the lipase gene were screened for by their negative phenotype on Spirit Blue Agar. Localization of Tn5 insertions that inactivate lip in pLIP1 was determined by ClaI-BglIl restriction patterns. Assay of esterase activity Assays were performed with p-nitrophenylbutyrate, p-nitrophenylcaprylate, p-nitrophenyllaurate, p-nitrophenylpalmitate, and p-nitrophenylstearate (Sigma) as the substrates. The reaction was carried out at 37°C in 0.2 M phosphate buffer pH 7.4. 1 unit of enzyme activity corresponds to the liberation of 1 ~mol of p-nitrophenol per rain (E = 0.016 /zM-1). Absorbance was measured continuously at 420 nm with a Beckman DU-8 Spectrophotometer.

DNA sequencing DNA sequencing was performed by the dideoxy chain termination method [29], with [35S]dATP (Amersham, > 1000 C i / m m o l ) as the radioactive label. Sequencing reactions were carried out on single strand and double strand plasmid DNA with a Sequenase kit (USB Corporation, USA) with synthetic oligonucleotide primers, in accordance with the manufacturer's instructions. Tn5 insertion mutants in the lip gene were used, after systematic deletion of one of the two IS50 flanking Tn5 transposon, in order to initiate the sequencing reactions from the extremity of the remaining IS. The PCR was run for 35 cycles with the following parameters: denaturation, 1 min at 94°C; annealing, 2 min at 50°C and elongation, 3 rain at 72°C. Prior to the sequencing reactions, the PCR products were purified from an agarose gel, dissolved in 5 #I water and denatured by adding 1 #l NaOH 1 M. After 10 min incubation at room temperature, 1 /~1 of HCI 1 M was added, directly followed with 1 p.l of primer (20 ng/p.l) and 2 /~I of 5 × sequencing buffer (U.S. Biochemicals). Annealing was carried out for 15 rain at 37°C. Sequence analysis Computer analysis of nucleotide and amino acid sequences were carried out with P C G E N E software. Results

Screening of the genomic library A genomic library of B. subtilis QBl133 was constructed in the bacteriophage EMBIA as described under Materials and Methods. The efficiency of plating on both strains NM538 and NM539 indicated that more than 95% of the viable phage particles were recombinants. Phages from this bank were plated on Spirit Blue Agar medium, using E. coli NK5830 as the indicator strain. Blue plaques, indicating hydrolysis of the substrate, were obtained at a frequency of about 1.1 • 1 0 - 4 . Large scale lysates of positive clones were prepared. Digestion of DNA preparations with BamHI, Sail and EcoRI (inserts into AEMBL4 are flanked with EcoR! sites) allowed the distinction between two different kinds of inserts. The clones were designated ALipl and ALip2. EcoRI fragments from both inserts were subcloned into plasmid vectors pBR322 and pBR328. Transformants of E. coli BJ5183 were screened on Spirit Blue Agar medium. Positive clones (Tc r, Ap ~ and Cm ~ were obtained with both ALipl and ALip2 DNA preparations and the respective plasmids were kept as pL1P1 (7.7 kb), pLIP2 (10.4 kb) and pLIP3 (6.7 kb). The three clones were tested on Rhodamine B medium [22] in order to detect true lipolytic activity. It appeared that only pLIP1 and pLIP3 conferred a li-

256 Plasmid Phenotype Lip

A

H H

H i H H

N E I i ,avuJuu N E

H

N

I

E

I

pLIP 1 pLIP 113 pLIP 115 pLIP3

H

+ + ÷

N

Iii

B f

'

0,7kb--~

E \

/

C

.,_

I

\ lip

E i

yJ-

Fig. 1. (A) Physical maps of the inserts of pLIPI, pL1P3 and derived plasmids. Thick lines indicate the position of the lip gene on the inserts. Restriction sites: E, EcoRl; H, Hindlll; N, Ncol. BamHl and Pstl do not cleave in any of the inserts. The hatched box indicates the position of the probe used in Southern blot hybridizatkms. (B) Positions of Tn5 insertions which inactivate the lip gene. (C) Sequencing strategy of the lip fragment: positions of the overlapping DNA regions sequenced on both strands are indicated by arrows.

