In Section 5.3, MMF4 was shown to cause a significant reduction in luminescence produced by the L3+mmyR strain, something not seen at either of the other MARE operators. It was therefore decided to investigate further into this result, adding 100 µM MMF2 and MMF5 to L3+mmfR and inspecting for changes in luminescence. The assay was carried out using the same methods as were used for L1+mmyR strains and results are shown in Figure 5.10, Figure 5.11 and Table 5.7. Data has also been compared to luminescence produced by L3+mmfR
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Figure 5.10. Luminescence produced by the lux operon under the control of mmyBp and MmyR in the absence and presence of 100 µM MMF2, 4 or 5 compared to a positive control over 72 hours
Average light production is calculated as a relative ratio of luminescence produced by L3+mmyR with no MMFs (giving this sample a value of 1). Strains used: L3+mmfR –
luxCDABE under the control of mmyBp and mmfR under the control of ermEp* (pKMS01), L3+mmyR – luxCDABE under the control of mmyBp and mmyR under the control of
ermEp* (pKMS03), L3+pCC4 – luxCDABE under the control of mmyBp, no repressors.
Unless otherwise specified, all data points are for the L3+mmyR strain.
Figure 5.11. Bar chart of luminescence produced by the lux operon under the control of mmyBp and MmyR in the absence and presence of 100 µM MMF2, 4 or 5 compared to a positive control at 48 hours
Average light production is calculated as a relative ratio of luminescence produced by L3+mmyR with no MMFs (giving this sample a value of 1).
Strains used: Same as Figure 5.10. Unless otherwise specified, all data points are for the
0 10 20 30 40 50 60 70 80 0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 Time (hours)
Ratio of luminescence produced compared
to L3+ mmyR with no MMFs no MMFs MMF2 MMF4 MMF5 L3+mmfR no MMFs L3+pCC4 no MMFs L3+mmyR no MMFs MMF2 MMF4 MMF5 L3+no MMFsmmfR L3+pCC4 no MMFs 0 1 2 3 4
Ratio of luminescence produced
Effect of different molecules on luminescence by L1:MmyR
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Table 5.7. A t-test analysis of luminescence produced by the lux operon under the control of mmyBp and MmyR in the presence of 100 µM MMF2, 4 or 5 at 48 hours
Average light production is calculated as a relative ratio of luminescence produced by L3+mmyR with no MMFs (giving this sample a value of 1). The p-value was also calculated based on L3+mmyR with no MMFs. Data for L3+mmfR is included as a comparison to represent more significant repression seen in this kind of assay.
MMF added to
L1+mmyR p-value difference? Significant
Average light production at 48 hr (R.R) MMF2 (100 µM) 0.5222 FALSE 0.88 MMF4 (100 µM) 0.0016 TRUE 0.56 MMF5 (100 µM) 0.0002 TRUE 0.39 L3+mmfR (no MMF) <0.0001 TRUE 0.26
The findings of the t-test analysis in Table 5.7 showed that both MMF4 and MMF5 caused a significant decrease in luminescence of the L3+mmyR strain, with up to a 61% reduction in luminescence seen compared to no MMFs. This compares to a reduction in luminescence of 74% for L3+mmfR versus L3+mmyR indicating that the presence of MMF5 brings MmyR repression almost to the level of that seen for MmfR. MMF2 did also cause a slight decrease in luminescence but it was not enough of one to be classed as statistically significant. Therefore, the greater the binding potential of the MMF to MmfR the greater the apparent decrease in luminescence it brought about in the L3+mmyR strain as well as the lower the p- value. A higher concentration of MMF2 therefore may reveal more significant results if trialled.
It should be noted that due to time constraints, L3+pCC4 had not been trialled with all five of the MMFs as a control during the preliminary investigations in Chapter 3. However, when trialled with L1+pCC4, none of the MMFs produced a significant change in luminescence at a 100 µM concentration. It cannot be said with certainty that this is also the case with the L3+pCC4 strain but it reduces the chances that the MMFs are bringing about a change in luminescence for the L3+pCC4 strain in the absence of MmyR/MmfR. There is also currently no experimental evidence of this extra MMF transcriptional regulation. Therefore, until further research is done, MMF interactions with MmyR rather than any other transcriptional control are the most reasonable explanation for the changes in luminescence seen in Table 5.7 and Figure 5.11.
Based on the analysis from Table 5.7 and Figure 5.11 therefore, it is possible that at least some of the MMFs can bind to MmyR to an extent and, in combination with the DNA binding sequence at the L3 MARE operator, cause MmyR to work better as a repressor. It has
been shown by amino acid sequence analysis that MmyR does not have the same ligand- binding pocket as MmfR (Figure 5.1). However, if MmyR was indeed binding the MMFs at the L3 MARE operator, it appears to lead to a conformational that may help it better bind the DNA instead of its release. It is not inconceivable therefore that the ligand-binding site could vary considerably between MmfR and MmyR and yet they can still both interact with the MMFs, with the differences in the structure of the binding pocket resulting in the opposite effect that the MMFs have on each.
When looking at the levels of repression achieved by L3+mmyR in the presence of the MMFs, there is never more than a 60% reduction in luminescence compared to when no MMFs are present. The impact of this leaky repression in the wild type system is not known. It must not be forgotten however, that the mmyR promoter was shown to be the strongest of the five studied in the methylenomycin cluster (Figure 3.16), indicating that relatively high levels of MmyR are produced in the absence of repression. In this assay, MmyR repression is limited by the strength of the ermE* promoter. MmyR also appears to not regulate itself at the L2 MARE operator, potentially leading to even greater levels produced in a wild type system compared to MmfR, which can repress its own production.
If the MMFs are promoting the binding of MmyR at the L3 MARE operator then the implications would be the repression of the mmyBQEDXCAPK and mmmYF operons, where
mmyB codes for a pathway specific transcriptional activator and all other genes code for enzymes thought to be used in methylenomycin biosynthesis. If there are greater levels of MmyR produced in a wild type system than this synthetic system then there may be total repression of the production of MmyB, which could be enough to stop the entire biosynthetic pathway from being expressed.
In Section 5.3, it was found that MmyR caused the greatest repression at the L3 MARE operator, followed by the L1 MARE operator, with no binding shown at the L2 operator. It is not totally clear why MmyR did not also work as a better repressor at the L1 operator in the presence of the MMFs. It is possible that the weaker binding of MmyR at 24 bp L1 MARE operator sequence means that the addition of 100 µM MMFs was not enough to cause a significant change in luminescence. If this is the case, a higher concentration of MMFs being added to the L1+mmyR may reveal higher levels of repression. Alternatively, when MmyR binds to the L1 MARE operator it may do so in a conformation that makes it harder for the MMFs to enter or interact with its ligand-binding pocket. The operator at the L3 intergenic region shares less than 63% identity (137) with the one at the L1 intergenic region, it is possible therefore that MmyR is in a slightly different conformation when bound at each of
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