Sequence-specific DNA uptake in Haemophilus transformation.

Abstract

Haemophilus cells efficiently take up Haemophilus DNA from the medium during transformation but do not take up other DNA. To study the mechanism of this specificity an 8.1-kilobase (kb) fragment of H. parainfluenzae DNA was cloned in the Escherichia coli-pBR322 host-vector system and reisolated the DNA fragment for use as a defined probe. The 5''-32P end-labeled 8.1-kb DNA is efficiently absorbed by competent Haemophilus cells whereas vector DNA present in the mixture is not, implying that the 8.1-kb DNA contains sequence-specific recognition sites that are needed for DNA uptake. Absorbed DNA can be recovered from cells as a 32P-labeled duplex of unaltered size for several minutes after uptake. The number and location of uptake sites in the 8.1-kb DNA were determined by constructing a restriction endonuclease cleavage map and assaying fragments for uptake. Only 2 small fragments retain the ability to be absorbed. These fragments, 120 and 140 base pairs long, are 3.8 kb apart on the 8.1-kb fragment. Each of these fragments contains a short common sequence, perhaps 8-12 base pairs long, that is the actual recognition site. DNA competition assays, with the 8.1-kb DNA as a standard, showed that about 600 copies of the uptake sites are present in the Haemophilus genome.