Transposon Tn10-like tetracycline resistance determinants in Haemophilus parainfluenzae

Abstract

Tetracycline resistance (Tcr) determinants from three different strains of Haemophilus parainfluenzae expressed 10-fold higher levels of resistance when mated into Escherichia coli. No plasmid was found in any of the E. coli recipients, even in matings in which a plasmid was identified in the donor Haemophilus sp. The Tcr determinant from Haemophilus sp. caused instability of resident plasmids in the recipient E. coli: all plasmids were lost within 30 generations in antibiotic-free media. However, by serial subculture in antibiotics, stable resident plasmids were obtained which carried the Tcr determinant from Haemophilus sp. and were transferable by conjugation and transformation among E. coli strains. All Haemophilus determinants hybridized with a probe for the Tcr determinant on Tn10, which bears inducible Tcr. However, Haemophilus determinants were constitutively resistant to tetracycline in the Haemophilus donors and in the E. coli recipients. This constitutive expression was recessive to wild-type Tn10 in the same cell, indicating that the constitutive phenotype resulted from the absence of an active repressor. Restrictive enzyme analysis of various E. coli plasmid derivatives bearing a Tcr determinant from Haemophilus sp. demonstrated that the inserted DNA was of similar size (8.95 to 9.35 kilobases), close to that of Tn10. Heteroduplex analysis and DNA:DNA hybridization confirmed that the Tcr determinant from Haemophilus sp. had greater than 90% homology with the Tn10 determinant, including the DNA sequence for the repressor.