A DNA sequence upstream of the tet(O) gene is required for full expression of tetracycline resistance

Abstract

The DNA sequences upstream of the tet(O) and tet(M) open reading frames (ORFs) (ca. 300 bp) were found to share a higher degree of homology than those of the tet(O) and tet(M) ORFs themselves. A transcription initiation site for tel(O) was located by primer extension analysis. Campylobacter coli was found to use a promoter sequence different from that used by Escherichia coli. The sequence upstream of tet(O) was shown to be required in cis for high-level resistance to tetracycline.The class 0 tetracycline (TC) resistance determinant [tet(O)] has been identified in Campylobacter spp. (18,20), Streptococcus mutans (9), and Enterococcus faecium (1). The determinant has been cloned and expressed in Escherichia coli (9,18,20). The deduced amino acid sequence of Tet(O) is very similar to that of Tet(M) (76% identity [13]), which has been found in a wide variety of gram-positive and gram-negative bacteria (21).The sequences of several tet(O) and tet(M) genes have been determined (3,9,11,13,14,16,18); however, little is known about the resistance mechanism or the control of expression of these genes. We believe that genetic studies of both tet(O) and tet(M) are necessary to understand the biochemical basis of Tet(O) and Tet(M) action as well as the reason for the ability of these resistance determinants to be expressed in a wide variety of bacteria. Previous DNA hybridization experiments with the tet(O) and tet(M) determinants suggested that DNA sequences flanking the tet(O) and tet(M) open reading frames (ORFs) share a higher degree of homology than the protein-coding sequences themselves (13,20). By comparing the sequencing data of the tet(O) and tet(M) genes (11, 13) with the results of Southern hybridization (20), the homologous sequences were located at the 5' end of the tet(O) ORF. However, attempts to delete this sequence by using nuclease Bal 31 or exonuclease III failed when transformants of Tcr phenotype were selected (26). These results suggested that the Tet(O) protein specified by the tet(O) ORF may not be the only factor required for resistance to TC.In this study, we determined the nucleotide sequences upstream of tet(O) and tet(M). They were found to be highly homologous, and their possible function was investigated. The transcription start point for tet(O) was also located. MATERIALS AND METHODSStrains and culture conditions. E. coli JM107 (23) and HB101 (12) were routinely used as plasmid hosts, and E. coli BW313 (8) was used for site-directed mutagenesis. Campylobacter coli UA585 (27) and Campylobacterjejuni UA466 (20) were also employed. The plasmids and phages used in this study were pUC13 (23), pUC118 (24), M13mp19 (23), M13K07 (24), pACYC177, pACYC184 (5), pT7-5 (19), pUOA2 (21; Fig. 1), pUOA11 (27, Fig. 1 In vitro-transcribed RNA was also used as a template for primer extension. The 4.4-kb EcoRV-PstI fragment of pUOA2A ( Fig. 1) was inserted into pT7-5 between the SmaI and PstI sites and subsequently named pT7-UOA2A. The tet(O) mRNA was transcribed from this plasmid by the T7 RNA polymerase (Bo...

Tetracycline Antibiotics: Mode of Action, Applications, Molecular Biology, and Epidemiology of Bacterial Resistance

“…This is consistent with the hypothesis that the tet(M) genes have come from gram-positive bacteria and illustrates the spread of gram-positive genes into gram-negative species (227). This information, along with that of Wang and Taylor (306), is consistent with the hypothesis that tet(O) and tet(S) genes may also be regulated. However, the regulation of tet(Q) expression is unclear since most of the work has been done to determine the regulation of self-transfer and mobilization of both coresident plasmids and unlinked integrated elements, rather than expression of the Tet(Q) protein (152,256,257).…”

“…The expression of both Tet(M) and Tet(O) proteins appears to be regulated. Wang and Taylor (306) have suggested that the 400-bp region directly upstream from the coding region of the tet(O) gene was needed for full expression of the gene; however, the function of the region is not understood (292). Burdett (29) reported that the amount of Tet(M) protein increased when streptococci carrying the determinant were exposed to tetracycline.…”

Incidence of Antibiotic Resistance inCampylobacter jejuniIsolated in Alberta, Canada, from 1999 to 2002, with Special Reference totet(O)-Mediated Tetracycline Resistance

“…The upstream region of tet(O) is known to be important for full expression of Tc r (57). To determine if the upstream region of the tet(O) gene played a role in mediating high-level Tc r , the tet(O) gene was cloned from C. jejuni clinical isolate 25-01 (tetracycline MIC ϭ 512 g/ml) incorporating increasing portions of its upstream region into the shuttle vector pRY107 (Fig.…”

The Butyrivibrio fibrisolvens tet (W) Gene Is Carried on the Novel Conjugative Transposon Tn B1230 , Which Contains Duplicated Nitroreductase Coding Sequences

“…This organization is characteristic of attenuation terminator sequences (http://www.bork.embl-heidelberg .de/Docu/attenuation/index.html). Similar inverted repeat sequences are located upstream of the ribosome protection genes (24) and tet(M) (21). The expression of tet(M) depends on the synthesis of a 28-amino-acid leader peptide encoded immediately upstream of the tet(M) ribosome binding site (21).…”