Tn916 transposition in Haemophilus influenzae Rd: preferential insertion into noncoding DNA.
The availability of completely sequenced genomes has created an opportunity for high throughput mutational studies. Using the conjugative transposon Tn916, a pilot project was initiated to determine the efficiency of gene disruption in the first completely sequenced bacterium, Haemophilus influenzae Rd strain KW20. DNA was isolated from Tn916-mutagenized cells, and the point of transposon insertion was determined by inverse PCR, DNA sequencing, and mapping to the wild-type genome sequence. Analysis of the insertion sites at the nucleotide level demonstrated a biased pattern of insertion into regions rich in stretches of A's and T's. Although Tn916 integrated at multiple dispersed positions throughout the chromosome, 9 of 10 insertion events occurred in noncoding, intergenic DNA. It was determined that the intergenic DNA was over 5% more A + T-rich than that of protein coding sequences. This suggests that A + T-rich sequences similar to the Tn916 insertion site would be more likely to reside in the intergenic DNA. In an effort to identify other likely sites for transposon integration, a hidden Markov model of the consensus target insertion site was derived from the Tn916-H. influenzae junction fragments and searched against the entire genome. Eighty percent of the 30 highest-scoring predicted Tn916 target sites were from intergenic, nonprotein-coding regions of the genome. These data support the hypothesis that Tn916 has a marked preference for insertion into noncoding DNA for H. influenzae, suggesting that this mobile element has evolved to minimize disruption of host cell function on integration.
Nelson
K E
KE
Institute for Genomic Research, Rockville, Maryland, USA.
Richardson
D L
DL
Dougherty
B A
BA
D001483:Base Sequence; D004251:DNA Transposable Elements / Q000235:genetics*; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics*; D006193:Haemophilus influenzae / Q000235:genetics*; D008390:Markov Chains; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010865:Pilot Projects; D016415:Sequence Alignment
D004251:DNA Transposable Elements; D004269:DNA, Bacterial