In Chapter 4 it was seen that when any of the five MMFs were added to the L1+mmfR strain, there was an apparent release of MmfR and an increase in luxCDABE expression. MMF4 was added to the 11NY strain and the effects on luminescence observed. The results from this assay can be found in Figure 6.3. Data for 11NY was compared to the M145 negative control and L1+pCC4 positive control.
Figure 6.3. Relative level of luminescence produced by the lux operon under the control of mmfLp and MmfR (under its own auto-regulatory control) in the presence and
absence of 400 nM MMF4 over time
The level of luminescence was calculated as a ratio compared to that of the M145 strain
The 11NY strain was more representative of the wild type system than L1+mmfR, i.e. mmfR is being controlled by its native promoter rather than ermEp*. Despite this, it can be seen from Figure 6.3 that the expression of the lux genes was not inducible upon the addition of MMFs in the 11NY strain. In the presence of MMF4, the levels of luminescence remain very close to those produced by the M145 negative control. The reason for this is again likely to be the auto-regulatory nature of MmfR. Upon the release of MmfR from the operator by MMF4, there is only a short window of lux expression before more MmfR is also made, repressing the expression of the luciferase genes almost immediately. Finding this very short window of luminescence was not experimentally practical.
A concentration of 400 nM MMF4 was used for the 11NY data collected, this is the same concentration as was used for the experiment displayed in Figure 3.10 where there was an observable increase in luminescence by L1+mmfR in the presence of MMF4. As the L1+mmfR strain has mmfR under the control of the constitutive ermE* promoter, it means that there are much longer lasting effects of de-repression by the MMFs. MmfR is produced at a relatively constant rate, where expression is presumed to be unaffected by the addition of the MMFs and is independent of its own repressive activity.
Trials where the concentration of the MMFs was increased did not show any more release of MmfR in the 11NY strains (data not shown here).
0 1 2 3 4 5 6 7 8 9 10 0 10 20 30 40 50 60 Time (days) Ratio of luminescence 11NY inducibility M145 L1_noMMF 11NY_noMMF 11NY_400 nM MMF4
It is unclear how these findings on the lack of observable effect of the addition of the MMFs in the 11NY strain is representative of what actually occurs in the wild type system. Studies with mmfLHP mutants have shown that the addition of MMFs will induce the production of methylenomycin (Table 6.1).(71) There are a number of possible explanations on how this release of MmfR occurs in the wild type system when it was not seen in this reporter system. The first is that there was a small window of MmfR release (and therefore increased luminescence) but due to readings only being taken every 24 hours, this period of luxCDABE
expression was missed. A small window of expression may be enough to result in the expression of mmyB and thereby switching on the entire biosynthetic cluster. Another possible explanation is that in the wild type system it is a very specific threshold concentration of the MMFs needed to release MmfR. A concentration too far above or below this specific threshold level may not alter the MmfR/MMF/operator feedback loops in a way that results in methylenomycin production. This precise threshold concentration of MMFs is not known but if more time was available, a gradient of different MMF concentrations could be trialled with the 11NY strain to check this hypothesis (see Figure 6.4). A final consideration as to how MmfR is released in the wild type system is that other factors may also be at play, altering the window of time during which MmfR is released. In the M145 strain used in this assay, none of the methylenomycin BGC is present except those genes added in the lux vectors and so any extra regulation by this cluster would also be lacking.
Figure 6.4. Schematic of proposed assay to measure the effects of a gradient of MMFs on the repression of the lux operon when mmfR expression is under auto-regulatory control
The lux operon is under the control of mmfLp and the L1 MARE operator. If there is a narrow threshold window of MMF concentration that will induce the strains containing the 11NY vector, this
6.4
Effect of the MMFs on MmyR When Under MARE
Operator Regulation
When MMF4 was trialled with the sp105 strain, no obvious change in luminescence was observed (data not shown here). This is consistent with the data reported in Section 5.4 where the addition of MMF4 produced no significant changes in luminescence for strains containing the L1 intergenic region with mmyR under the control of ermEp*. Having mmyR under the control of the L1 MARE operator and mmfRp would not be expected to change this phenotype as there should be no change in the ligand-binding pocket of MmyR.
Although MmyR may not directly control the expression of its own gene, there is a possibility that it has an indirect regulatory effect on its own activity. This hypothesis relates to putative interactions with the MMFs and will now be discussed.
In Chapter 5, a reduction in lux expression was observed for mmyR strains in the presence of 100 µM MMF2, 4 and 5 at the L3 MARE operator. It appears therefore that in some cases, the MMFs may improve the repressive ability of MmyR. This however was a property not seen for MmyR binding at the L1 MARE operator. It is possible that weaker binding of MmyR seen at the 24 bp L1 MARE operator sequence is not strong enough for the addition of 100 µM MMFs to cause a significant change in luminescence. If this is the case, a higher concentration of MMFs added to the L1+mmyR may reveal higher levels of repression. Alternatively, MmyR may bind to the two operator sites in a slightly different conformation. The L1 and L3 MARE operators share less than 63% identity (137) and it is possible that this difference could have enough of an effect on the MmyR tertiary structure to alter the way that the MMFs can enter its ligand-binding pocket.
There is a biological explanation for why the wild type system may be set up so that at the L1 MARE operator, MMFs would not enhance MmyR binding. This is due to the genes actually being regulated at the L1 MARE site; mmfR and mmfLHP. In particular, mmfLHP which code for MmfLHP, the enzymes used in the assembly of the MMFs.
If the MMFs could bind to MmyR at the L1 MARE operator and make it a better repressor, it would create a negative feedback loop. A MMF-induced enhancement of MmyR repression would switch off the production of more MMFs. This decrease in the concentration of the MMF ligands would then lead to MmyR becoming a less efficient repressor again and a return the system to the over-production of the MMFs. This would then become a feedback cycle alternating between enhanced and reduced MmyR activity, something that would clearly not be productive whilst trying to switch off methylenomycin production. It is
at the L1 MARE operator in the presence of the MMFs. The more MmyR represses these genes, the more it would be down regulating its own effects and thus preventing further repression. The hypothesised different conformations of MmyR therefore may make it indirectly auto-regulatory, based on its resultant control over the levels of MMFs produced, which are possibly needed for its full repressive activity at the L3 MARE operator.
This inference is entirely hypothetical however, and purely based on there results collected in this projects investigation. It does appears however that the different MARE operator sequences are more important in controlling the promiscuous effects of MmyR than MmfR, the latter of which appears to be less selective and have a more similar role at each operator site.
It is very hard to shed light on the exact role of MmyR, which proves to be much more elusive and complex than MmfR regulation. One area that may help develop hypotheses about MmyR activity (and MmfR) is that of a mathematical model of the methylenomycin regulatory system.