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Microbiology 144 (1998), 2525-2530; DOI  10.1099/00221287-144-9-2525
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An in silico evaluation of Tn916 as a tool for generalized mutagenesis in Haemophilus influenzae Rd

Sarah L. Hosking1, Mary E. Deadman2, E. Richard Moxon2, John F. Peden3, Nigel J. Saunders2 and Nicola J. High1,*

University of Manchester, School of Biological Sciences, 1.800, Stopford Building, Oxford Road, Manchester M13 9PT, UK
Molecular Infectious Diseases Group, University of Oxford, Institute of Molecular Medicine, John Radcliffe Hospital, Headington, Oxford OX3 9DS, UK
Molecular Biology Data Centre, IMSU, University of Oxford, Headington, Oxford, UK

ABSTRACT

The transposon Tn916 was evaluated as a tool for generalized mutagenesis of the genome of Haemophilus influenzae. This was achieved in silico by searching the genome sequence of H. influenzae Rd for the published Tn916 target site consensus sequence 5' TT/ATTTT(N)6AAAAAA/TA. This search identified 16 putative target sites. In subsequent experiments, integration of Tn916 did not occur at any of these sites. Using the nucleotide sequences of these observed integration sites, a new consensus sequence, 5' TTTTT(N)xAAAAA (4x7), was derived. This sequence reflects the curve-twist-curve DNA topology which is a feature common to all Tn916 integration sites. A search of the H. influenzae Rd genome using the new consensus sequence identified 167 potential target sites, representing approximately 1% of the total genome. Only 80 of these sites were located within ORFs. The presence of such a limited number of target sites places severe constraints on the use of Tn916 as a tool for generalized mutagenesis of the genome of H. influenzae.

*Author for correspondence: Nicola J. High. Tel: +44 161 275 5749. Fax: +44 161 275 5656.

Keywords: Haemophilus influenzae Rd, Tn916 mutagenesis, genome sequence






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