Integration and amplification of the Bacillus sp cellulase gene in the Bacillus subtilis 168 chromosome

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1 J. Gen. Appl. Microbiol., 44, (1998) Short Communication Integration and amplification of the Bacillus sp cellulase gene in the Bacillus subtilis 168 chromosome Kyung Hwa Jung, Dae-Hee Lee, Ki-Hong Yoon, 1 and Seung-Hwan Park* Bacterial Molecular Genetics Research Unit, Korea Research Institute of Bioscience & Biotechnology (KRIBB), P.O. Box 115, Yusong, Taejon , Korea 1 Department of Food Biotechnology, Woosong University, San 7 6, Jayang-dong, Dong-ku, Taejon , Korea (Received December 4, 1997; Accepted February 16, 1998) Key Words Bacillus sp ; Bacillus subtilis 168; cellulase gene, cels; chromosomal integration; gene amplification; homologous recombination * Address reprint requests to: Dr. Seung-Hwan Park, Bacterial Molecular Genetics Research Unit, Korea Research Institute of Bioscience & Biotechnology (KRIBB), P.O. Box 115, Yusong, Taejon , Korea. To overproduce Bacillus sp cellulase for industrial use, two strategies can be considered for increasing the gene copy number in Bacillus: (i) transformation with a replicative plasmid harboring cloned genes, and (ii) integration and/or amplification of cloned genes in the Bacillus chromosome. In B. subtilis, it is known that replicative plasmids harboring cloned genes can be prone to either segregational or structural instability (Bron, 1990). Furthermore, antibiotics cannot be used in large-scale production for both economical and safety reasons although drug resistance is usually applied to construct and maintain recombinant strains in laboratories. A promising way to avoid these problems may be to use integrative vectors for introducing cloned genes into the Bacillus chromosome. Others have achieved integration by a single (Campbell-like) or double crossover recombination event by using the appropriate circular or linear vector DNA (Niaudet et al., 1985; Vosman et al., 1986; Young, 1983). Multiple copies of the integrated genes can also be obtained by selecting spontaneous amplification of the integrates or by repeating the integration procedure (Albertini and Galizzi, 1985; Declerck et al., 1988; Kallio et al., 1987; Shendye and Rao, 1993; van der Laan et al., 1991; Young, 1984). Moreover, such repeated DNA sequences in the Bacillus chromosome are more stable than when carried on a replicative plasmid although the significant stability of the amplified structures appeared to be variable. In recent studies, Tangney et al. (1995) have developed a new strategy for the integration and stable amplification of cloned DNA sequences in the chromosome of poorly transformable bacilli including B. licheniformis. The method is based on the presence of two plus origins of replication in the same orientation on the same plasmid, which provides the possibility of forming two progeny vectors. We have previously reported the cloning and nucleotide sequence of the cellulase gene of Bacillus sp , cels, and also an examination of cels gene expression on a replicative plasmid in B. subtilis DB104 (Jung et al., 1996). In this study, for integration and amplification of the cels gene in the B. subtilis chromosome, we constructed a recombinant plasmid, psi7-s, by modifying a vector system developed by Tangney et al. (1995). Its restriction map is shown in Fig. 1. This plasmid based on puc19 contains three distinct fragments derived from pub110; a 1.43 kb EcoRI-HaeIII fragment including both a plus origin of replication ( ori) and a gene for the replication initiation protein (rep gene), a 0.97 kb EcoRI-NsiI fragment including both a ori and a truncated rep gene, and a kanamycin-resistance gene. Plasmid psi7-s also contains a 1.68 kb EcoRI-HindIII fragment of pjcmc (Jung et al., 1996) harboring the cels gene modified with a strong B. subtilis promoter (Kim and Pack, 1993) and a synthetic Shine-Dalgarno sequence. Plasmid pub110 replicates by the rolling-circle mode. The minimal replication function consists of a ori and a replication initiation protein (Rep) which has a strand nicking-closing activity at ori sequences.