pase-positive phenotype to E. coli while pLIP2 probably carried a gene coding for an esterase. Restriction maps of pLIP1 and pLIP3 are shown in Fig. 1A. It was confirmed by Southern hybridizations that the 0.7 kb EcoRI-Ncol fragment of pLIP1 (see Fig. 1)hybridized to a single band of PstI-digested genomic DNA from B. subtilis QBl133 (data not shown).

Difference in substrate specificity between the enzymes In order to detect a possible effect of substrate chain length on the rate of hydrolysis by the two enzymes, esterase activity was measured in the cytoplasmic fraction of both clones in E. coil with pnitrophenyl esters derived from different chain length fatty acids (conditions of reaction and units are defined under Materials and Methods). As shown in Table II, the enzyme encoded by plasmid pLIPI as well as the lipase secreted by B. subtilis hydrolysed esters of different fatty acid chain length at approximately the same rate excepted a slight preference for p-nitrophenyl caprylate (Cs). This result suggests that the lip gene cloned in E. coil codes for a lipase similar to that present in the supernatant of B. subtilis cultures. On the other hand, the enzyme encoded by plasmid pLIP2, carrying a presumed esterase gene, preferentially hydrolysed esters with short chain fatty acids. It should be pointed out that p-nitrophenylpalmitate and pnitrophenylstearate are not water soluble substrates. The rather low rate of hydrolysis of these substrates by est-encoded enzyme combined with its lipase-negative phenotype on Rhodamine B plates warrants its classification as an esterase rather than a lipase.

Position of the lip gene on pLIPI Preliminary localization of the lip gene was performed by subcloning fragments of the insert. Plasmids subclones and the corresponding phenotypes are shown in Fig. 1. Direction of transcription has not been determined. Yet, the lip gene is probably transcribed from its own active promoter since the level of gene expression is comparable in plasmid pLIP10 where the insert is inverted (not represented). In order to determine more precisely the size and position of the cloned gent, Tn5 mutagenesis of plasmid pLIP1 was performed in E. coli strain NK5830 with phage A467 as the vector of Tn5 transposon. Fig. 1B shows maps of Tn5 insertions which inactivated the lip gene. It can be seen that 47 insertions which confer a lipase-negative phenotype delineate a 0.7 kb DNA region. DNA sequence of the lip gene in pLIP1 and pLIP3 Using plasmid pLIP1 and taking into account the position of the gene determined by Tn5 insertions, the lipase gene (lip) was sequenced by the methods described. Overlapping DNA regions were sequenced on both strands (see Fig. IC) and each nucleotide was read between three and six times on sequencing gels. A single open reading frame of 591 bp was identified in the sequence. However, the ATG initiation codon was preceded by a very poor Shine-Dalgarno sequence and no sequence resembling a standard signal peptide could be found in the N-terminal region of the encoded protein. These results led us to determine the complete sequence of lip on an independent E. coil clone containing plasmid pLIP3 (Fig. 1). The sequence obtained is presented in Fig. 2. The only difference between the two sequences is a frameshift created by the addition, in the nucleotide sequence of lip in pLIPl, of a single

TABLE II

Fatty acid chain-length .~ecificiO, of the enzymes coded by genes lip atul est Substrate ~

Specific activity ( m U / m g protein) 1, pLl P 1

B. subtilis

pLl P2

supernatant pNP-butyrate (C 4) pNP-caprylate (C8) pNP-laurate (C12) pNP-palmitate (C 1~,) pNP-stearate ( C ~ )

85 209 58 27 5l

22 51 27 16 16

2315 1852 16 4 0.5

~' All assays contained the substrate at 3 raM. C4_12 were dissolved in ethanol at 100-fold final concentration. C16 and C1~ were emulsified by sonication at final concentration in phosphate citrate buffer containing 0.05% Triton X-100. t, 1 unit is defined as the amount of enzyme which liberates 1 #mol of p-nitrophenol m i n - i at 37°C.

