C. difficile CD37
This experiment was carried out to investigate the effect of the disruption of the start codon of orf12 in the mobility of Tn916. In this filter mating experiment, C. difficile CD37 was used as the recipient. The frequency of transfer [ standard deviation (SD)] detected for BS34A (contains wild type Tn916) was 1.74 x 10-6 ( 2.28 x 10-6) per recipient. As for BS79A which contains Tn916Δorf12, the transfer frequency detected was 4.14 x 10-6 ( 4.07 x 10-6) per recipient. From these results, it was concluded that the transfer of Tn916Δorf12 is not appreciably different to that of the wild type Tn916, despite the disruption of the start codon of orf12. The presence of Tn916 in C. difficile CD37 was confirmed by PCR amplification of tet(M) gene (Figure 3.17).
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Figure 3.17 PCR amplification of tet(M) gene in C. difficile CD37. Panel A: tet(M) gene in CD37 transconjugants (BS34A x CD37). Panel B: tet(M) gene in CD37 transconjugants (BS79A x CD37). Lane 1: Hyperladder I; Lane 2: Genomic DNA of B. subtilis BS34A (positive control); Lane 3: Genomic DNA of B. subtilis BS79A (positive control); Lanes 4-8: Putative transconjugants carrying tet(M) gene; Lane 9: Genomic DNA of C. difficile CD37 (negative control); Lane 10: No DNA.
3.4 Discussion
In this study, an in-depth investigation concerning the role of orf12 and its translation in regulating the transcription of Tn916 was carried out using B. subtilis construct containing 2 bp mutation disrupting the start codon of orf12. The experiments were performed in the presence and absence of tetracycline, which was previously shown to be involved in the transcriptional regulation of this element (Su et al., 1992; Celli and Trieu-Cuot, 1998). In agreement with previous reports, the involvement of tetracycline in the transcriptional regulation of Tn916 was again highlighted in this study. In growth conditions where tetracycline is present (abf-Tc and Tc-Tc), both the wild type (BS34A) and the mutated strain [BS79A (contains Tn916Δorf12)] demonstrated a slower growth rate compared to their respective tetracycline-free culture (Tc-abf and abf-abf) counterparts, especially during the mid-logarithmic phase where cells were rapidly dividing (Figure 3.13 and Figure 3.14). It is possible that at this stage, the presence of tetracycline has partially inhibited the growth while Tet(M) takes time to protect enough ribosomes to allow increased translation of tet(M) gene. As this happens, the cells are able to grow normally, indicated by comparable growth rates with tetracycline-free cultures towards the end of the logarithmic phase and the start of stationary phase. The ability of Tet(M) to protect ribosomes was demonstrated by Burdett (1986) although it remains a matter of speculation of the average time required to complete the process.
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Interestingly, results presented also suggests that tetracycline exerted a more pronounced effect to the growth profile when added to the cultures during monitoring of growth (abf-Tc) rather than exposure at the onset (Tc-Tc). Pre- exposing the cultures with tetracycline for overnight has assisted the cells to maintain the resistance during the second exposure, which led to a higher growth rate when compared with the non pre-exposed cells. The significance of pre- exposing the cells to tetracycline has been highlighted in a number of studies. For instance, Nesin et al. (1990) have demonstrated that pre-exposure of cells to tetracycline not only increase the tetracycline resistance but also caused an increase in the level of mRNA transcripts for tet(M). Pre-exposing the cultures with tetracycline has also been shown to enhance conjugal transfer of Tn916 between Bacillus species as reported by Showsh and Andrews (1992).
In the presence of tetracycline, BS79A (contains Tn916Δorf12) demonstrated a slower growth rate compared to the wild type BS34A (Figure 3.15), which is probably due to the defective start codon of orf12. The absence of the start codon is expected to permanently stop the translation of orf12, thus led to the maximal formation of the transcriptional terminator structures. Therefore minimal read- through of transcription is expected to occur into tet(M) gene, representing the basal level of transcription through these terminators. This hypothesis is supported by previous study by Burdett (1986) who demonstrated that the transcription into tet(M) without the translation of the leader peptide is possible as the ribosomes could still bind to the tet(M) Shine-Dalgarno site. The model proposed for tet(M)
regulation requires a basal level of Tet(M) to be produced in order to protect a few of the ribosomes in the cell in case of exposure to tetracycline. This basal level of expression is investigated in detail in the next chapter.
As comparison of the exponential growth rates was insufficient to detect any biological fitness cost of BS34A (contains wild type Tn916) and BS79A (contains Tn916Δorf12), therefore a more sensitive and accurate evaluation was carried out using competition experiments. Results suggest that BS79A is 7.12% more fit than BS34A in the absence of selective pressure, indicating that the 2 bp mutation at the start codon of orf12 does not confer any fitness cost to BS79A in the absence of antibiotic. In fact, it appears that there is a slight fitness benefit to this strain in the absence of antibiotic. Although the reasons for this remain elusive, we can hypothesise that the level of translation of orf12 in the wild type will be more than zero, as it will be in BS79A therefore more terminator structures will be destroyed in the wild type when compared to BS79A. This means the wild type is likely producing a higher basal amount of Tet(M) and other Tn916 proteins.
In the presence of tetracycline, the fitness of BS79A (contains Tn916Δorf12) reduced relative to BS92A (BS34AΔamyE) after 2-3 h of growth (Table 3.3). As synthesis of Tet(M) is limited in BS79A due to the defective start codon of orf12, therefore it is suspected that the ability of the cells to compete is substantially reduced especially during mid-logarithmic phase where cells are rapidly dividing, this is what we detected in our experiments.
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Additionally, it was shown that in the presence of tetracycline, Tn916Δorf12 was transferred at a comparable frequency as the wild type Tn916 despite the mutation in the start codon. It has been shown previously in this study that BS34A (contains wild type Tn916) and BS79A (contains Tn916Δorf12) grow at different rate in the mid-exponential phase (Figure 3.16), hence transfer frequency was expected to be lower in the mutant, however this does not seems to be the case. The reason that contributed to this result are unclear, however it is suspected it could be due to the basal level of transcriptional read-through of tet(M) and the downstream genes (xis and int) is high enough for the transfer of the mutant Tn916orf12, although this needs to be experimentally proven. It also suspected that the concentration of tetracycline might affect the frequency of transfer as shown in a number of studies. Although in this study tetracycline was used at a concentration of 10 µg ml-1, which was previously shown to enhance the transfer of Tn916 (Showsh and Andrews, 1992), in most cases, tetracycline was used at a sub-inhibitory concentration as reported by Doucet-Populaire et al. (1991) and Ammor et al. (2008). Another reason, and probably the correct one, is that the filter-mating experiments are not refined enough to detect small differences in the transfer frequency.
3.5 Conclusions
In this chapter the 2 bp mutation in the start codon of orf12 of Tn916 has been shown to have an effect on the growth of the cells [BS79A (contains Tn916Δorf12)] compared to the strain carrying wild type Tn916 (BS34A). However, no appreciable difference could be detected in the frequency of transfer to C. difficile CD37. Furthermore this is the first demonstration that the basal level of transcription of tet(M) and downstream genes is enough to provide resistance to inhibitory concentrations of the antibiotic (10 µg ml-1) and enough to allow transfer.
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