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

Abstract

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.

Circularization of Tn916 is required for expression of the transposon‐encoded transfer functions: characterization of long tetracycline‐inducible transcripts reading through the attachment site

“…2). The 0.24, 2.37 and 3.2 kb hybridizing bands have been characterized previously (Su et al, 1992) and probably correspond to transcripts that initiated at the tetM promoter (P tet ) and ended at the palindromes pal orf 12 (0.24 kb), pal orf 6 (2.37 kb) and pal orf10 (3.2 kb) respectively. Accordingly, the 0.24 kb transcript disappeared when the RNAs extracted from both strains were probed with tetM.…”

“…1. characterized previously (Su et al, 1992), Tn916 contained at least three additional promoters that were tentatively located between orf10 and orf7 (P orf7 ), orf8 and xis (P xis ) and orf10 and orf9 (P orf9 ). We studied the activity of each of these promoters plus that of P tet following cloning, in the proper orientation, of the corresponding DNA fragments upstream from the promoterless spoVG-lacZ gene of pTCV-lac.…”

High Prevalence of tetM as Compared to tetK Amongst Methicillin-Resistant Staphylococcus aureus (MRSA) Isolates from Hospitals in Perak, Malaysia

“…This shows that the prevalence of tetM resistant determinant roughly tripled that of tetK (38.4%) in these MRSA samples. Among the tetracycline resistance genes, tetM prominence in MRSA is hypothesized to be due to the exposure to tetracycline which increases the rate of excision and transfer of Tn916 carrying tetM gene (26). As suggested by some researchers, tetM is relatively stable and can persist in a population for a longer period of time.…”

Tales of conjugation and sex pheromones

“…Regulation of its expression appears to involve a transcription-attenuation mechanism. 95 (3) Near the left end of Tn916 are determinants for an integrase and excisase, as well as related controlling genes. [96][97][98] In the right portion of the transposon are genes related to conjugation, including an origin of transfer oriT (Fig.…”