Translational control of tetracycline resistance and conjugation in the Bacteroides conjugative transposon CTnDOT

Abstract

The tetQ-rteA-rteB operon of the Bacteroides conjugative transposon CTnDOT is responsible for tetracycline control of the excision and transfer of CTnDOT. Previous studies revealed that tetracycline control of this operon occurred at the translational level and involved a hairpin structure located within the 130-base leader sequence that lies between the promoter of tetQ and the start codon of the gene. This hairpin structure is formed by two sequences, designated Hp1 and Hp8. Hp8 contains the ribosome binding site for tetQ. Examination of the leader region sequence revealed three sequences that might encode a leader peptide. One was only 3 amino acids long. The other two were 16 amino acids long. By introducing stop codons into the peptide coding regions, we have now shown that the 3-amino-acid peptide is the one that is essential for tetracycline control. Between Hp1 and Hp8 lies an 85-bp region that contains other possible RNA hairpin structures. Deletion analysis of this intervening DNA segment has now identified a sequence, designated Hp2, which is essential for tetracycline regulation. This sequence could form a short hairpin structure with Hp1. Mutations that made the Hp1-Hp2 structure more stable caused nearly constitutively high expression of the operon. Thus, stalling of ribosomes on the 3-amino-acid leader peptide could favor formation of the Hp1-Hp2 structure and thus preclude formation of the Hp1-Hp8 structure, releasing the ribosome binding site of tetQ. Finally, comparison of the CTnDOT tetQ leader regions with upstream regions of five tetQ genes found in other elements reveals that the sequences are virtually identical, suggesting that translational attenuation is responsible for control of tetracycline resistance in these other cases as well.

FIG. 1.

Diagram of the RNA secondary structure of Pq and the locations of mutations that affect the translation of three possible leader peptides within the tetQ leader sequence and/or disrupt the secondary structure. The RNA secondary structure was predicted by the MFOLD version 3.1 program of Zucker (http://www.bioinfo.rpi.edu/applications/mfold/rna.forml.cgi). The asterisk indicates the transcription initiation site at −130 bp upstream of the tetQ start codon that was identified by Bayley et al. (1). In panel A, three possible leader peptides (LPs) are indicated by solid or dashed lines. The first putative peptide is only 3 amino acids long and starts at bp −126 relative to the AUG of tetQ. The triangles and LP designations indicate the positions of stop codons that were inserted in the sequence at positions −71 (LP7) and −48 (LP8) relative to the tetQ start codon (Table 1). The effect of each mutation in the tetQ operon promoter on the tetracycline regulation of the uidA (GUS) reporter gene fused within the tetQ is shown in Table 1. In panel B, the site-directed mutations in Hp1 and Hp8 sequences are indicated. YP161 contains the 5-nucleotide changes in Hp8 cloned in the GUS fusion, and YP226 contains the same YP161 Hp8 sequences with complementary changes in the Hp1 sequence to restore the hairpin formation, as indicated by the brackets. The mutations in Hp1 on YP226 also increased the coding sequence of the tripeptide (indicated by the solid line) from 3 to 8 amino acids (indicated by the dotted line extension). There are three additional site-directed mutations involving the tripeptide sequence, which are in addition to the two shown in panel A. LP10 changes the sequences of the first two codons to stop codons, and this mutation also interferes with the pairing of the Hp1-Hp8 adjacent to the AUG (position 0) of tetQ. LP11 shortens the mini-peptide to only 2 amino acids by mutating the third codon, CAG, to UAG. LP14 changes the sequences of the 2nd and 3rd amino acids with minor effects on the pairing of Hp1 and Hp8. The effects of the mutations on the GUS activity, plus and minus tetracycline, compared to that of the wild-type promoter are shown in Table 1.

FIG. 2.

