Manipulation of topoisomerase expression inhibits cell division but not growth and reveals a distinctive promoter structure in Synechocystis

Abstract

In cyanobacteria DNA supercoiling varies over the diurnal cycle and is integrated with temporal programs of transcription and replication. We manipulated DNA supercoiling in Synechocystis sp. PCC 6803 by CRISPRi-based knockdown of gyrase subunits and overexpression of topoisomerase I (TopoI). Cell division was blocked but cell growth continued in all strains. The small endogenous plasmids were only transiently relaxed, then became strongly supercoiled in the TopoI overexpression strain. Transcript abundances showed a pronounced 5’/3’ gradient along transcription units, incl. the rRNA genes, in the gyrase knockdown strains. These observations are consistent with the basic tenets of the homeostasis and twin-domain models of supercoiling in bacteria. TopoI induction initially led to downregulation of G+C-rich and upregulation of A+T-rich genes. The transcriptional response quickly bifurcated into six groups which overlap with diurnally co-expressed gene groups. Each group shows distinct deviations from a common core promoter structure, where helically phased A-tracts are in phase with the transcription start site. Together, our data show that major co-expression groups (regulons) in Synechocystis all respond differentially to DNA supercoiling, and suggest to re-evaluate the long-standing question of the role of A-tracts in bacterial promoters.

Self-sustained rhythmic behavior ofSynechocystisPCC 6803 under continuous light conditions in the absence of light-dark entrainment

“…Sugar feeding blocked the effect of resetting the clock by a dark pulse, even in the absence of light (62). However, 24h rhythms without LD-entrainment have been observed not only for plants, microalgae, and cyanobacteria in bioreactor-setups (6, 68, 69) but also for non-photosynthetic prokaryotes like E. coli (55).…”

“…The loss of pattern may result from the inability to detect glycogen fluctuations in the backscatter signal rather than a disruption of circadian rhythms. Similarly, 24-hour rhythms in Synechocystis under LL disappeared after manipulation of topoisomerase expression, which increased glycogen content and optical density without affecting cell numbers (6).…”

“…For enzymatic digestion, samples were incubated at 60°C for 2 h. For the spectrometric glycogen determination, the Sucrose/D-Glucose Assay Kit from Megazyme (K-SUCGL) was applied according to the manufacturer’s specifications, but omitting the fructosidase reaction step and scaling down the total reaction volume to 850 μL according to Behle and Dietsch et al . 2022 (6). Absorbance at 510 nm was measured using a BMG Clariostar Photospectrometer.…”

“…The counted events in electric field cell counting are a mixture of live and dead cells, as well as cell debris and background signals from the growth medium. Only counts with a diameter between 1.5 and 5 μm were considered for the experiment (6). We conducted six replicates for Synechocystis WT and four replicates for the Δ kaiABC .…”

“…Cyanobacteria, a monophyletic group of photoautotrophic prokaryotes, have been used for decades to study fundamental processes such as photosynthesis and gene regulation, and are attractive hosts for biotechnological production (5, 6). They are well-suited model-organisms for studying circadian rhythms and the clock’s connection to metabolism (7–9).…”