Genetic and functional analyses of the mob operon on conjugative transposon CTn341 from Bacteroides spp

Abstract

Bacteroides are Gram-negative anaerobes indigenous to the intestinal tract of humans, and they are important opportunistic pathogens. Mobile genetic elements, such as conjugative transposons (CTns), have contributed to an increase in antibiotic resistance in these organisms. CTns are self-transmissible elements that belong to the superfamily of integrative and conjugative elements (ICEs). CTn341 is 52 kb; it encodes tetracycline resistance and its transfer is induced by tetracycline. The mobilization region of CTn341 was shown to be comprised of a three-gene operon, mobABC, and the transfer origin, oriT. The three genes code for a nicking accessory protein, a relaxase, and a VirD4-like coupling protein, respectively. The Mob proteins were predicted to mediate the formation of the relaxosome complex, nick DNA at the oriT, and shuttle the DNA/protein complex to the mating-pore apparatus. The results of mutational studies indicated that the three genes are required for maximal transfer of CTn341. Mob gene transcription was induced by tetracycline, and this regulation was mediated through the two-component regulatory system, RteAB. The oriT region of CTn341 was located within 100 bp of mobA, and a putative Bacteroides consensus nicking site was observed within this region. Mutation of the putative nick site resulted in a loss of transfer. This study demonstrated a role of the mobilization region for transfer of Bacteroides CTns and that tetracycline induction occurs for the mob gene operon, as for the tra gene operon(s), as shown previously.

FIG. 1.

Genetic map of the mob operon and RT-PCR evidence showing linkage between genes. (A) Map showing mob genes and functional domains of the Mob proteins. The regions used for the construction of complementing plasmids are shown below the map. The functional domains of the Mob proteins are shown above the map. The red bar above MobB indicates the location of the relaxase/mobilization nuclease domain (Pfam accession number 03432), and the red bar above MobC shows the TraG/TraD superfamily domain (Pfam accession number 02534). The Walker boxes are shown by the inverted triangles above the red bar, and the five MobC transmembrane domains are indicated by black diamonds above the map. (B) Design and results of RT-PCR experiment, showing the locations of the primers used and the agarose gel of the amplified products. The cDNA (shown in red) was generated by reverse transcription of the mob mRNA using primers located toward the end of the message in the mobC gene. The arrowheads to the right of the gel indicate the sizes of the expected 621- and 400-bp products. Lane A, 1-kb-ladder molecular-size marker; B, cDNA-1 plus primer set mobA/B; C, cDNA-2 plus primer set mobA/B; D, pFD699 DNA-positive control with primer set mobA/B; E, cDNA-1 plus primer set mobB/C; F, pFD699 DNA-positive control with primer set mobB/C. All primers are listed in Table S1 in the supplemental material.

FIG. 2.

Northern blot analysis of mob operon region. Fifty micrograms of RNA was separated on formaldehyde agarose gels, transferred to nylon membranes, and hybridized to ³²P-labeled probes from the mob operon as indicated. (A) RNA samples from B. fragilis cells containing CTn341 were either induced (+Tc) or not induced (−Tc) and probed with a 4-kb probe containing the entire mob operon. (B) RNA sample from tetracycline-induced B. fragilis containing CTn341 plus a multicopy plasmid with the cloned mob operon. Lanes are labeled to indicate induction where appropriate, and the full-length 4-kb mRNAs are shown by the arrows to the right of the blots. The apparent bands (*) at about 1.5 and 2.5 kb are a commonly observed compression artifact caused by the 16S and 23S rRNA. These artifacts are exacerbated by the extensive RNA degradation.

FIG. 3.

Quantitative RT-PCR analysis of the mobA, mobB, and traA genes in rteA and rteB mutants. Overnight cultures of the wild-type (BTSpFD699), rteA (BTSJo68), and rteB (BTSJo69) strains were subcultured and grown to mid-log phase either with or without 1 μg/μl tetracycline (1). RNA was isolated from the cultures, and two independent sets of RNA were used for analysis, using primers for the mobA, mobB, or traA gene as indicated. Each PCR measurement was performed in triplicate. The sigma-54 modulation protein gene was used as the reference gene. The results are expressed as the fold induction relative to the results with no tetracycline. Error bars represent standard deviations.

FIG. 4.

Identification of the CTn341 oriT region. Three fragments from the mob operon region (shown by the numbered lines over the map) were PCR amplified, inserted into the mobilization-deficient plasmid pFD1112, and tested for the ability to mediate transfer of the plasmid in mating-out assays, as described in the text. Transfer frequencies were determined and are shown to the right. Based on these results, subfragments (shown below the map) were amplified and tested in the mating-out assay, and the results showed that there was a common 120-bp region just upstream of mobA (see parallel dashed vertical lines). Transfer frequencies are the number of transconjugants per input donor cell (means of the results of triplicate experiments).

FIG. 5.

Identification of the CTn341 nick site. (A) Location of the oriT region between mobA and the divergently transcribed traA. DNA sequence showing four of the putative nick sequences (underlined and in upper case) that were mutated. The number 2 nick site shows the putative GC cleavage nick site with the arrowhead under the sequence, and the imperfect inverted repeat sequence is shown by the dashed arrows over the sequence. (B) Alignment of Bacteroides nick sites and generation of consensus. The Tn5520 nick site, Tn4555 nick site, and nick site homologues in the CTn341 oriT region are shown. Site-directed mutagenesis of the GC of homologue 2 resulted in a loss of transfer, indicating that it is the nick site of CTn341. Mutations of the other sites did not result in loss of the transfer phenotype.