Abstract
Bifidobacteria are the main component of the human microflora. We constructed a temperature-sensitive (Ts) plasmid by random mutagenesis of the Bifidobacterium–Escherichia coli shuttle vector pKKT427 using error-prone PCR. Mutant plasmids were introduced into Bifidobacterium longum 105-A and, after screening approximately 3,000 colonies, candidate clones that grew at 30 °C but not at 42 °C were selected. According to DNA sequence analysis of the Ts plasmid, five silent and one missense mutations were found in the repB region. The site-directed mutagenesis showed only the missense mutation to be relevant to the Ts phenotype. We designated this plasmid pKO403. The Ts phenotype was also observed in B. longum NCC2705 and Bifidobacterium adolescentis ATCC15703. Single-crossover homologous-recombination experiments were carried out to determine the relationship between the length of homologous sequences encoded on the plasmid and recombination frequency: fragments greater than 1 kb gave an efficiency of more than 103 integrations per cell. We performed gene knockout experiments using this Ts plasmid. We obtained gene knockout mutants of the pyrE region of B. longum 105-A, and determined that double-crossover homologous recombination occurred at an efficiency of 1.8 %. This knockout method also worked for the BL0033 gene in B. longum NCC2705.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Biswas I, Gruss A, Ehrlich SD, Maguin E (1993) High-efficiency gene inactivation and replacement system for gram-positive bacteria. J Bacteriol 175:3628–3635
Chen XY, Dego OK, Almeida RA, Fuller TE, Luther DA, Oliver SP (2011) Deletion of sua gene reduces the ability of Streptococcus uberis to adhere to and internalize into bovine mammary epithelial cells. Vet Microbiol 147:426–434
Datsenko KA, Wanner BL (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci U S A 97:6640–6645
Florez AB, Ammor MS, Alvarez-Martin P, Margolles A, Mayo B (2006) Molecular analysis of tet (W) gene-mediated tetracycline resistance in dominant intestinal Bifidobacterium species from healthy humans. Appl Environ Microbiol 72:7377–7379
Fuchs TM, Klumpp J, Przybilla K (2006) Insertion–duplication mutagenesis of Salmonella enterica and related species using a novel thermosensitive vector. Plasmid 55:39–49
Fukuda S, Toh H, Hase K, Oshima K, Nakanishi Y, Yoshimura K, Tobe T, Clarke JM, Topping DL, Suzuki T, Taylor TD, Itoh K, Kikuchi J, Morita H, Hattori M, Ohno H (2011) Bifidobacteria can protect from enteropathogenic infection through production of acetate. Nature 469:543–547
Guzman LM, Belin D, Carson MJ, Beckwith J (1995) Tight regulation, modulation, and high-level expression by vectors containing the arabinose P-BAD promoter. J Bacteriol 177:4121–4130
Hamilton CM, Aldea M, Washburn BK, Babitzke P, Kushner SR (1989) New method for generating deletions and gene replacements in Escherichia coli. J Bacteriol 171:4617–4622
Hirashima K, Iwaki T, Takegawa K, Giga-Hama Y, Tohda H (2006) A simple and effective chromosome modification method for large-scale deletion of genome sequences and identification of essential genes in fission yeast. Nucleic Acids Res 34:e11
Horton RM, Hunt HD, Ho SN, Pullen JK, Pease LR (1989) Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. Gene 77:61–68
Humphreys GO, Willshaw GA, Smith HR, Anderson ES (1976) Mutagenesis of plasmid DNA with hydroxylamine: isolation of mutants of multi-copy plasmids. Mol Gen Genet 145:101–108
Lee JH, Karamychev VN, Kozyavkin SA, Mills D, Pavlov AR, Pavlova NV, Polouchine NN, Richardson PM, Shakhova VV, Slesarev AI, Weimer B, O’Sullivan DJ (2008) Comparative genomic analysis of the gut bacterium Bifidobacterium longum reveals loci susceptible to deletion during pure culture growth. BMC Genomics 9. doi:10.1186/1471-2164-9-247
Maguin E, Duwat P, Hege T, Ehrlich D, Gruss A (1992) New thermosensitive plasmid for gram-positive bacteria. J Bacteriol 174:5633–5638