2 108 JUNG et al. Vol. 44 Fig. 1. Restriction map of psi7-s and diagrammatic representation of the integration and amplification of the cels gene in the chromosome of B. subtilis 168. The numbers denote DNA size in kilobases. Abbreviations: rep, gene for replication initiation protein; rep, truncated rep gene; cels gene, cellulase gene cloned from Bacillus sp ; Km, kanamycin-resistance gene; Ap, β-lactamase gene; P, B. subtilis promoter; ori, plus origin of replication for pub110; ori, replication origin for puc19. During rolling-circle plasmid replication, the Rep protein produces a nick at ori, and a single strand of DNA is produced. This is subsequently converted to double-stranded DNA, which is nicked again at the newly generated ori sequence and then ligated in the cell (Bron, 1990). However, psi7-s contains a functional rep gene and two separate ori sequences in the same orientation on a single plasmid. Due to these direct repeats, the replication of this plasmid in Bacillus can give rise to two different progeny plasmids as shown in Fig. 1. One of the progeny plasmids, psi7-s1, can replicate autonomously in Bacillus since this plasmid contains a ori sequence and a functional rep gene, but psi7-s1 has no selection marker for Bacillus. The other progeny plasmid, psi7-s2, contains kanamycin-resistance gene, but psi7-s2 cannot replicate in Bacillus since this vector has no functional rep gene. In addition, the nucleotide sequence of the cels gene showed 99% homology with that of the cellulase gene of B. subtilis 168 (Jung et al., 1996).

3 1998 Chromosomal integration and amplification of the cels gene 109 Under these conditions, Km-resistant colonies should arise only by homologous recombination between the cels gene on psi7-s2 and a cellulase gene on the chromosome of B. subtilis 168, as depicted in Fig. 1. We transformed B. subtilis 168 with psi7-s by a competent method (Cutting and van der Horn, 1990) and obtained four colonies grown on LB agar plates containing 20 µg/ml kanamycin (Km). All transformants produced large halos as compared to that of the parent strain grown on LB agar plates containing 0.5% carboxymethylcellulose (CMC) (data not shown). Transformants grown on LB agar plates containing 20 µg/ml Km were first re-streaked for single colonies onto the same agar plates and subsequently grown overnight in SpC broth without Km (Cutting and van der Horn, 1990). This single overnight incubation was enough to eliminate the autonomously replicating plasmids from these strains. All transformants were plasmid-free as evident from these facts: (i) free plasmids could not be found on an EtBr-stained agarose gel, and (ii) plasmid preparations could not transform E. coli to ampicillin resistance. The presence of psi7- S2 in the chromosome of B. subtilis 168 was confirmed by Southern hybridization analysis (data not shown). One strain among these transformants, designated BS7S-20, was used to amplify the cels gene within the chromosomal DNA. Spontaneous amplification of the integrated cels gene could be selected by increasing the level of antibiotic in the growth medium. The BS7S-20 strain was grown in 20 ml of SpC with 20 µg/ml Km. From this culture, 200 µl aliquots were subcultured into 20 ml of SpC containing Km with concentrations increasing stepwise from 20 up to 100 µg/ml. From a culture grown in 100 µg/ml Km, 200 µl aliquots were removed and subcultured again, but this time in the range of 100 1,000 µg/ml Km. The cultures were fully grown even at the highest concentration of Km. In preliminary assays on LB agar plates containing 0.5% CMC, the culture in 600 µg/ml Km, designated BS7S-600, showed noticeably enhanced cellulase activity suggesting that it harbored the amplified psi7-s2 integrates. To verify that the integrated cels gene was actually amplified within the chromosome of strain BS7S-600, restriction analysis was performed with the chromosomal DNA prepared from the culture. When the chromosomal DNA of strain BS7S-600 was digested with EcoRI and separated in an agarose gel, two prominent bands with sizes of 3.19 and 0.62 kb could be seen in an EtBr-stained agarose gel (Fig. 2A). These bands could not be seen in a DNA sample of B. subtilis 168. Southern analysis was performed using the cels gene and Km r gene as DNA probes, and we observed that both 3.19 and 0.62 kb EcoRI fragments showed positive signals for the cels gene probe (Fig. 2B), while only the 3.19 kb EcoRI fragment harboring the Km r gene showed a positive signal for the Km r gene probe (Fig. 2C). The sum of the sizes of those two fragments, 3.81 kb, is equal to the size of plasmid psi7-s2. This is an indication that the amplified sequence has a unit length corresponding to the size of the integrated plasmid. No free plasmids were detected in the B. subtilis 168 transformants. From these results, we concluded that parental plasmid psi7-s had given rise to non-replicative psi7-s2, which had integrated by a Campbell-like recombination between the homologous cellulase genes; the resulting chromosome structures then progressed spontaneous amplification within the chromosome, as depicted in Fig. 1 (Albertini and Galizzi, 1985; Declerck Fig. 2. Restriction analysis of the chromosomal DNAs prepared from B. subtilis strains (A), and Southern hybridization with the DIG-labeled cels gene (B) and the Km r gene (C), respectively. Chromosomal DNAs were prepared by a standard method described in Cutting and van der Horn (1990). After complete digestion with EcoRI, the chromosomal DNAs were electrophoresed on a 0.8% agarose gel followed by Southern hybridization according to the DIG system described in the supplier s manual (Boehringer Mannheim, Germany). Lane 1, EcoRI-digests of B. subtilis 168; lane 2, EcoRI-digests of BS7S-20; lane 3, EcoRI-digests of BS7S-600; M, DIG-labeled DNA molecular-weight marker VII (Boehringer Mannheim).

