Abstract
Selective markers employed in classical mutagenesis methods using natural genetic transformation can affect gene expression, risk phenotypic effects, and accumulate as unwanted genes during successive mutagenesis cycles. In this chapter, we present a protocol for markerless genome editing in Streptococcus mutans and Streptococcus pneumoniae achieved with an efficient method for natural transformation. High yields of transformants are obtained by combining the unimodal state of competence developed after treatment of S. mutans with sigX-inducing peptide pheromone (XIP) in a chemically defined medium (CDM) or of S. pneumoniae with the competence-stimulating peptide (CSP) together with use of a donor amplicon carrying extensive flanking homology. This combination ensures efficient and precise integration of a new allele by the recombination machinery present in competent cells.
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References
Khan R, Rukke HV, Ricomini AP, Fimland G, Arntzen MO, Thiede B, Petersen FC (2012) Extracellular identification of a processed type II ComR/ComS pheromone of Streptococcus mutans. J Bacteriol 194:3781–3788
Mashburn-Warren L, Morrison DA, Federle MJ (2010) A novel double-tryptophan peptide pheromone controls competence in Streptococcus spp. via an Rgg regulator. Mol Microbiol 78:589–606
Reck M, Tomasch J, Wagner-Dobler I (2015) The alternative sigma factor SigX controls bacteriocin synthesis and competence, the two quorum sensing regulated traits in Streptococcus mutans. PLoS Genet 11, e1005353
Son M, Ghoreishi D, Ahn SJ, Burne RA, Hagen SJ (2015) Sharply tuned pH response of genetic competence regulation in Streptococcus mutans: a microfluidic study of the environmental sensitivity of comX. Appl Environ Microbiol 81:5622–5631
Son MJ, Ahn SJ, Guo Q, Burne RA, Hagen SJ (2012) Microfluidic study of competence regulation in Streptococcus mutans: environmental inputs modulate bimodal and unimodal expression of comX. Mol Microbiol 86:258–272
Chang JC, LaSarre B, Jimenez JC, Aggarwal C, Federle MJ (2011) Two group A streptococcal peptide pheromones act through opposing Rgg regulators to control biofilm development. PLoS Pathog 7, e1002190
Desai K, Mashburn-Warren L, Federle MJ, Morrison DA (2012) Development of competence for genetic transformation of Streptococcus mutans in a chemically defined medium. J Bacteriol 194:3774–3780
Johnston C, Campo N, Berge MJ, Polard P, Claverys JP (2014) Streptococcus pneumoniae, le transformiste. Trends Microbiol 22:113–119
Cato A Jr, Guild WR (1968) Transformation and DNA size: I. Activity of fragments of defined size and a fit to a random double cross-over model. J Mol Biol 37:157–178
Morrison DA, Guild WR (1972) Transformation and deoxyribonucleic acid size: extent of degradation on entry varies with size of donor. J Bacteriol 112:1157–1168
Morrison DA, Khan R, Junges R, Amdal HA, Petersen FC (2015) Genome editing by natural genetic transformation in Streptococcus mutans. J Microbiol Methods 119:134–141
Tovpeko Y, Morrison DA (2014) Competence for genetic transformation in Streptococcus pneumoniae: mutations in sigmaA bypass the comW requirement. J Bacteriol 196:3724–3734
Szewczyk E, Nayak T, Oakley CE, Edgerton H, Xiong Y, Taheri-Talesh N, Osmani SA, Oakley BR (2006) Fusion PCR and gene targeting in Aspergillus nidulans. Nat Protoc 1:3111–3120
Sharp PM, Bailes E, Grocock RJ, Peden JF, Sockett RE (2005) Variation in the strength of selected codon usage bias among bacteria. Nucleic Acids Res 33:1141–1153
Korbie DJ, Mattick JS (2008) Touchdown PCR for increased specificity and sensitivity in PCR amplification. Nat Protoc 3:1452–1456
Acknowledgments
This work was partially supported by the National Science Foundation, grant no. MCB-1020863, by the Faculty of Dentistry, University of Oslo, and by the Norwegian surveillance system for antibiotic resistance in microbes (Norsk overvåkingssystem for antibiotikaresistens hos mikrober—NORM). We thank Kunal Desai for assistance with exploratory experiments.
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Junges, R., Khan, R., Tovpeko, Y., Ã…mdal, H.A., Petersen, F.C., Morrison, D.A. (2017). Markerless Genome Editing in Competent Streptococci. In: Seymour, G., Cullinan, M., Heng, N. (eds) Oral Biology. Methods in Molecular Biology, vol 1537. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6685-1_14
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DOI: https://doi.org/10.1007/978-1-4939-6685-1_14
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