257 60 GTCCAAATGACGGAAGCTACCTGTTGACGCTGCTGTAAAACCGGAGCAAAGGGGATTGTA 120 TTTGCCGGTTCTGGGAACGGGTCTTTATCTGATGCAGCCGAAAAAGGGGCGGACAGCGCA 180 GTCAAAAAAGGCGTTACAGTGGTGCGCTCTACCCGCACGGGAAATGGTGTCGTCACACCA 240 AACCAAGACTATGCGGAAAAGGACTTGCTGGCATCGAACTCTTTAAACCCCCAAAAAGCA 300 CGGATGTTGCTGATCGTTGCTCTTACCAAAACAAA~ATCCTCAAAAAATCCAAGCTTAT 360 TTCAATGAGTATTGAAGAAAAGAAGGCGAATAAGCCTTCTTTTTTTTGGCTTTTTAGGAC oooooooooooooooooooooooooooooo

420

CAATAATGACCTCTGAATCTTAAAATTTCTTTAAAAATAAGCCAAAATTACCCTTTACTT 480 M AATTAATTTGGTAACGTAATATAATTGGAGAATTTGTTACAAAA/kAAGGAGGATATTATG lO

RBS

+ + + + o . o . . o . . . K F V K R R I I A L V T I L M L S V T S AAATTTGTAAAAAGAAGGATCATTGCACTTGTAACAATTTTGATGCTGTCTGTTACATCG

. . . .

/

540

.0

L F A L Q P S K A E H N P V V M V H CTGTTTGCGTTGCAGCCGTCAGCAAAAGCCGCTGAACACAATCCAGTCGTTATGGTTCAC 660 G I G G A S F N F A G I K S Y L V S Q G GGTATTGGAGGGGCATCATTCAATTTTGCGGGAATTAAGAGCTATCTCGTATCTCAGGGC 720 W S R D K L Y A V D F W D K T G T N Y N TGGTCGCGGGACAAGCTGTATGCAGTTGATTTTTGGGACAAGACAGGCACAAATTATAAC 780 N G P V L S R F V Q K V L D E T G A K K AATGGACCGGTATTATCACGATTTGTGCAAAAGGTTTTAGATGAAACGGGTGCGAAAAAA 840 V D I V A H S M G G A N T L Y Y I K N L GTGGATATTGTCGCTCACAGCATGGGGGGCGCGAACACACTTTACTACATAAAA~TCTG 900 D G G N K V A N V V T V G G A N R L T T GACGGCGGAAATAAAGTTGCAAACGTCGTGACGGTTGGCGGCGCGAACCGTTTGACGACA 960 G K A L P G T D P N Q K I L Y T S I Y S GGCAAGGCGCTTCCGGGAACAGATCCAAATCAAAAGATTTTATACACATCCATTTACAGC 1020 S A D M I V M N Y L S R L D G A R N V Q AGTGCCGATATGATTGTCATGAATTACTTATC~GATTAGATGGTGCTAGAAACGTTCAA 1080 I H G V G H I G L L Y S S Q V N S L I K ATCCATGGCGTTGGACACATCGGCCTTCTGTACAGCAGCCAAGTCAACAGCCTGATTAAA 1140 E

G

L

N

G

G

G

Q

N

T

N

******

GAAGGGCTGAACGGCGGGGGCCAGAATACGAATTAATGAAAAACAAAACCTTGAAGAATG 1200 C~ATTCTTCAAGGTTATTCCGCTTTCGGCACAATGGTTTTCGCAGCCGTATCGTGAACGG 1260 FTTGTTTTTTCTTCGTAAATGCGGCAGTCAAATAGATCAGGCGGGAGAACACATGCACCC 1320 ACGCTATCAGGTAACGGACAATGGCTTGCGGGAA