"tet(U)" is not a tetracycline resistance determinant

Abstract

The enterococcal plasmid pKQ10 has been reported to carry a poorly characterized tetracycline resistance determinant designated tet(U). However, in a series of studies intended to further characterize this determinant, we have been unable to substantiate the claim that tet(U) confers resistance to tetracyclines. In line with these results, bioinformatic analysis provides compelling evidence that "tet(U)" is in fact the misannotated 3' end of a gene encoding a rolling-circle replication initiator (Rep) protein.

Fig 1

(A) Plasmid map of pKQ10, with annotation of open reading frames (ORFs) according to Ridenhour et al. (4). Based on the analysis presented in this study, we propose that both ORFs in fact form part of a larger, single ORF encoding a Rep protein (see below). (B) Alignment of a frameshift-corrected pKQ10 Rep sequence (pKQ10_ fs-rep) with related Rep proteins. Addition of a single nucleotide in the stop codon of ORF2 in conjunction with a single nucleotide substitution upstream of ORF1 in pKQ10 restores a contiguous gene that when translated is recognized as part of the Rep_trans superfamily (Pfam PF02486). The arrows above the alignment correspond to the ORFs indicated in panel A. The consensus sequence of the prototype pT181 RepC protein active site is indicated in boldface, with the conserved catalytic tyrosine underlined (3). Additional conserved residues important for Rep nicking activity are highlighted in gray (8). Values in square brackets indicate percentage amino acid identity over the region aligned with pKQ10 Rep.