CHARACTERIZATION AND HETEROLOGOUS EXPRESSION OF THE TETL GENE AND IDENTIFICATION OF ISO-ISS1 ELEMENTS FROM ENTEROCOCCUS-FAECALIS PLASMID PJH1

The tetracycline-resistance (TCR) determinant of the Enter ococcus fae calis plasmid pJH1 has been identified and located on a 2.2-kb RsaI-Ec oRI fragment, The fragment was cloned in Escherichia coli, and specifi ed TCR in this host. The nucleotide (nt) sequence of the cloned fragme nt showed the presence of an open reading frame (ORF) of 1374 bp, desi gnated tetL. The nt sequence of tetL from pJH1 was identical to that o f the tetL present on pLS1 from Streptococcus agalactiae. Upstream of the pJH1 tetL, part of another ORF was found that, except for two sing le-nt substitutions, was identical to an iso-ISS1 element from Lactoco ccus lactis. Hybridization studies indicated the presence of several I SS1-like elements in plasmid pJH1, but not on the En. faecalis chromos ome. To study its usefulness as a marker in Gram(+) organisms, the pJH 1 tetL was cloned on the broad-host-range plasmid pNZ124, resulting in pNZ280, that was found to give resistance to 40 mu g Tc/ml in L c. la ctis and Bacillus subtilis.