4 110 JUNG et al. Vol. 44 et al., 1988; Shendye and Rao, 1993; van der Laan et al., 1991; Young, 1984). Gene amplification is viewed as an adaptive response (i.e., a response to stress). Selection for resistance to increase the level of antibiotic, to which the integrated plasmid confers resistance, may further increase the copy number through unequal recombination between identical sequences on nascent daughter chromosomes (Albertini and Galizzi, 1985; Anderson and Roth, 1977; Camerini-Otero and Hsieh, 1995; Ives and Bott, 1990; Petes and Hill, 1988). The B. subtilis 168 derivatives containing additional cels genes were grown in antibiotic-free LB medium, and expression of the cellulase gene was observed (Fig. 3). The cellulase activity in the culture supernatant of strain BS7S-600 reached a maximum level of 2.7 unit/ml when the cell growth reached an OD 600 of 2.6, while the parent strain and strain BS7S-20 showed maximum cellulase activity with 0.1 unit/ml and 0.4 unit/ml, respectively. Strain BS7S-600, selected during the amplification procedure, showed about 27 times higher cellulase activity than that of B. subtilis 168. The increase in cellulase production in BS7S-600 was considered to be the result of cels gene amplification, although we did not determine the copy number of the cels gene on the chromosome of BS7S-600. Strains BS7S-600 and B. subtilis DB104 containing pjcmc, a recombinant plasmid harboring the cels gene (Jung et al., 1996), were grown in antibiotic-free LB medium to compare the stability of cellulase production between the amplified strain and the plasmidcontaining strain without selection pressure (Fig. 4). Six successive cycles of cultures were performed under non-selective conditions. Bacillus strains were grown in Km-free LB medium for 12 h at 37 C and then 1,000-fold diluted in 50 ml of fresh medium to start a new cycle. After each of these cycles, 5 ml aliquots of the cultures were centrifuged to assay the cellulase activity of the supernatants and to analyze the DNA fragments containing the cels gene. As shown in Fig. 4A, the cellulase production by strain BS7S-600 appeared to be rather unstable since about 80% of the cellulase activity was lost after six culturing cycles. This might be owed to a decrease in the number of gene copies as culturing proceeded under nonselective conditions (Fig. 4B and C). In spite of this instability, cellulase production by the amplified strain appeared more stable than that by the plasmid-containing strain, since the loss of cellulase activity in the Fig. 3. Cellulase production by the derivatives of B. subtilis 168 during growth in antibiotic-free LB medium. Cellulase activities were determined by measuring the amount of reducing sugars using dinitrosalicylic acid (Miller et al., 1960). The reaction mixtures containing 0.5% (w/v) carboxymethylcellulose in 50 mm sodium phosphate buffer (ph 7.0) were incubated at 50 C for 15 min. One unit of enzyme activity was defined as the amount of enzyme that produced 1 µmol of reducing sugar per minute. Symbols: Growth ( ) and enzyme production ( ) by strain BS7S- 600; growth ( ) and enzyme production ( ) by strain BS7S-20; growth ( ) and enzyme production ( ) by B. subtilis 168. Fig. 4. Stability of cellulase production by BS7S-600 ( ) and B. subtilis DB104 containing pjcmc ( ) under non-selective conditions (A), and restriction analysis (B) and Southern hybridization analysis (C) of the BS7S-600 chromosomal DNAs. (A) BS7S-600 and B. subtilis (pjcmc) were streaked on agar plates supplemented with 600 µg/ml and 50 µg/ml Km, respectively. With fresh colonies, these Bacillus strains were grown in Km-free LB broth for 12 h at 37 C and then 1,000-fold diluted in 50 ml of fresh medium to start a new cycle. (B) and (C) Chromosomal DNAs were prepared from each culture of BS7S-600. The EcoRI-digests of BS7S-600 chromosomal DNAs were hybridized with the DIG-labeled cels gene. Lanes 1 6, EcoRI-digests of the chromosomal DNAs prepared from each culture of BS7S-600; M, DIG-labeled DNA molecular-weight marker VII (Boehringer Mannheim).

