Tetracycline-associated transcriptional regulation of transfer genes of the Bacteroides conjugative transposon CTnDOT

Abstract

Many human colonic Bacteroides spp. harbor a conjugative transposon, CTnDOT, which carries two antibiotic resistance genes, tetQ and ermF. A distinctive feature of CTnDOT is that its excision and transfer are stimulated by tetracycline. Regulation of the genes responsible for excision has been described previously. We provide here the first characterization of the regulation of CTnDOT transfer (tra) genes. Reverse transcription-PCR analysis of the region containing the tra genes showed that these genes are regulated at the transcriptional level. Surprisingly, increased production of tra gene mRNA in tetracycline-stimulated cells was mediated by the proteins encoded by the excision genes. Previous studies have shown that expression of the excision gene operon is controlled by the regulatory protein RteC. Accordingly, it was possible that RteC was also regulating tra gene expression and that the excision proteins were only accessory proteins. However, placing the excision gene operon under the control of a heterologous promoter showed that the excision proteins alone could activate tra gene expression and that RteC was not directly involved. We also found a second level of tra gene control. The transfer of CTnDOT was inhibited by a DNA segment that included only a portion of the 3' end of one of the excision genes (exc). This segment contained a small open reading frame, rteR. By replacing the codons encoding the first two amino acids of the putative protein product of this open reading frame with stop codons, we showed that the rteR gene might encode a small regulatory RNA. RteR acted in trans to reduce the number of tra transcripts in a way that was independent of the excision proteins. The repressive effect of RteR was not the result of decreased stability of the tra mRNA. Instead, RteR appears to be modulating the level of tra gene expression in some more direct fashion. The complex regulatory system that controls and links the expression of CTnDOT excision and transfer genes may be designed to ensure stable maintenance of CTnDOT in nature by reducing the fitness toll it takes on the cell that harbors it.

FIG. 1.

Map of the CTnDOT excision region, central regulatory region, and transfer region. A solid line under a given region denotes the presence of those genes in the corresponding plasmid or chromosomal insertion (Ω). An arrow indicates the placement of a given gene in CTnDOT. An arrowhead linked to the horizontal line indicates a promoter. A dashed line indicates that the region is not included.

FIG. 2.

RT-PCR experiments were used to determine if the CTnDOT genes traA to traQ might be part of an operon by demonstrating that an RNA spans each region where one ORF ends and another begins. Panel A is a diagram indicating which RT-PCR products (short bars) were detected. All the genes in the transfer operon comprising traA to traQ tested positive for an RT-PCR product. Panel B shows some typical results of the RT-PCR analysis of the tra operon. Cells grown in the absence (−) and presence (+) of tetracycline (Tc) were tested in each case. RT-PCR analysis of the sigma 70 gene was used as an internal control to verify the presence of mRNA.

FIG. 3.

Promoter and leader region of the tra operon. Primer extension results are shown in panel A. The product obtained with tetracycline induction (+tc) is indicated by an arrow to the side of the gel, along with the surrounding sequence. No product was present in the absence of tetracycline induction (−tc). In panel B, the locations of the TIS and the start codon of traA in the region upstream of the tra operon are shown. The putative tra promoter is labeled as −33 and −7.

FIG. 4.

Excision of CTnERL with and without pGRW3. The minus signs at the bottom of the figure denote that cells were grown in the absence of tetracycline (Tc). The plus signs denote that cells were grown in the presence of tetracycline. Excision triggered by pGRW3, the plasmid carrying the construct with the heterologous promoter, was compared with excision triggered by CTnERL (carrying the wild-type [WT] promoter only). All cells were grown in the presence of maltose, the inducer of the heterologous promoter.

FIG. 5.

Constitutive expression of rteR. Panel A shows the Northern blot for BT4007 cells grown in the presence and absence of tetracycline. The probe was for RNA synthesized in the region encoding RteR. The 100-nt hybridizing band appears in samples both from cells grown in medium containing tetracycline (+Tc) and from cells grown in medium containing no tetracycline (−Tc). In the +Tc lane, there are weak, larger bands that are believed to represent breakdown products from the exc transcript. Note that these are not present in the −Tc lane, as expected due to the fact that the transcription of exc is elevated only in cells exposed to tetracycline. Panel B shows the results of primer extension to detect the transcriptional start site of the RteR gene. Products were obtained from cells grown in the absence of tetracycline, as well as from cells grown in the presence of tetracycline. The G represents the transcription start site of the RteR gene shown in the sequence to the right. The product is observed in both the (+)Tc and (−)Tc lanes. The sequence containing rteR is shown in panel C. The putative ORF which was originally suggested to be rteR is shown, and the ATG sequence which was mutated into a stop codon is indicated. The transcriptional start site determined by primer extension as described in the legend to panel B is labeled, and an arrow shows where a sequence of 100 nt would end, as indicated by the results in panel A. The stop codon for exc is also shown. The exact 3′ end of RteR has not yet been determined.

FIG. 6.

Results of qualitative RT-PCR stability assays for traG mRNA with (+) and without (−) the RteR repressor (on pKSO1) present. (A and C) The traG mRNA concentrations with (A) and without (C) the RteR repressor present were compared at 15, 30, and 60 min relative to time zero (the addition of rifampin). (B and D) As a control, the sigma 70 mRNA concentrations with (B) and without (D) the RteR repressor present were also compared at time zero and at 15, 30, and 60 min.