Investigation of the regulatory interactions and dynamics between the circadian clock and sigma factors in the cyanobacterium Synechococcus elongatus

Circadian clocks are biological pacemakers that oscillate with a circa 24 h period. Many organisms across different taxa have evolved circadian clocks, which allow the anticipation of daily events caused by the Earth’s rotation and provide an adaptive advantage. The molecular mechanisms underlying circadian clocks have been studied extensively. However, how the circadian clock couples to multiple other biological processes in individual cells and what the physiological consequences of this coupling are remains unclear. To help address this, we studied the coupling of the circadian clock to sigma factor expression in Synechococcus elongatus PCC 7942 (S. elongatus), a fresh-water unicellular cyanobacterium. The core of the circadian clock of S. elongatus is composed of only three proteins, KaiA, KaiB and KaiC, but generates a 24 h expression rhythm in multiple downstream genes, including sigma factors. Sigma factors are required for promoter recognition and transcription initiation in bacteria, and there are 10 different sigma factors in S. elongatus. In order to investigate the coupling of cellular processes in S. elongatus, we needed to find suitable fluorescent protein (FP) pairs that allow multiplex imaging. We first developed and characterised new FP reporters from a total of six candidate FPs. We validated the new FP reporters by generating multiplexed reporters of the cyanobacterial clock. Next, using the new FP reporters and quantitative single-cell microscopy, we found that the expression of the group 2 sigma factor rpoD4 was not only modulated by the circadian clock but also pulsed at cell division. By simultaneously tracking the expression dynamics of rpoD4 and of a reporter for the essential cell division protein FtsZ, we could correlate their levels in individual cells. We then investigated the mechanism of rpoD4 pulsing at cell division and its potential function. Using promoters of different lengths we showed that the cis-regulatory elements of rpoD4 transcription are located within 100 bp upstream of its start codon. In addition, deletion of each alternative sigma factor or the known rpoD4 regulator NtcA did not abolish the rpoD4 pulsing dynamics. By analysing single cells grown on agarose pads and in liquid cultures, we found rpoD4 was required for maintaining the correct cell size at division and the correct free-running period of the circadian clock. To summarise, our study revealed intriguing sigma factor pulsing dynamics that receive input and feedback into the circadian and cell division oscillators in cyanobacteria.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2022

Author dc:creator

• Ye, Chao

Advisor dc:contributor.advisor

• Locke, James

• Circadian Clock
• Cyanobacteria
• Fluorescent Protein Reporters
• Sigma Factors

Licence

• https://www.rioxx.net/licenses/all-rights-reserved/

Language dc:language

eng

DOI dc:identifier.doi

https://doi.org/10.17863/CAM.90964

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/343540