| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1 Biological Science Laboratories, Kao Corporation, 2606 Akabane, Ichikai, Tochigi 321-3497, Japan
2 Graduate School of Information Science, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0101, Japan
Correspondence
Shenghao Liu
liu.shenghao{at}kao.co.jp
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
|---|
). The complete sequencing and annotation of the B. subtilis genome (Kobayashi et al., 2003; Kunst et al., 1997) has facilitated the construction of artificial mutant strains for industrial and scientific uses (Harwood & Wipat, 1996). Multiple mutations are required in many cases. Mutant selection relies mostly on antibiotic-resistance markers; however, the number of markers available for use in B. subtilis is limited. To circumvent this problem, several methods have been developed to produce unmarked mutations in B. subtilis (Bloor & Cranenburgh, 2006; Brans et al., 2004; Fabret et al., 2002; Leenhouts et al., 1996; Zhang et al., 2006) and other micro-organisms (Bloor & Cranenburgh, 2006; Leenhouts et al., 1996; Malaga et al., 2003; White et al., 2007). A method of selectable marker gene excision by Xer recombination has been reported recently, in E. coli and B. subtilis. The native Xer site-specific recombinase, RipX/CodV in B. subtilis, recognizes the dif sites and acts to resolve the two directly repeated dif sites to a single site (Bloor & Cranenburgh, 2006). Leenhouts et al. (1996) have described a method to generate unlabelled gene replacements in Lactococcus lactis and B. subtilis, using a conditional replication plasmid as the integration vector and E. coli lacZ as the reporter. However, the selections of the unmarked mutants were conducted with negative selection. The method involving the upp cassette, described by Fabret et al. (2002), tends to result in many false-positive colonies in our experience, so that it is not adequate for the efficient construction of multiple mutants. Two other methods use, respectively, the heterologous genes blaI from Bacillus licheniformis and mazF from E. coli for counter selection (Brans et al., 2004; Zhang et al., 2006), and specific direct repeat DNA sequences, used for homologous recombination, are introduced into the genome during every round of manipulation. For multiple modifications of the B. subtilis genome, dozens of mutations might be performed. If many direct repeat sequences are left in the genome, intra-genomic homologous recombinations are more likely to occur, which might result in the deletion or inversion of genomic regions. The purpose of this study is to construct a marker-free multiple mutation system by positive selection, without heterologous DNA fragments remaining in the target sequence.
We describe here a method that involves a selective marker cassette carrying an antibiotic-resistance gene and a gene for a repressor active in B. subtilis cells. Upstream and downstream sequences from the target locus are placed in front of the cassette, which is then inserted into the upstream region of the target locus by homologous recombination (Fig. 1). This cassette is designed to be removed later, and can be used subsequently for another round of mutation. Another antibiotic-resistance gene, whose expression is controlled by the repressor, is used as a counter-selective marker for excision of the selective marker cassette along with the target DNA sequence. The sequence downstream of the target locus, similar to the downstream sequence introduced with the selective marker cassette, acts as a specific site for homologous recombination, which makes it possible for no heterologous DNA sequences to remain at the mutation locus. When the selective marker cassette is removed, the counter-selective marker gene is released from repression, so that antibiotic resistance is expressed. The counter-selective marker remains in the B. subtilis genome after the deletion of the target locus and is used again for the next round of deletion manipulation.
|
, 2001; Sa-Nogueira & Mota, 1997), for use in the selective marker cassette, and the promoter of the araABDLMNPQ-abfA operon (Sa-Nogueira et al., 1997) for use in the counter-selective marker cassette. This new mutation system will be convenient for the introduction of multiple mutations, and we discuss its use in large-scale deletion mutations of the B. subtilis genome. |
METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
|---|
. All B. subtilis recombinant strains were B. subtilis 168 derivatives. The primers used for PCR amplification were synthesized by Sigma Genosys and are listed in Table 2.
|
|
).
DNA manipulation techniques.
The isolation and manipulation of recombinant DNA was performed using standard techniques. Enzymes were commercial preparations and were used as specified by the supplier (Takara). Chromosomal DNA was prepared using the Microbial Genomic DNA Isolation kit (MO Bio). PCRs were performed in final volumes of 50 µl containing 1.25 U Pyrobest DNA polymerase (Takara), 1 mM MgSO4, 0.2 mM dNTP and 0.4 µM of each primer. The amplification program consisted of 30 cycles of 10 s at 98 °C, 30 s at 55 °C, 1 min for every 1 kb of amplified product at 72 °C, followed by a final 5 min at 72 °C. All PCRs were performed in a Perkin-Elmer 9700 thermal cycler. The PCR products were analysed by electrophoresis in 1 % (w/v) agarose gels. PCR products were purified with a PCR Purification kit (MO Bio).