Effects of deletions in the Hp2-Hp7 region. The hypothetical secondary structure of the tetQ leader region is shown. The transcriptional start site determined by Bayley et al. (1) is indicated by an asterisk. The sequences of hairpin structures (Hps) that were deleted are indicated by dotted or dark lines. In panel A, the solid line above and between Hp1 and Hp8 indicates the sequences remaining in the SM Del-3 construct that leaves only the 3 nucleotides, AUA, between the hairpin formed by Hp1 and Hp8. Del Hp2-5 deletes all the sequences in the left loop, and SM Del-1 removes the sequences of Hp6 and Hp7. The effects of the deletions on the resultant GUS activities of the Pq::GUS fusions, with and without tetracycline induction, are shown in Table 1. In panel B, deletions of various sequences in the left loop are shown. The deletions are indicated by various solid and dashed lines, and the effects of these deletions on the GUS activity of the Pq::GUS fusions are shown in Table 1.

FIG. 3.

Putative hairpin structure formed by Hp1 and Hp2. If the formation of Hp1 and Hp8 as a hairpin is prevented, the MFOLD version 3.1 program predicts the formation of a hairpin between Hp1 and Hp2. The calculated energy of this putative structure is ΔG = −4.81 kcal/mol. Site-directed mutations to test the Hp1-Hp2 pairing are shown. YP239 contains the mutations in Hp1. In addition to the base substitutions in Hp1 in YP239, complementary base substitutions were made in Hp2 to construct YP241. The mutations in Hp2 reestablish the Hp1-Hp2 pairing with GC base pairs instead of AU base pairs. The effects of the mutated Hp1 and Hp2 regions on the regulation of the GUS activity in the Pq::GUS fusions are shown in Table 1.

FIG. 4.

DNA sequence alignment of the tetQ leader and promoter sequences from different alleles of tetQ. The ClustalW sequence alignment of tetQ leader regions found in Bacteroides and Prevotella spp. is shown. In all cases, the ATG start codon of tetQ (boldface) is numbered 0. The negative numbers above the sequence indicate the relative upstream positions of the nucleotides to the ATG of tetQ of CTnDOT (theta). The asterisks below the aligned sequences indicate that all residues at that position were identical. The consensus transcription initiation sites (1) are indicated by the asterisk at −130. The horizontal arrows labeled Hp1, Hp2, and Hp8 indicate the positions of the two stems of the hairpin that are important for tetracycline control in the case of the TnDOT tetQ operon. The 3-amino-acid leader peptide region (shaded) is identical in all sequences. PIQ is a tetQ leader sequence from Prevotella intermedia (accession no. U73497), B1126Q is a tetQ leader sequence from Bacteroides fragilis (accession no. Z21523), theta is the CTnDOT tetQ leader sequence (accession no. X58717), BFQ is a tetQ leader from B. fragilis (accession no. Y08615), pRRI4 is a tetQ leader sequence from a plasmid that was isolated from P. ruminicola (accession no. L33696), and 7853Q is the tetQ leader sequence from the Bacteroides CTn, CTn7853 (19).

FIG. 5.

Proposed model for tetracycline regulation of the tetQ-rteA-rteB operon. In the absence of tetracycline (step I), ribosomes bind to the mRNA and translate the leader tripeptide (dashed line). The region containing the putative ribosome binding site for tetQ (rbs; shaded box) is tied up in the Hp1-Hp8 hairpin and is thus not available for ribosome binding. In the presence of tetracycline (indicated by an asterisk), those ribosomes that have tetracycline bound to them stall on the leader peptide mRNA (step II), allowing alternate mRNA structures to form (e.g., Hp1-Hp2), thus making the ribosome binding site for tetQ available. The tetQ gene can then be translated, presumably by those ribosomes that do not have bound tetracycline. Once TetQ is made, it alters the conformation of the ribosome and actually causes the release of bound tetracycline (5) and the conformation changes reduce the number of ribosomes that can bind tetracycline (step III). The effect of the altered ribosomes on the translation of Pq, indicated by the arrow (step IV), is the return of the situation in step I. Because the ribosomes are no longer sensitive to tetracycline, the mRNA structures which embed the ribosome binding site can be reformed. Thus, the alternate structures shown in steps II and III are once again prevented from forming. The percentage of ribosomes protected by interaction with TetQ will then determine the level of promoter modulation observed. This fact would explain why the concentration of TetQ produced from a multicopy vector would have a stronger negative regulatory effect than the amount of TetQ produced from a single copy of tetQ in the chromosome.