Matsumura H, Takeuchi A, Kano Y (1997) Construction of Escherichia coli Bifidobacterium longum shuttle vector transforming B. longum 105-A and 108-A. Biosci Biotechnol Biochem 61:1211–1212
Missich R, Sgorbati B, Leblanc DJ (1994) Transformation of Bifidobacterium longum with pRM2, a constructed Escherichia-coli B. longum shuttle vector. Plasmid 32:208–211
Mitsuoka T (1990) Bifidobacteria and their role in human health. J Ind Microbiol 6:263–267
Nakamura J, Kanno S, Kimura E, Matsui K, Nakamatsu T, Wachi M (2006) Temperature-sensitive cloning vector for Corynebacterium glutamicum. Plasmid 56:179–186
Nallapareddy SR, Singh KV, Murray BE (2006) Construction of improved temperature-sensitive and mobilizable vectors and their use for constructing mutations in the adhesin-encoding acm gene of poorly transformable clinical Enterococcus faecium strains. Appl Environ Microbiol 72:334–345
Rossi M, Brigidi P, Rodriguez A, Matteuzzi D (1996) Characterization of the plasmid pMB1 from Bifidobacterium longum and its use for shuttle vector construction. Res Microbiol 147:133–143
Sakaguchi K, He J, Funaoka N, Tani S, Hobo A, Mitsunaga T, Kano Y, Suzuki T (2012) The pyrE gene as a bidirectional selection marker for Bifidobacterium longum 105-A. Appl Environ Microbiol (Submitted: AEM07703-11)
Salminen S, Ouwehand A, Benno Y, Lee YK (1999) Probiotics: how should they be defined? Trends Food Sci Technol 10:107–110
Sanchez H, Cozar AC, Martinez-Jimenez MI (2007) Targeting the Bacillus subtilis genome: an efficient and clean method for gene disruption. J Microbiol Methods 70:389–394
Schell MA, Karmirantzou M, Snel B, Vilanova D, Berger B, Pessi G, Zwahlen MC, Desiere F, Bork P, Delley M, Pridmore RD, Arigoni F (2002) The genome sequence of Bifidobacterium longum reflects its adaptation to the human gastrointestinal tract. Proc Natl Acad Sci U S A 99:14422–14427
Suzuki T, Yasui K (2011) Plasmid artificial modification. In: Williams JA (ed) Strain engineering-methods and protocols. Springer, New York, pp 309–326
Suzuki T, Itoh A, Ichihara S, Mizushima S (1987) Characterization of the sppA gene coding for protease IV, a signal peptide peptidase of Escherichia coli. J Bacteriol 169:2523–2528
Takamatsu D, Osaki M, Sekizaki T (2001) Thermosensitive suicide vectors for gene replacement in Streptococcus suis. Plasmid 46:140–148
Yakhnin H, Babitzke P (2004) Gene replacement method for determining conditions in which Bacillus subtilis genes are essential or dispensable for cell viability. Appl Microbiol Biotechnol 64:382–386
Yasui K, Kano Y, Tanaka K, Watanabe K, Shimizu-Kadota M, Yoshikawa H, Suzuki T (2009a) Improvement of bacterial transformation efficiency using plasmid artificial modification. Nucleic Acids Res 37:e3
Yasui K, Tabata M, Yamada S, Abe T, Ikemura T, Osawa R, Suzuki T (2009b) Intra-species diversity between seven Bifidobacterium adolescentis strains identified by genome-wide tiling array analysis. Biosci Biotechnol Biochem 73:1422–1424
Zakataeva NP, Nikitina OV, Gronskiy SV, Romanenkov DV, Livshits VA (2010) A simple method to introduce marker-free genetic modifications into the chromosome of naturally nontransformable Bacillus amyloliquefaciens strains. Appl Microbiol Biotechnol 85:1201–1209
Funding
This research was partially supported by a Grant-in-Aid for Scientific Research (C), 20510189, from the Ministry of Education, Culture, Sports, Science and Technology of Japan, the Iijima Memorial Foundation for the Promotion of Food Science and Technology (2011), and C19 Kiyomi Yoshizaki research grant (2011).
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Sakaguchi, K., He, J., Tani, S. et al. A targeted gene knockout method using a newly constructed temperature-sensitive plasmid mediated homologous recombination in Bifidobacterium longum . Appl Microbiol Biotechnol 95, 499–509 (2012). https://doi.org/10.1007/s00253-012-4090-4
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00253-012-4090-4