To spore or not to spore
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Transcript of To spore or not to spore
TO SPORE OR NOT TO SPORE…
“Chromosomal Arrangement of Phosphorelay
Genes Couples Sporulation and DNA Replication”
Narula et al., 2015Cell 162, 328-337
Joshua Gefen
…that is the question• Is there any describable model for sporulation
gene expression during replication? In the sporulation network there is a transient gene dosage
imbalance during replication.
• What is the composition of the feedback loops in the sporulation gene network?
This paper suggests that there is a pulsed response that couples sporulation and DNA replication.
Sporulation gene translocations which eliminate pulses then lead to sporulation defects.
Some of the experiments indicate that negative feedback leads to pulsatile activation of sporulation master regulator.
Key Terms• DNA Replication– Ori, Terminus, Gene Dosage
• Vegetative Growth– Vegetative Division,
Cell Growth,
• Sporulation– KinA-E, Spo0A, Spo0B,
Spo0F, Phosphorelay
1. 0A Activity Pulses Follow the Completion of DNA Replication
Fluorescently tagged DnaN for replication detection
Using FACS, P0A expression level was measured with a cfp tag
Computation of 0A activity, defined as production rate of the reporter protein, was enabled by the calculation of cell growth rate, inferred from cell elongation rate, and the expression level of P0A-cfp
Tr time of replicationTp time of peak promoter activity
Conclusions• A single pulse of 0A activity is produced every
cell cycle in starvation conditions.• Tp > Tr for the vast majority of the cell cycles,
implying that 0A activity peaks occur after DNA replication is complete.
• (Veening et al., 2009) pulsing Sda (inhibitor of the same kinase as 0A) causing pulsatile response of 0A.
• (Levine et al., 2012) deletion of sda does not completely abolish pulsing.
There is something more…
2. A Hidden Negative Feedback Loop in the Phosphorelay
•A mathematical model describing with and without substrate inhibition of KinA by 0F (phosphotransfer-ase)
monotonic vs. non-monotonic
• Experimental results confirm the non-monotonic model
•KinA is the major cytoplasmatic sporulation kinase
•Mathematical modelling shows the ultrasensitive dependence of [0AP] on the [0F]T/[KinA]T ratio
•Evolutionarily conserved chromosomal locations of KinA and 0Fs in relation to the unconserved lacl chromosomal location.
3. Chromosomal Arrangement of Phosphorelay Genes Provide a Pulse-Triggering Perturbation -
Simulation• Translocated 0F • IPTG inducible
More Modelling Predictions
Chromosomal Arrangement of Phosphorelay Genes Provide a Pulse-Triggering Perturbation
– Experimental Results
Comparison of Pulsing and Non-pulsing responses
Lack of 0A~P Pulsing Leads to Sporulation Defects
Discussion1. A key design feature that of this model is the 0F negative feedback loop.
2. The negative feedback plays a critical role in coupling 0A~P pulsing to DNA replication.3. Alternative explanation, 0A~P-AbrB-Spo0E negative feedback loop is unlikely.
4. Another alternative explanation, Sda negative feedback loop does provide the same outcome for the DNA replication coupled 0A~P pulses but…
Thank you
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Supplementary Figure 1
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Supplementary Figure 3
Supplementary Figure 4
Supplementary Figure 5
Table S1
Table S2
Table S3