Construction of strains AR1 and AR2.
Strain AR1, in which the coding region of araA was substituted with a promoterless neo gene (Fig. 2), was constructed from strain 168 as follows. The 0.8 kb promoterless neo gene fragment was amplified from pUB110 (McKenzie et al., 1986) using the primers Nmf and Nmr. Approximately 1.0 kb of each of the flanking sequences upstream (UParaA) and downstream (DNaraA) of the coding region of araA was amplified by PCR from the B. subtilis genome, using the primers araA/N5f and araArv, and araAfw and araA/N3r, respectively. The UParaA and DNaraA fragments overlapped with neo by 20 bp at the upstream and downstream ends, respectively. These three PCR fragments were then ligated in the order UParaA-neo-DNaraA by splicing by overlapped extension PCR (SOE-PCR) (Ho et al., 1989; Liu et al., 2007) using the primer set araAfw2 and araArv2. In order to minimize the minor products of PCR ligation, these primers were designed using sequences 12 bp from the ends of a similar product that would be amplified using the araAfw and araArv primers. The 2.8 kb PCR product was then introduced into B. subtilis cells by competent cell transformation, and an Nm-resistant transformant was selected from an LB agar plate containing 0.4 % L-arabinose (Sa-Nogueira et al., 1997) and designated AR1 (Fig. 2).
|
) using the primers spf and spr. Approximately 1.0 kb of each of the flanking sequences upstream (UParaR) and downstream (DNaraR) of araR was amplified by PCR from the B. subtilis genome using the primers araRfw and araR/Spr, and araR/Spf and araRrv, respectively. The UParaR fragment and DNaraR fragments overlapped with spm by 20 bp at the upstream and the downstream ends, respectively. The three PCR fragments were then ligated in the order UParaR-spm-DNaraR by SOE-PCR (Ho et al., 1989; Liu et al., 2007) using the primer set araRfw2 and araRrv2. The 3.2 kb PCR product was then used for transformation of strain AR1, and an Sp-resistant transformant was selected and designated AR2 (Fig. 2
).
Construction of the selective marker cassette.
The selective marker cassette containing the Cm-resistance gene (cat) and araR was constructed by PCR amplification. The 0.9 kb cat fragment was amplified from pC194 (Horinouchi & Weisblum, 1982) using the primers cat/csbCDf and catr (Table 2). The 1.3 kb araR fragment, including the promoter and coding regions, was amplified from the B. subtilis genome using the primers araRf/Cm and araRr. The resulting fragment overlapped by 20 bp with the downstream end of the cat fragment. The cat and araR fragments were then ligated by SOE-PCR (Ho et al., 1989; Liu et al., 2007), using the primers cat/csbCDf and araRr, to obtain the 2.1 kb selective marker cassette.
Construction of the counter-selective marker cassette.
The 1.1 kb DNA fragment Para-neo (Fig. 2) was amplified from the AR1 genome using the primers Paraf and Nmr, for use as the counter-selective marker cassette.
Transformation with PCR fragments.
Transformation of B. subtilis 168 with PCR fragments was performed using competent cells, as described by Anagnostopoulos & Spizizen (1961).
DNA sequence determination.
The DNA sequences of cassettes, and the junctions of upstream and downstream regions of deletion targets in the deletion mutants were determined. The dideoxy chain-termination method was used, with a BigDye Terminator v3.1 Cycle Sequencing kit and a 3730XL DNA sequencer (Applied Biosystems). The deletion junctions were amplified from mutants and the PCR products were used as templates.
|
RESULTS AND DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
|---|
) and the divergently arranged araE/araR genes (Sa-Nogueira & Ramos, 1997; Sa-Nogueira & Mota, 1997), are induced by L-arabinose and negatively controlled by AraR (Mota et al., 1999, 2001). We selected the B. subtilis araR gene and one of its target promoters as candidates for utilization in the selective and counter-selective marker cassettes in this study for the following reasons. First, the arabinose utilization pathway is not essential for energy production, and a deficiency in the pathway does not affect the growth of B. subtilis cells in the absence of arabinose. Second, the introduction of a DNA sequence derived from B. subtilis into the B. subtilis genome should have no effect on cell growth and chromosome replication. Third, the mechanism of AraR regulation has been extensively studied in recent years.