5 1998 Chromosomal integration and amplification of the cels gene 111 cultures of the plasmid-containing strain was faster than that in the amplified strain. Furthermore, strain BS7S-600 still harbored the amplified structure even after six successive culturings while plasmid preparation from the sixth culture of B. subtilis DB104 (pjcmc) did not show the pjcmc plasmid bands on an EtBr-stained agarose gel (data not shown). With the plasmid system, about 90% of the cellulase activity was lost, and cellulase activity was eventually lowered to below that of the amplified strain after only three culturing cycles. Gene integration may be advantageous for large-scale production because of the gene dosage effect, efficient gene expression, efficient secretion of the gene products and higher stability of the integrates as compared to plasmid-containing strains (Kallio et al., 1987; Shendye and Rao, 1993). This work was supported by a grant from the Ministry of Science and Technology of Korea (HS1720). References Albertini, A. M. and Galizzi, A. (1985) Amplification of a chromosomal region in Bacillus subtilis. J. Bacteriol., 162, Anderson, P. R. and Roth, J. R. (1977) Tandem genetic duplications in phage and bacteria. Annu. Rev. Microbiol., 31, Bron, S. (1990) Plasmids. In Molecular Biological Methods for Bacillus, ed. by Harwood, C. R. and Cutting, S. M., John Wiley & Sons Ltd., UK, pp Camerini-Otero, R. D. and Hsieh, P. (1995) Homologous recombination proteins in prokaryotes and eukaryotes. Annu. Rev. Genet., 29, Cutting, S. M. and van der Horn, P. B. (1990) Genetic analysis. In Molecular Biological Methods for Bacillus, ed. by Harwood, C. R. and Cutting, S. M., John Wiley & Sons Ltd., UK, pp Declerck, N., Joyet, P., Coq, D. L., and Heslot, H. (1988) Integration, amplification and expression of the Bacillus licheniformis α-amylase gene in Bacillus subtilis chromosome. J. Biotechnol., 8, Ives, C. L. and Bott, K. F. (1990) Characterization of chromosomal DNA amplifications with associated tetracycline resistance in Bacillus subtilis. J. Bacteriol., 172, Jung, K. H., Chun, Y. C., Lee, J.-C., Kim, J. H., and Yoon, K.-H. (1996) Cloning and expression of a Bacillus sp cellulase gene. Biotechnol. Lett., 18, Kallio, P., Palva, A., and Palva, I. (1987) Enhancement of α-amylase production by integrating and amplifying the α-amylase gene of Bacillus amyloliquefaciens in the genome of Bacillus subtilis. Appl. Microbiol. Biotechnol., 27, Kim, J. H. and Pack, M. Y. (1993) Overproduction of extracellular endoglucanase by genetically engineered Bacillus subtilis. Biotechnol. Lett., 15, Miller, G. L., Blum, R., Glennon, W. E., and Burton, A. L. (1960) Measurement of carboxymethylcellulase activity. Anal. Biochem., 2, Niaudet, B., Jannière, L., and Ehrlich, D. D. (1985) Integration of linear, heterologous DNA molecules into the Bacillus subtilis chromosome: Mechanism and use in induction of predictable rearrangements. J. Bacteriol., 163, Petes, T. D. and Hill, C. W. (1988) Recombination between repeated genes in microorganisms. Annu. Rev. Genet., 22, Shendye, A. and Rao, M. (1993) Chromosomal gene integration and enhanced xylanase production in an alkalophilic thermophilic Bacillus sp. (NCIM 59). Biochem. Biophys. Res. Commun., 195, Tangney, M., Jørgensen, P. L., Diderichsen, B., and Jørgensen, S. T. (1995) A new method for integration and stable DNA amplification in poorly transformable bacilli. FEMS Microbiol. Lett., 125, van der Laan, J. C., Gerritse, G., Mulleners, L. J. S. M., van der Hoek, R. A. C., and Quax, W. J. (1991) Cloning, characterization, and multiple chromosomal integration of a Bacillus alkaline protease gene. Appl. Environ. Microbiol., 57, Vosman, B., Kooistra, J., Olijve, J., and Venema, G. (1986) Integration of vector-containing Bacillus subtilis chromosomal DNA by a Campbell-like mechanism. Mol. Gen. Genet., 204, Young, M. (1983) The mechanism of insertion of a segment of heterologous DNA into the chromosome of Bacillus subtilis. J. Gen. Microbiol., 129, Young, M. (1984) Gene amplification in Bacillus subtilis. J. Gen. Microbiol., 130,