Characterization of a novel tetracycline resistance that functions only in aerobically grown Escherichia coli

Abstract

A tetracycline resistance (Tcr) gene that was found originally on two Bacteroides plasmids (pBF4 and pCP1) confers tetracycline resistance on Escherichia coli, but only when it is grown aerobically. Using maxicells, we have identified a 44-kilodalton protein which is encoded by the region that carries the Tcr gene and which may be the Tcr gene product. Localization experiments indicate that this 44-kilodalton protein is cytoplasmic. To determine whether the tetracycline resistance gene is expressed under anaerobic conditions, we have constructed a protein fusion between the Tcr gene and lacZ. In strains of E. coli carrying the fusion, beta-galactosidase activity was the same when the cells were grown under anaerobic conditions as when the cells were grown under aerobic conditions. This indicates that the tetracycline resistance gene product is made under anaerobic conditions but does not work. The failure of the Tcr protein to function under anaerobic conditions was not due to a requirement for function of the anaerobic electron transport system, because neither nitrate nor fumarate added to anaerobic media restored tetracycline resistance. Inhibition of the aerobic electron transport system with potassium cyanide did not prevent growth on tetracycline of cells containing the Tcr gene. A heme-deficient mutant, E. coli SHSP19, which carries the Tcr gene, was still resistant to tetracycline even when grown in heme-free medium. These results indicate that functioning of the Tcr gene product is not dependent on the aerobic electron transport system. Thus the requirement for aerobic conditions appears to reflect a requirement for oxygen. Spent medium from an E. coli strain carrying the Tcr gene, which was grown in medium containing tetracycline (50 micrograms/ml), did not inhibit growth of a tetracycline-susceptible strain of E. coli. Thus, the Tcr gene product may be detoxifying tetracycline.

Cloning and characterization of a Bacteroides conjugal tetracycline-erythromycin resistance element by using a shuttle cosmid vector

“…Freshly prepared chromosomal DNA from B. thetaiotaomicron DOT and B. uniformis 1006 was partially digested with Sau3A under conditions that gave a visible widening of the chromosomal band on a stained agarose gel but did not give extensive (25) pBR328-based vector containing 0.9-kbp ClaIl-to-EcoRI fragment of Tn4351 (see Fig. 2D) (28) a Abbreviations for bacterial phenotypes Strr, streptomycin resistance; Rift, rifampin resistance; Thy-, thymidine requiring; Tpmr, trimethoprim resistance due to Thy-phenotype. Abbreviations for plasmid, transposon, or conjugal element phenotypes: Tpr, trimethoprim resistance; Smr, streptomycin resistance; Knr, kanamycin resistance; Tcr tetracycline resistance; Emr, erythromycin resistance; Apr, ampicillin resistance; Tra+, capable of self-transfer; Mob', can (+) and cannot (-) be mobilized; Rep+, can replicate.…”

Novel aerobic tetracycline resistance gene that chemically modifies tetracycline

“…To check for toxicity of the extracellular fluid, an additional 10-ml portion of cell suspension was removed after 2, 4, and 8 h of incubation. Toxicity of the medium to Tcs E. coli was tested as previously described (14). By comparing the concentrations of each filtrate dilution which showed growth of Tcs E. coli with the original solution concentration, it was possible to estimate the amount of active tetracycline remaining in the extracellular fluid.…”

“…To confirm that tetracycline had been detoxified, a separate portion of supernatant fluid was filter sterilized through a 0.22-,umpore-size membrane filter (Millipore Corp., Bedford, Mass. ), and dilutions were assayed for ability to support growth of Tcs E. coli EM24 (MIC, 3 ,ug/ml) (14). The lyophilized powder was extracted by using 1/10 the original volume of absolute ethanol.…”

“…E. coli EM24 (14) carrying either pBR328 (Tcr; 4) or pBS2 (*Tcr; 14) was grown on either Luria broth (LB) or M9 minimal medium (9) supplemented with glucose (40 mM), Casamino Acids (20 mM; Difco Laboratories, Detroit, Mich.), magnesium sulfate (1 MM), and thiamine (1 ,uM). Tetracycline was added to the growth medium to final concentrations of 10 to 100 ,ug/ml.…”

“…Not only did this gene fail to confer resistance to tetracycline on Bacteroides, but it conferred resistance on Escherichia coli only under aerobic growth conditions. Recently, we showed that this gene, which we designated *Tcr to differentiate it from other tetracycline resistance (Tc) genes, was expressed in E. coli under anaerobic conditions (14). This finding indicated that the *Tcr gene product required oxygen to function.…”