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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