Development of an in vivo Himar1 transposon mutagenesis system for use in Streptococcus equi subsp. equi.
Abstract: Streptococcus equi subsp. equi is the causative agent of the equine disease strangles. In this study we describe the development of an in vivo Himar1 transposon system for the random mutagenesis of S. equi and, potentially, other Gram-positive bacteria. We demonstrate efficient and random transposition of a modified mini-transposon onto the chromosome by Southern blot analysis and insertion site sequencing. Non-haemolytic mutants were isolated at a frequency of 0.2%, and acapsular mutants at a frequency of 0.04%. Taken together, these data demonstrate that in vivo Himar1 mutagenesis can be used for genomic-scale mutational analysis of S. equi, and is likely to be applicable to the study of other streptococci.
Publication Date: 2004-09-11 PubMed ID: 15358426DOI: 10.1016/j.femsle.2004.08.003Google Scholar: Lookup
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- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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The researchers developed a new genetic manipulation method using a tool called an in vivo Himar1 transposon to cause random mutations in Streptococcus equi subsp. equi, a bacterium that causes strangles in horses.
Development of the Himar1 Transposon System
- The researchers focused their efforts on determining the viability of an in vivo Himar1 transposon system. This system was used for random mutagenesis, meaning it was designed to inflict random genetic changes in the bacterium Streptococcus equi subsp. equi.
- Streptococcus equi subsp. equi is a Gram-positive bacterium that is responsible for a disease commonly found in horses known as strangles. The development of a system that allows for this organism’s mutagenesis could potentially provide valuable insights into how the disease operates and evolves within its host. The main goal of this research was to attempt to develop such a system, using a technique known as transposon mutagenesis.
Evidence of Efficient and Random Transposition
- The researchers tested the efficiency of the transposition process (how well the template DNA strand is transferred to the bacterium’s DNA) and whether it was truly random. This was achieved by verifying the transposition through Southern blot analysis and insertion site sequencing.
- The results showed that the system managed to successfully transpose a modified mini-transposon onto the chromosome of the bacterium. This suggests that the in vivo Himar1 system can be used effectively for transposon mutagenesis in this bacterium, leading to random genetic changes.
Isolation of Mutants and Potential Applications
- The researchers were able to isolate specific mutants after the mutagenesis process, specifically non-haemolytic mutants, which were found at a frequency of 0.2%, and acapsular mutants, found at a frequency of 0.04%. This numerical information indicates the effectiveness of the in vivo Himar1 system for creating specific, targeted mutations in these bacteria.
- The study concludes that in vivo Himar1 mutagenesis can be harnessed for genomic-scale mutational analysis of S. equi. Furthermore, the research suggests potential applicability of this method for studying genetic changes on a larger scale in other streptococci, opening the door to future research in other related bacteria.
Cite This Article
APA
May JP, Walker CA, Maskell DJ, Slater JD.
(2004).
Development of an in vivo Himar1 transposon mutagenesis system for use in Streptococcus equi subsp. equi.
FEMS Microbiol Lett, 238(2), 401-409.
https://doi.org/10.1016/j.femsle.2004.08.003 Publication
Researcher Affiliations
- Department of Clinical Veterinary Medicine, Centre for Veterinary Science, University of Cambridge, Madingley Road, Cambridge CB3 0ES, UK.
MeSH Terms
- DNA Transposable Elements / genetics
- DNA, Bacterial / chemistry
- DNA, Bacterial / genetics
- Electroporation
- Genetic Vectors / chemistry
- Genetic Vectors / genetics
- Mutagenesis, Insertional / methods
- Plasmids
- Streptococcus equi / chemistry
- Streptococcus equi / genetics
- Streptococcus suis / chemistry
- Streptococcus suis / genetics
Citations
This article has been cited 10 times.- Bossé JT, Li Y, Leanse LG, Zhou L, Chaudhuri RR, Peters SE, Wang J, Maglennon GA, Holden MTG, Maskell DJ, Tucker AW, Wren BW, Rycroft AN, Langford PR. Rationally designed mariner vectors for functional genomic analysis of Actinobacillus pleuropneumoniae and other Pasteurellaceae species by transposon-directed insertion-site sequencing (TraDIS).. Anim Dis 2021;1(1):29.
- Ramos Y, Rocha J, Hael AL, van Gestel J, Vlamakis H, Cywes-Bentley C, Cubillos-Ruiz JR, Pier GB, Gilmore MS, Kolter R, Morales DK. PolyGlcNAc-containing exopolymers enable surface penetration by non-motile Enterococcus faecalis.. PLoS Pathog 2019 Feb;15(2):e1007571.
- Neamat-Allah AN, Damaty HM. Strangles in Arabian horses in Egypt: Clinical, epidemiological, hematological, and biochemical aspects.. Vet World 2016 Aug;9(8):820-6.
- Liu R, Zhang P, Su Y, Lin H, Zhang H, Yu L, Ma Z, Fan H. A novel suicide shuttle plasmid for Streptococcus suis serotype 2 and Streptococcus equi ssp. zooepidemicus gene mutation.. Sci Rep 2016 Jun 3;6:27133.
- Hooven TA, Catomeris AJ, Akabas LH, Randis TM, Maskell DJ, Peters SE, Ott S, Santana-Cruz I, Tallon LJ, Tettelin H, Ratner AJ. The essential genome of Streptococcus agalactiae.. BMC Genomics 2016 May 26;17:406.
- Varahan S, Harms N, Gilmore MS, Tomich JM, Hancock LE. An ABC transporter is required for secretion of peptide sex pheromones in Enterococcus faecalis.. mBio 2014 Sep 23;5(5):e01726-14.
- Picardeau M. Transposition of fly mariner elements into bacteria as a genetic tool for mutagenesis.. Genetica 2010 May;138(5):551-8.
- Brochet M, Rusniok C, Couvé E, Dramsi S, Poyart C, Trieu-Cuot P, Kunst F, Glaser P. Shaping a bacterial genome by large chromosomal replacements, the evolutionary history of Streptococcus agalactiae.. Proc Natl Acad Sci U S A 2008 Oct 14;105(41):15961-6.
- Forquin MP, Tazi A, Rosa-Fraile M, Poyart C, Trieu-Cuot P, Dramsi S. The putative glycosyltransferase-encoding gene cylJ and the group B Streptococcus (GBS)-specific gene cylK modulate hemolysin production and virulence of GBS.. Infect Immun 2007 Apr;75(4):2063-6.
- Felsheim RF, Herron MJ, Nelson CM, Burkhardt NY, Barbet AF, Kurtti TJ, Munderloh UG. Transformation of Anaplasma phagocytophilum.. BMC Biotechnol 2006 Oct 31;6:42.
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