The nucleotide sequence of the tetracycline resistance determinant tetM from Ureaplasma urealyticum

136

129

The nucleotide sequence of the tetracycline resistance determinant tet(M), located on conjugative transposon Tn916 ofEnterococcusfaecalis, was determined and found to encode a 72,486-dalton protein exhibiting a high degree of homology with other tet(M) determinants. A short open reading frame corresponding to a 28-amino-acid peptide and containing a number of inverted repeat sequences was noted immediately upstream of tet(M), suggesting that regulation might occur by a mechanism involving transcriptional attenuation. Transcription analyses found this to indeed be the case, showing that the expression oftet(M) resulted from an extension of a small transcript representing the upstream leader region into the resistance determinant. Exposure of cells to tetracycline resulted in a significant increase in the amount of tet(M) transcription; this increase could be explained on the basis of increased transcriptional read-through from the upstream transcript. A model suggesting how transcriptional attenuation might operate in this system is presented.Tn916 (16.4 kb) is the prototype of a large family of conjugative transposons commonly found in enterococci and streptococci (7,9). These elements exhibit a broad host range, and essentially all of them carry a tetracycline resistance determinant of the tet(M) variety, as originally designated by Burdett et al. (6). The tet(M) gene product has been found to associate with ribosomes and mediate resistance at the level of protein synthesis (3, 5). Burdett recently purified the protein (5) and found it to consist of a single polypeptide with a molecular weight of approximately 68,000. Interestingly, it was found to have a ribosome-dependent GTPase activity. Tet(M) was also reported to be present in increased amounts when cells were grown in the presence of tetracycline (3).In this report, we present the results of a nucleotide sequence analysis of tet(M) and flanking DNA and, in addition, provide evidence that the regulation of expression occurs at the level of transcription. After we had completed the sequence analysis, Burdett (4) reported the results of an independent investigation of the same sequence. Although our data are in good agreement (a few minor differences), we wish to point out here features of the sequence that we believe are related to gene expression. Aspects of the structure of the region immediately upstream of tet(M), along with our mRNA analyses, support the view that the regulation of Tet(M) biosynthesis involves transcriptional attenuation. A plausible model pointing out the mechanism by which this regulation may occur is presented. MATERIALS AND METHODSBacterial strains and plasmids. Table 1 lists the bacterial strains and plasmids used in this study. Figure 1 illustrates the construction of pBAfo, pAM1005, pAM1006, and pAM1008. Plasmid pAM620 is a derivative of pACYC184 * Corresponding author.

“…The nucleotide sequence reported here for tet(M) exhibits significant homology with other tet(M) determinants (25,27,33) as well as tet(O) determinants (21,23). Nesin et al (27) (33).…”

Section: Discussionmentioning

confidence: 60%

Characterization of the tet(M) determinant of Tn916: evidence for regulation by transcription attenuation

Clewell

1992

136

129

“…These sequences also exhibit significant homology with the sequence upstream of tet(O) from C. coli (18) and with the sequences upstream of tet(M) from Tn916 (4), TnJS45 (13), Staphylococcus aureus (14), and Ureaplasma urealycum (16 Another potential promoter sequence (P2) was identified at 281 nucleotides upstream of the ATG start codon, of which the -35 and -10 sites are 100% identical to the E. coli consensus sequences. Transcription from P2 was demonstrated in E. coli but not in C. coli by primer extension experiments (Fig.…”

Section: Discussionmentioning

confidence: 90%

“…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.…”

mentioning

confidence: 99%

“…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.…”

mentioning

confidence: 99%

See 1 more Smart Citation

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

Wang

Taylor

1991

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...

“…Recently, a 2.7-kb SstI fragment carrying a tetracycline resistance (Tcr) gene was cloned from the Bacteroides thetaiotaomicron clinical isolate DOT (38,44). The cloned gene appeared to be the only Tcr gene in the strain of origin because a gene disruption made by using the 0.9-kb EcoRI-EcoRV fragment, which is shown in the present report to be internal to the Tcr gene, made the strain susceptible to tetracycline (44).…”

mentioning

confidence: 71%

A Bacteroides tetracycline resistance gene represents a new class of ribosome protection tetracycline resistance

Nikolich

Shoemaker

Salyers

1992

105

The ribosome protection type of tetracycline resistance (Tcr) has been found in a variety of bacterial species, but the only two classes described previously, Tet(M) and Tet(O), shared a high degree of amino acid sequence identity (greater than 75%). Thus, it appeared that this type of resistance emerged recently in evolution and spread among different species of bacteria by horizontal transmission. We obtained the DNA sequence of a Tcr gene from Bacteroides, a genus of gram-negative, obligately anaerobic bacteria that is phylogenetically distant from the diverse species in which tet(M) and tet(O) have been found. The Bacteroides Tcr gene defines a new class of ribosome protection resistance genes, Tet(Q), and has a deduced amino acid sequence that was only 40% identical to Tet(M) or Tet(O). Like tet(M) and tet(O), tet(Q) appears to have spread by horizontal transmission, but only within the Bacteroides group.