Mutagenesis of Streptococcus equi and Streptococcus suis by transposon Tn917.
Genetic tools for studying streptococci are much less sophisticated than those that are available for many other bacterial genera. In this paper, we describe the development of a transposon mutagenesis system that we have used to mutate two important veterinary streptococci, Streptococcus equi and Streptococcus suis. The system uses a temperature-sensitive suicide vector to deliver Tn917 via electroporation, transposing Tn917 into the chromosomal DNA of the two streptococci. The transposon insertions can be rescued from the streptococcal chromosomes by plasmid rescue and selection in E. coli, with subsequent insertion site analysis by DNA sequencing. Transposition appeared to have occurred in an essentially random fashion when chromosomal DNA of S. suis and S. equi mutants was analysed by Southern blotting. However, when analysis of 60 S. equi mutants was carried out using the S. equi genome sequence database, 60% of transposon insertions had occurred within a 15 kb region of the genome whereas the other insertions appeared to have occurred essentially randomly. This finding suggests that Southern blot analysis for assessing the randomness of transposon libraries may need to be interpreted with caution. However, this observation notwithstanding, the Tn917 based system described in this paper will facilitate the study of S. suis and S. equi.
Slater
J D
JD
Department of Clinical Veterinary Medicine, Centre for Veterinary Science, University of Cambridge, Madingley Road, Cambridge CB3 0ES, UK.
Allen
A G
AG
May
J P
JP
Bolitho
S
S
Lindsay
H
H
Maskell
D J
DJ
D000818:Animals; D015139:Blotting, Southern / Q000662:veterinary; D004251:DNA Transposable Elements / Q000235:genetics*; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D018274:Electroporation; D005822:Genetic Vectors / Q000737:chemistry / Q000235:genetics; D006736:Horses; D016254:Mutagenesis, Insertional / Q000379:methods; D010957:Plasmids; D017422:Sequence Analysis, DNA; D018502:Streptococcus equi / Q000737:chemistry / Q000235:genetics*; D017011:Streptococcus suis / Q000737:chemistry / Q000235:genetics*; D013552:Swine
D004251:DNA Transposable Elements; D004269:DNA, Bacterial