5.8. Estrategias de medios no tradicionales
5.8.4. Otros medios no tradicionales o no convencionales
Previous studies of MmyR homologues such as ScbR2 have shown that this pseudo GBL receptor will bind to and be released by endogenous antibiotics, potentially a form of cross regulation between pathways.(138) It would appear logical that either an MMF or the end product of the methylenomycin biosynthetic cluster; methylenomycin A, or one of its precursors may bind to MmyR. In this case however, with limited repression seen in the apo form, MmyR may only be ‘activated’ as a repressor by the cognate antibiotic. Binding to methylenomycin A or a precursor could be a method to prevent the over production of the antibiotic, something that would be potentially toxic to the streptomycetes as well as a drain on cell resources if the antibiotic is not needed anymore. As yet, there has been no experimental evidence of this negative feedback loop.
Both methylenomycin A and the precursor methylenomycin C (Figure 5.6) trialled with the L1+mmyR strain to look for any significant change in luminescence produced. This could be either an increase in luminescence if these small molecules cause the release of MmyR or a reduction in luminescence if they are ‘activating’ MmyR as a repressor.
Figure 5.6. Chemical structures of methylenomycin furans, methylenomycin A and its precursor; methylenomycin C
Methylenomycin A and C have lengthy purification procedures which do not yield large amounts of product. For this reason, preliminary trials were run where the methylenomycins were added in different concentrations to sterile filter paper discs on top of a lawn of
L1+mmyR, in an effort to decrease the volumes of these small molecules needed. From this,
the aim was that more thorough trials could be designed and run when more was known about optimal concentration of the potential ligands. The methylenomycin molecules were diluted
in the group to not be toxic to Streptomyces. Figure 5.7 shows these L1+mmyR plates with methylenomycin C and A added.
Figure 5.7. CCD camera images of luminescence produced by a lawn of S. coelicolor M145 containing the lux operon under the control of mmfLp and MmyR in the presence of methylenomycin A and C compared to a DMSO control
Methylenomycin A and C were used diluted in DMSO at a concentration of 5.5 mM and 6.0 mM respectively and added to sterile paper dots on a lawn of L1+mmyR. Equivalent amounts of DMSO were added as a negative control.
As can be seen in Figure 5.7, there does not appear to be any obvious difference in luminescence when adding methylenomycin A or C when compared to a DMSO control. However, it was very hard to spread an even lawn of Streptomyces over such a large plate, meaning that any small changes in luminescence may be missed due to a non-uniform lawn of streptomycetes present. This investigation was therefore carried forward to tests involving quantitative data taken from 12-well plates as was done in all previous investigations with the luciferase assay. The data for this investigation can be see in Figure 5.8 and Figure 5.9. Due to the lack of methylenomycins available however, the number of trials that could be run were limited and not every possibility could be tested.
Figure 5.8 compares luminescence produced by the L1+mmyR strain in the presence and absence of methylenomycin A or C compared to the L1+mmfR strain over five time points in 72 hours.
coelicolor
Figure 5.8. Luminescence produced by luxCDABE, under the control of mmfLp and MmyR, in the presence of different concentrations of methylenomycin A and C
Average light production is calculated as a relative ratio of luminescence produced by L1+mmyR with no potential ligand added (giving this sample a value of 1).
Strains used: L1+mmfR – luxCDABE under the control of mmfLp and mmfR under the control of ermEp* (pKMS01), L1+mmyR – luxCDABE under the control of mmfLp and
mmyR under the control of ermEp* (pKMS03). Unless otherwise specified, all data points are for the L1+mmyR strain.
The data collected from the methylenomycins show more promise in having interactions with MmyR at the L1 MARE operator than the MMFs. As can be seen in Figure 5.8, there is a general decrease in the levels of luminescence in the presence of methylenomycin A and C compared to those seen for the MMFs in Figure 5.4.
