Evidence that a novel tetracycline resistance gene found on two Bacteroides transposons encodes an NADP-requiring oxidoreductase

Abstract

Two transposons, Tn4351 and Tn4400, which were originally isolated from the obligate anaerobe Bacteroides fragilis, carry a tetracycline resistance (Tcr) gene that confers resistance only on aerobically grown Escherichia coli. This aerobic Tcr gene, designated tetX, has been shown previously to act by chemically modifying tetracycline in a reaction that appears to require oxygen. We have now obtained the DNA sequence of tetX and 0.6 kb of its upstream region from Tn4400. Analysis of the DNA sequence of tetX revealed that this gene encoded a 43.7-kDa protein. The deduced amino acid sequence of the amino terminus of the protein had homology with a number of enzymes, all of which had in common a requirement for NAD(P). In an earlier study, we had observed that disrupted cells, unlike intact cells, could not carry out the alteration of tetracycline. We have now shown that if NADPH (1 mM) is added to the disrupted cell preparation, alteration of tetracycline occurs. Thus, TetX appears to be an NADP-requiring oxidoreductase. Tn4400 conferred a fivefold-lower level of tetracycline resistance than Tn4351. This finding appears to be due to a lower level of expression of the tetX on Tn4400, because the activity of a tetX-lacZ fusion from Tn4400 was 10-fold lower than that of the same fusion from Tn4351. A comparison of the sequence of the tetX region on Tn4351 with that on Tn4400 showed that the only difference between the upstream regions of the two transposons was a 4-base change 350 bp upstream of the start of the tetX coding region. The 4-base change difference creates a good consensus -35 region on Tn4351 that is not present on Tn4400 and could be creating an extra promoter.An unusual tetracycline resistance (Tcr) gene has been found on two closely related Bacteroides transposons, Tn4351 and Tn4400 (6,16,21). This gene encodes a cytoplasmic protein which detoxifies tetracycline (21, 22). Because the mechanism of this resistance differs from those of the efflux-type resistances (tetA to E and tetL) and the ribosomal protection-type resistances (tetM to 0), we have designated this gene tetX (23). Although tetX was found originally in Bacteroides fragilis strains, it does not confer resistance on these organisms. tetX does confer resistance on Escherichia coli but only when the bacteria are grown under aerobic conditions (6). Here we report the DNA sequence and deduced amino acid sequence of tetX and show that TetX shares amino acid homology with NAD(P)-requiring oxidoreductases.We have also compared the DNA sequence of the coding and upstream regions of tetX from Tn4400 with the corresponding regions on Tn4351. Previously, Smith and Gonda (20) used hybridization analysis to show that the two transposons were virtually identical. However, we had noted that the tetracycline MIC associated with Tn4351 was about fivefold higher than the MIC associated with Tn4400 for cells grown under the same conditions (23). It was possible that sequence differences in the coding regions of the tetX genes caused the TetX of Tn4400 to be ...

Fate of antimicrobials and antimicrobial resistance genes in simulated swine manure storage

“…In contrast, the two tet genes tested in the study mostly respond to chlortetracycline. The tet(X) gene encodes an enzyme which modifies and inactivates the tetracycline molecule (Speer et al, 1991), while the tet(Q) gene encodes a cytoplasmic protein that protect the ribosomes from the action of tetracycline (Chopra and Roberts, 2001). The specificity of the tet genes might explain their overlapping trends with the chlortetracycline compound (Fig.…”

Tetracyclines in veterinary medicine and bacterial resistance to them

“…This gene codes for a 44-kDa cytoplasmic protein that chemically modifies tetracycline in the presence of oxygen and NADPH (Speer et al, 1991). This gene has been discovered on two Bacteroides transposons, Tn4351 and Tn4400, and has been found to share considerable amino acid homology with a number of NADPH-requiring oxidoreductases (Speer et al, 1991).…”

“…Three different tetracycline resistance mechanisms have been described: (1) active efflux of the antibiotic (Franklin and Snow, 1971), (2) ribosomal protection (Burde�, 1986), as the most common resistance mechanisms, and (3) enzymatic inactivation of the drug (Speer et al, 1991). All these mechanisms are based on the acquisition of one or several tetracycline resistance determinants, which are widely distributed among bacterial genera (Schnappinger and Hillen, 1996).…”

“…Two genes, tet(X) and tet(37), code for enzymes which inactivate tetracyclines (Speer et al, 1991;DiazTorrez et al, 2003). Mechanisms of otr(C) and tet(U) encoded resistance are unknown (Levy et al, 1999).…”

Two Different Tetracycline Resistance Mechanisms, Plasmid-Carried tet (L) and Chromosomally Located Transposon-Associated tet (M), Coexist in Lactobacillus sakei Rits 9

“…Usually, these genes code for energy-dependent efflux systems or for proteins that protect the bacterial ribosomes from the blockage of protein synthesis (9,10,37). In rare cases, Tc r is mediated through direct inactivation of the antibiotic (40) or by mutations in the 16S rRNA that prevent the binding of tetracycline to the ribosome (38).…”