To evaluate the stringency of regulation by AraR of an antibiotic-resistance gene under the control of the ara operon promoter, we replaced the araA gene with the promoterless Nm-resistance gene (neo) to produce the strain AR1 (Fig. 2). AR1 was selected for Nm resistance in the presence of 0.4 % (w/v) L-arabinose, and confirmed to be sensitive to Nm in the absence of L-arabinose (Table 3). Next, the araR gene in AR1 was deleted and replaced with the Sp-resistance gene spm, to produce the Sp-resistant transformant AR2 (Fig. 2). AR2 was confirmed to be resistant to Nm whether L-arabinose was present in the medium or not (Table 3). It was encouraging to find that the neo gene fused to the ara promoter (Para-neo) was constitutively expressed in the absence of the araR gene. These results indicated that the expression of Para-neo could be stringently controlled by araR. In other words, the expression of Para-neo could be switched on or off, depending on the presence or absence of araR in the genome, under growth conditions without L-arabinose.
|
The 1.1 kb DNA fragment of Para-neo (Fig. 2) was amplified from the AR1 genome using the primers Paraf and Nmr, to be used as the counter-selective marker cassette. It was ligated between upstream and downstream regions (
1 kb each) from the araR gene by SOE-PCR. The 3.1 kb fragment thus obtained was introduced into B. subtilis 168 cells by competent cell transformation, and mutants were selected on an LB agar plate containing Nm. An Nm-resistant transformant was confirmed by PCR and designated AR3 (Fig. 2). Deletion of araR made it possible for the Para-neo gene in the counter-selective marker cassette to be expressed, because of the absence of the AraR repressor. However, if the araR gene were reintroduced into the AR3 strain, we would expect the expression of Para-neo to be repressed, as shown in Table 3. In other words, a selective marker cassette containing araR and an antibiotic-resistance gene would transform strain AR3 to be sensitive to Nm. Then, deletion of the selective marker cassette from the transformant could be positively selected for on the basis of resistance to Nm. Therefore it would be possible to introduce multiple mutations into strain AR3 by the repeated process of introduction (along with target gene sequences) and rescue of the selective marker cassette containing araR.
Markerless deletions of genomic regions from AR3
Strain AR3 was used as a starting strain for markerless deletions. First, we tried to delete the 3.8 kb region of iolS–csbC, as shown in Fig. 1. Approximately 1.0 kb of each of the flanking sequences upstream of iolS (UP) and downstream of csbC (DN) was amplified by PCR from the B. subtilis genome using the primers iolfw and iol/csbCUr, and csbC-Df and csbCrv, respectively. These primers were designed so that the downstream end of the UP fragment overlapped with the upstream end of the DN fragment by 20 bp. The UP and DN fragments were ligated by SOE-PCR (Ho et al., 1989), using the primers iolfw and csbCrv2, to obtain a 2.0 kb fragment. The 2.1 kb selective marker cassette consisting of the cat and araR genes was constructed by PCR as described in Methods. A 1.0 kb fragment (labelled G in Fig. 1) from the upstream region of the iolS gene (the upstream end in the deletion target region) was amplified by PCR from the B. subtilis genome using the primers iol/CRf and iolrv. These three fragments of 2 kb, 2.1 kb (the selective marker cassette) and 1 kb, which overlapped one another by 20 bp, were then ligated by SOE-PCR (Ho et al., 1989) using the primer set iolfw2 and iolrv2. The resulting 5.1 kb PCR fragment was used to transform strain AR3 by the competent cell method (Anagnostopoulos & Spizizen, 1961). A transformed strain was obtained by selection for Cm resistance, and designated AR31 (Fig. 1). Introduction of the selective marker cassette together with the DN fragment into the genome immediately upstream of the deletion target was verified by PCR analysis (Fig. 3). Thus, in the genome of strain AR31, a DN fragment was located at each end of the region consisting of the selective marker cassette and the deletion target (Fig. 1). We verified that AR31 was sensitive to Nm as a consequence of the integration into the genome of the selective marker cassette containing araR (Table 3).
|
). This was the expected size of the fragment after the deletion of the iolS–csbC region. The csbCrv primer is located 70 bases downstream of the DN fragment in the B. subtilis genome (Fig. 4a), so that the UP-DN fragment of AR31 (Fig. 1) cannot be amplified as a 2 kb fragment using the primers iolfw and csbCrv. Thus, it appeared that the iolS–csbC region was successfully deleted from the genome, together with the selective marker cassette, by intra-genomic homologous recombination at the two DN fragments, as shown in Fig. 1. The deletion mutant of AR31 was designated AR31d.
