A new Bacteroides conjugative transposon that carries an ermB gene

Abstract

The erythromycin resistance gene ermB has been found in a variety of gram-positive bacteria. This gene has also been found in Bacteroides species but only in six recently isolated strains; thus, the gene seems to have entered this genus only recently. One of the six Bacteroides ermB-containing isolates, WH207, could transfer ermB to Bacteroides thetaiotaomicron strain BT4001 by conjugation. WH207 was identified as a Bacteroides uniformis strain based on the sequence of its 16S rRNA gene. Results of pulsed-field gel electrophoresis experiments demonstrated that the transferring element was normally integrated into the Bacteroides chromosome. The element was estimated from pulsed-field gel data to be about 100 kb in size. Since the element appeared to be a conjugative transposon (CTn), it was designated CTnBST. CTnBST was able to mobilize coresident plasmids and the circular form of the mobilizable transposon NBU1 to Bacteroides and Escherichia coli recipients. A 13-kb segment that contained ermB was cloned and sequenced. Most of the open reading frames in this region had little similarity at the amino acid sequence level to any proteins in the sequence databases, but a 1,723-bp DNA segment that included a 950-bp segment downstream of ermB had a DNA sequence that was virtually identical to that of a segment of DNA found previously in a Clostridium perfringens strain. This finding, together with the finding that ermB is located on a CTn, supports the hypothesis that CTnBST could have entered Bacteroides from some other genus, possibly from gram-positive bacteria. Moreover, this finding supports the hypothesis that many transmissible antibiotic resistance genes in Bacteroides are carried on CTns.

FIG. 1.

Schematic representation of the plasmid rescue experiment done to obtain additional flanking sequences and thus reach an end. BT4020 is the transconjugant obtained by mating WH207 with BT4001. CTnBST is the only known mobile element in this strain.

FIG. 2.

(A) PFGE of I-CeuI-digested recipient (lanes 3) and transconjugant (lanes 4 to 6) DNA was carried out in a 1% agarose-0.5× Tris-borate-EDTA gel run at 6 V/cm, with an initial pulse of 10 s and a final pulse of 120 s, for 30 h with a Bio-Rad CHEF-DR III PFGE apparatus. A Bio-Rad lambda ladder PFG marker (lanes 2) was used along with an S. cerevisiae chromosomal PFG marker (lanes 1). The approximate sizes of the bands of interest are indicated to the left of the figure in megabases. BT4001 is the recipient. All other strains shown are transconjugants containing the ermB element. A 300-kb band in the recipient shifted to 400 kb in the transconjugants, indicating that the element is integrated into the chromosome and is at least 100 kb in size. The 2.9-Mb band is in the well. (B) Southern blot of the PFG with I-CeuI-digested recipient and transconjugant DNA. The probe was the 3-kb HindIII region containing ermB plus flanking DNA. A λ HindIII probe was also used to detect the lambda PFG marker. No bands are visible in the lane for strain BT4001 because it does not have the ermB element. All other lanes contain strains that are transconjugants containing the ermB element. A 400-kb band hybridizes to the 3-kb HindIII probe, proving that the band shift seen in the PFG is due to integration of the ermB element. An additional band is seen in one lane, which might be due to partial digests or the presence of another copy of CTnBST.

FIG. 3.

Matches obtained when a BLAST search was performed with the 13 kb of DNA sequence obtained from CTnBST. The 1.7-kb sequence from the right end is 99% identical to a sequence found for C. perfringens (accession no. U18931.1) (1). The ermB and ermB(P) genes are nearly identical, as is orf3. Only part of direct repeat 2 (DR2) is present in CTnBST; orf298 is truncated, and only a copy of palB is present. Sections of DR2 consisting of palA and most of orf298 are truncated and are indicated by broken lines. A Tn10-like transposase gene (tpn) was found inserted into a methyltransferase-like (mte) gene. Truncated ORFs for rep, mob, and met were found. An ORF which had low identity to site-specific recombinases (ssr) was also found.

FIG. 4.

Results of Southern hybridization with probes from CTnBST. The strain from which the DNA was obtained is indicated above each lane. The DNA was digested with HindIII and PstI in all the lanes. The probes used were a 3-kb HindIII region from CTnBST (A) and a 10-kb HindIII-PstI region upstream of the 3-kb probe (B). The asterisk indicates the ermB-containing band. The band containing the probe is also indicated (↔). Weakly hybridizing bands are indicated by arrows. The positions of the lambda HindIII size standards are indicated in kilobases. The multiplier indicates a doublet seen in WH207 that was not seen BT4020 and BT4100. B, ermB; G, ermG; F, ermF.

FIG. 5.

(A and B) Southern blot analysis to estimate the distance to the ends from the available sequence. Lanes 1, 4, and 9, WH207 DNA; lanes 2, 3, 5 to 8, and 9 to 13, transconjugant DNA digested with NsiI, SacII, or KpnI as indicated in the figure. The 3-kb HindIII fragment was used as the probe in panel A, and the 10-kb PstI-HindIII fragment was the probe in panel B. (C) Schematic diagram indicating how the distances from the ends were estimated. Diagram 1, positions and sizes of the probes used. Diagram 2, positions of the restriction sites. P, PstI; SII, SacII; HIII, HindIII; N, NsiI; K, KpnI. The position of ermB is also indicated. Diagram 3, method for estimations of the distance to the ends made for each restriction enzyme with the data from the Southern blot in Fig. 4A and B. Diagram 4, consolidated figure schematic with a rectangle indicating the position of the known sequence used as the probe in the Southern hybridization. The left end is at least 20 kb from an available sequence, while the right end is at least 15 kb from an available sequence.