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

Abstract

The Bacteroides conjugal tetracycline resistance (Tcr) elements appear not to be plasmids. In many cases, resistance to erythromycin (Emr) is cotransferred with Tcr. Using a newly constructed shuttle cosmid, pNJR1, we cloned 44 to 50 kilobase pairs of a conjugal Tcr Emr element on overlapping cosmid clones. Cosmid libraries were made in Escherichia coli with DNA from the original clinical Bacteroides thetaiotaomicron DOT strain containing Tcr Emr-DOT or from a Bacteroides uniformis Tcr Emr-DOT transconjugant strain. The cosmid clones were mobilized from E. coli into B. uniformis in groups of 10 to 20 per filter mating, with selection for Tcr or Emr transconjugants. The Tcr and Emr genes were cloned both separately and together on 30-kilobase-pair fragments. Several of the Tcr clones also contained transfer genes that permitted self-transfer of the cosmid from B. uniformis donors to E. coli or B. uniformis recipients. Neither the Tcr nor the Emr gene conferred resistance on E. coli, and the transfer-proficient clones did not self-transfer out of E. coli. Southern blot analysis was used to compare DNA from independently isolated Bacteroides strains carrying conjugal Tcr or Tcr Emr elements and their respective B. uniformis transconjugants. Results of these analyses indicate that there are large regions of homology, including regions outside the Tcr and Emr genes, but that the elements are not identical. Some Tcr clones contained a region which hybridized to chromosomal DNA from the wild-type B. uniformis recipient strain that did not carry the Tcr Emr-DOT element. This region of homology appeared not to be a junction fragment. It was not required in a Bacteroides recipient for successful transfer of the Tcr Emr element. Although we are not sure we have cloned a junction fragment between the Tcr Emr-DOT element and the B. uniformis chromosome, the preliminary function and restriction map appears to be linear.

The region of a Bacteroides conjugal chromosomal tetracycline resistance element which is responsible for production of plasmidlike forms from unlinked chromosomal DNA might also be involved in transfer of the element

“…Several overlapping cosmid clones containing portions of the Tcr Emr DOT element (12) were tested for Plf activity (Fig. 4).…”

“…4). A third subclone, pAMS1, was constructed in pNJR5 (12) Plf activity, we created three different chromosomal insertions into this region: fQRDB1, fQRDB2, and QRDBT (Fig. 5).…”

“…Regions of the cosmid clones (12) were subcloned in the E. coli-Bacteroides shuttle vector pNJR5 (12) or pVAL1 (22). Frozen competent E. coli cells were transformed as previously described (14) with selection on LB with kanamycin (100 ,ug/ml) or ampicillin (100 pug/ml).…”

The mobilization regions of two integrated Bacteroides elements, NBU1 and NBU2, have only a single mobilization protein and may be on a cassette

“…Escherichia coli strains were grown in Luria broth or Luria broth agar plates. Bacteroides strains were grown in prereduced trypticase-yeast extract-glucose broth or agar (11,17).…”

“…Many Bacteroides clinical isolates carry large (Ͼ70-kbp) integrated self-transmissible elements, called conjugative transposons (2,11). These elements have also been called Tc r elements because most of them carry a tetracycline resistance (Tc r ) gene, tetQ.…”