|
), and the insertion was verified by PCR as shown in Fig. 3. Strain AR32 was then incubated to allow for the excision of the selective marker cassette, together with the 41.8 kb deletion target region, by homologous recombination. The deletion mutant, designated AR32d, was selected for using Nm resistance, which was caused by the deletion of araR in the selective marker cassette (Fig. 1). The putative AR32d colonies were tested for Cm sensitivity by replicating them on an LB plate containing Cm. Of 100 colonies plated, 98 were Cm sensitive. Six of these Cm-sensitive colonies were randomly selected for genomic DNA preparation and PCR analysis. The results (Fig. 3) indicated that the 41.8 kb deletion target was excised exactly as expected, together with the selective marker cassette.
The 2060- and 2000-base DNA sequences at the junctions between the upstream and downstream regions of the deletion targets in the AR31d and AR32d mutants were amplified by PCR using the primer sets iolfw and csbCrv, and hutMfw and csbCrv, respectively, and determined by DNA sequencing. The partial sequences were shown in Fig. 4. The 3.8 and 41.8 kb fragments between UP and DN were deleted as expected in AR31d and AR32d, respectively. The UP-DN fragments in AR31 and AR32 (Fig. 1) could not be amplified using the same primer sets as above under the PCR conditions for 2 kb fragment amplification, because the reverse primer of csbCrv was 70 bases downstream of the DN sequence. The results indicated that the target regions were deleted accurately at the expected sites.
The method described above has the following advantages: (1) both steps in the mutation strategy, including the introduction of the selective marker cassette and the excision of the target region along with the selective marker cassette, are positively selected for by antibiotic resistance (Cm and Nm resistance, respectively); (2) there is no heterologous DNA fragment left in the genome at the site of excision of the selective marker cassette; (3) it is possible to modify the genome repeatedly for an unlimited number of times. In this report, we tested the procedure using deletions of genetic loci. The same procedure can also be used for point mutations, substitutions, additions and other modifications of the B. subtilis genome. The deletion strategy described in this method has the important advantage that, due to the introduction of the downstream sequence (DN) from the deletion target (Fig. 1), it is possible for intra-genomic homologous recombination to occur, enabling the excision of the selective marker cassette. Our unpublished data indicate that regions of up to 50 kb can be excised by intra-genomic homologous recombination in the presence of homologous DNA sequences of at least 0.5 kb, with efficiencies of around 10–4 recombinants per cell plated. This recombination rate is much higher (about three to four orders of magnitude) than that of transformation with PCR fragments. If the DN fragment is not introduced together with the selective marker cassette, one more step of transformation with a PCR fragment must be conducted, in order to excise the selective marker cassette and the deletion target. In this case, even if the selective marker cassette is present, the Nm-resistant colonies occur with frequencies of 10–7 to 10–8, similar to the transformation efficiencies of PCR fragments. It is apparent that the selection for excision of the selective marker cassette will be difficult after transformation using a PCR fragment containing only the UP and DN fragments. Moreover, the selection of unmarked deletion mutants is from LB cultures, in which the viable cell count will be over 108 cells ml–1 after an incubation of 4 h. This implies that there will be 104 unmarked deletion mutants per millilitre in this LB culture. Thus, our results indicate that the method described above can be used simply, easily and precisely for multiple rounds of deletion mutagenesis of the B. subtilis genome, without any heterologous sequences remaining at the sites of mutagenesis.
|
ACKNOWLEDGEMENTS |
|---|
Edited by: W. Quax
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
|---|
Bloor, A. E. & Cranenburgh, R. M. (2006). An efficient method of selectable marker gene excision by Xer recombination for gene replacement in bacterial chromosomes. Appl Environ Microbiol 72, 2520–2525.
Brans, A., Filee, P., Chevigne, A., Claessens, A. & Joris, B. (2004). New integrative method to generate Bacillus subtilis recombinant strains free of selection markers. Appl Environ Microbiol 70, 7241–7250.