The effects of methylenomycin C and A on luminescence appear greatest in the first 27 hours but this fluctuates a lot, with different concentrations swapping in position in terms of greatest level of light produced. It was found in earlier investigations (see Chapter 3 and 4) that readings from the 48 and 72 hour time points had smaller standard deviations and the cell cultures appeared to have stabilised in growth. The methylenomycins were added at a concentration below the predicted lethal level but will likely still cause stress to the cells and so potentially disrupt their growth. This may explain why a general dip in luminescence was seen at 21 hours in Figure 5.8 before increasing again over the next few days. As a control, methylenomycin C was added to L1+pCC4 strains (data not shown here), this showed no significant difference in luminescence produced when compared to the same strain in the absence of methylenomycin C during the first four time points. There was however a decrease in luminescence at the 72-hour time point, possibly due to cell death caused by the antibiotic.
0 10 20 30 40 50 60 70 80 0.00 0.25 0.50 0.75 1.00 1.25 1.50 Time (hours)
Ratio of luminescence produced compared
to L1+
mmyR
with no MMFs
presence of different MMFs and methylenomycin
L1+mmyR no MMFs 2.2 µM MmA 2.4 µM MmC 7.2 µM MmC 12.0 µM MmC L1:mmfR no MMFs
time point, to avoid this potential cell death and the larger deviations at the earlier time points as well as allowing direct comparisons with all other data collected in previous chapters. The results from this analysis can be found in Figure 5.9 and the corresponding Table 5.6.
Figure 5.9. Bar chart of the effect on luminescence produced by luxCDABE, under the control of mmfLp and MmyR, by the presence of different concentrations of
methylenomycin A and C at 48 hours growth
Average light production is calculated as a relative ratio of luminescence produced by L1+mmyR with no potential ligand added (giving this sample a value of 1).
Strains used: Same as Figure 5.8. Unless otherwise specified, all data points are for the L1+mmyR strain.
Table 5.6. A t-test analysis of the effect on luminescence produced by luxCDABE, under the control of mmfLp and MmyR, by the presence of different small molecules at 48 hours growth
Average light production is calculated as a relative ratio of luminescence produced by L1+mmyR with no MMFs (giving this sample a value of 1). p-value was also calculated based on L1+mmyR with no MMFs. Data for L1+mmfR is included as a comparison to represent how more significant repression may be seen in the assay.
MMF added to
L1+mmyR p-value difference? Significant
Average light production at 48 hr (R.R) MMF1 (100 µM) 0.2008 FALSE 1.09 MMF2 (100 µM) 0.3266 FALSE 0.93 MMF3 (100 µM) 0.1041 FALSE 1.13 MMF4 (100 µM) 0.3122 FALSE 0.93 MMF5 (100 µM) 0.3327 FALSE 1.08 MmA (2.2 µM) 0.0003 TRUE 0.72 MmC (2.4 µM) 0.0017 TRUE 0.79 MmC (7.2 µM) 0.3645 FALSE 0.90 MmC (12 µM) 0.5558 FALSE 0.95 L1+mmfR (no MMF) <0.0001 TRUE 0.15 no MMFs 2.2 µM MmA 2.4 µM MmC 7.2 µM MmC 12.0 µM MmC L1:mmfR 0.0 0.5 1.0 1.5 2.0
Ratio of luminescence produced
Effect of different molecules on luminescence by L1:MmyR
coelicolor
Figure 5.9 and the corresponding t-test analysis from Table 5.6 reveal that both methylenomycin A and C will cause a significant change in luminescence produced by L1+mmyR. However, this was only the case for the lowest concentration of methylenomycin C, with the higher ones showing no significant effect on luminescence produced by L1+mmyR. Equivalent trials were also run with methylenomycin A but there was not enough compound available to get the full set of repeats and so these are not presented here. Nonetheless, the few trials that were run did indicate the same pattern, where only the lowest concentration of methylenomycin A caused a significant change in luminescence produced. The change produced even by the lowest concentrations of methylenomycin A and C are not large however, with a reduction in luminescence of less than 30% for L1+mmyR compared to the same strain with no compound being present. This compares to presence of MmfR (L1+mmfR) producing 85% less luminescence than the L1+mmyR strain (Table 5.6). It is still possible that methylenomycin A or C do cause the ‘activation’ of MmyR as a repressor but results are currently inconclusive and will likely remain so until the effect of methylenomycin concentration and its mechanism of antibiotic action is understood better.