Fabret, C., Ehrlich, S. D. & Noirot, P. (2002). A new mutation delivery system for genome-scale approaches in Bacillus subtilis. Mol Microbiol 46, 25–36.[CrossRef][Medline]
Guerout-Fleury, A. M., Shazand, K., Frandsen, N. & Stragier, P. (1995). Antibiotic-resistance cassettes for Bacillus subtilis. Gene 167, 335–336.[CrossRef][Medline]
Harwood, C. R. & Wipat, A. (1996). Sequencing and functional analysis of the genome of Bacillus subtilis strain 168. FEBS Lett 389, 84–87.[CrossRef][Medline]
Harwood, C. R., Coxon, R. D. & Hancock, I. C. (1990). The Bacillus cell envelope and secretion. In Molecular Biological Methods for Bacillus, pp. 327–390. Edited by C. R. Harwood & S. M. Cutting. New York: John Wiley & Sons.
Ho, S. N., Hunt, H. D., Horton, R. M., Pullen, J. K. & Pease, L. R. (1989). Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 77, 51–59.[CrossRef][Medline]
Horinouchi, S. & Weisblum, B. (1982). Nucleotide sequence and functional map of pC194, a plasmid that specifies inducible chloramphenicol resistance. J Bacteriol 150, 815–825.
Kobayashi, K., Ehrlich, S. D., Albertini, A., Amati, G., Andersen, K. K., Arnaud, M., Asai, K., Ashikaga, S., Aymerich, S. & other authors (2003). Essential Bacillus subtilis genes. Proc Natl Acad Sci U S A 100, 4678–4683.
Kunst, F., Ogasawara, N., Moszer, I., Albertini, A. M., Alloni, G., Azevedo, V., Bertero, M. G., Bessieres, P., Bolotin, A. & other authors (1997). The complete genome sequence of the Gram-positive bacterium Bacillus subtilis. Nature 390, 249–256.[CrossRef][Medline]
Leenhouts, K., Buist, G., Bolhuis, A., ten Berge, A., Kiel, J., Mierau, I., Dabrowska, M., Venema, G. & Kok, J. (1996). A general system for generating unlabelled gene replacements in bacterial chromosomes. Mol Gen Genet 253, 217–224.[CrossRef][Medline]
Liu, S., Endo, K., Ara, K., Ozaki, K. & Ogasawara, N. (2007). The accurate replacement of long genome region more than several hundreds kilobases in Bacillus subtilis. Genes Genet Syst 82, 9–19.[CrossRef][Medline]
Malaga, W., Perez, E. & Guilhot, C. (2003). Production of unmarked mutations in mycobacteria using site-specific recombination. FEMS Microbiol Lett 219, 261–268.[CrossRef][Medline]
McKenzie, T., Hoshino, T., Tanaka, T. & Sueoka, N. (1986). The nucleotide sequence of pUB110: some salient features in relation to replication and its regulation. Plasmid 15, 93–103.[CrossRef][Medline]
Mota, L. J., Tavares, P. & Sa-Nogueira, I. (1999). Mode of action of AraR, the key regulator of L-arabinose metabolism in Bacillus subtilis. Mol Microbiol 33, 476–489.[CrossRef][Medline]
Mota, L. J., Sarmento, L. M. & de Sa-Nogueira, I. (2001). Control of the arabinose regulon in Bacillus subtilis by AraR in vivo: crucial roles of operators, cooperativity, and DNA looping. J Bacteriol 183, 4190–4201.
Sa-Nogueira, I. & Mota, L. J. (1997). Negative regulation of L-arabinose metabolism in Bacillus subtilis: characterization of the araR (araC) gene. J Bacteriol 179, 1598–1608.
Sa-Nogueira, I. & Ramos, S. S. (1997). Cloning, functional analysis, and transcriptional regulation of the Bacillus subtilis araE gene involved in L-arabinose utilization. J Bacteriol 179, 7705–7711.
Sa-Nogueira, I., Nogueira, T. V., Soares, S. & de Lencastre, H. (1997). The Bacillus subtilis L-arabinose (ara) operon: nucleotide sequence, genetic organization and expression. Microbiology 143, 957–969.
White, A. P., Allen-Vercoe, E., Jones, B. W., DeVinney, R., Kay, W. W. & Surette, M. G. (2007). An efficient system for markerless gene replacement applicable in a wide variety of enterobacterial species. Can J Microbiol 53, 56–62.[CrossRef][Medline]
Zhang, X. Z., Yan, X., Cui, Z. L., Hong, Q. & Li, S. P. (2006). mazF, a novel counter-selectable marker for unmarked chromosomal manipulation in Bacillus subtilis. Nucleic Acids Res 34, e71
Received 22 January 2008;
revised 16 May 2008;
accepted 9 June 2008.
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
| J MED MICROBIOL | ALL SGM JOURNALS | |
