Bacterial cell cycle and growth phase switch by the essential transcriptional regulator CtrA

Abstract

11Many bacteria acquire dissemination and virulence traits in G1-phase. CtrA, an 12 essential and conserved cell cycle transcriptional regulator identified in the dimorphic 13 alpha-proteobacterium Caulobacter crescentus, first activates promoters in late S-14 phase and then mysteriously switches to different target promoters in G1-phase. We 15 uncovered a highly conserved determinant in the DNA-binding domain (DBD) of CtrA 16 uncoupling this promoter switch. We also show that it reprograms CtrA occupancy in 17 stationary cells inducing a (p)ppGpp alarmone signal perceived by the RNA 18 polymerase beta subunit. A simple side chain modification in a critical residue within 19 the core DBD imposes opposing developmental phenotypes and transcriptional 20 activities of CtrA. A naturally occurring polymorphism in the rickettsial DBD resembles 21 a mutation that drives CtrA towards activation of the dispersal (G1-phase) program in 22Caulobacter. Hence, we propose that this determinant dictates promoter 23 reprogramming during the growth transition of obligate intracellular rickettsia 24 differentiating from replicative cells into dispersal cells. 25 26 rod-shaped and dimorphic alpha-proteobacterium that undergoes an asymmetric cell 31 division into two unequally sized and polarized daughter cells: a motile and non-32 replicative dispersal (swarmer, SW) cell residing in G1-phase and a capsulated and 33 replicative (stalked, ST) cell. In C. crescentus, cell cycle progression is intimately tied 34 to polar remodeling via a circuit of transcriptional activators that direct sequential gene 35 expression programs 1 . The G1-phase program is implemented in the SW daughter cell 36 that inherits the new cell pole where the flagellum and adhesive pili are located. By 37 contrast, the old pole is inherited by the replicative ST cell that is engaged in DNA-38replication. As the cell cycle proceeds, the ST cell prepares for division, expresses the 39 late S-phase program and polarizes before dividing asymmetrically into a SW and ST 40 daughter cell ( Figure 1A). The transcriptional programs are not only temporally 41 ordered, but also spatially confined during cytokinesis, with the G1-phase program 42 being activated in the nascent SW chamber during cytokinesis, but not in the ST cell 43 chamber 1,2 . 44 Cell cycle analyses are facile with C. crescentus because the non-capsulated 45 G1-phase (SW) cells can be separated from capsulated S-phase (ST) cells by density 46 gradient centrifugation 3 . Replicative functions are acquired with the obligate G1àS-47 classes and recognized by the C-terminal DNA binding domain (DBD) of CtrA. At the 65 N-terminus, CtrA harbors a receiver domain (RD) with a phosphorylation site at a 66 conserved aspartate (at position 51, D51). Phosphorylation at D51 stimulates DNA 67 binding and is required for viability. The hybrid histidine kinase CckA directs a multi-68 component phosphoryl-transfer reaction to D51 of CtrA 10,11 . Though loss of CckA is 69 lethal, missense mutations in the RD were isolated in ...

Pulling the strings of cell cycle: a non-coding RNA, CcnA, modulates the master regulators CtrA and GcrA in Caulobacter crescentus

“…Other regulators of transcription intervene to fine tune the cell cycle-regulated transcription of genes that must be activated at specific phases of the cell cycle; for example, MucR and SciP regulate CtrA activity (Delaby et al, 2019; Fumeaux et al, 2014; Gora et al, 2010, 2010, 2013). The interconnections between DnaA, GcrA, CcrM and CtrA create an intricate network whose behavior emerges from the integration of multiple master regulatory inputs.…”

Specificity in glycosylation of multiple flagellins by the modular and cell cycle regulated glycosyltransferase FlmG

“…We determined the β-galactosidase activities from these reporters in WT cells and ctrA401 cells that have a missense mutation (encoding the T170I substitution) in ctrA, a partial loss of function mutation that renders cells temperature sensitive for growth at 37°C ( Quon et al, 1996 ). At the permissive temperature (30°C), ctrA401 cells grow well, albeit several CtrA-activated promoters show substantially reduced activity ( Quon et al, 1996 ; Delaby et al, 2019 ). We observed a similar reduction in activity of the promoter probe reporters of flmG , flmA, and neuB in ctrA401 cells ( Figure 6B ).…”

The DUF1013 protein TrcR tracks with RNA polymerase to control the bacterial cell cycle and protect against antibiotics

“…When needed, sucrose was added at 0.3% final concentration and vanillate at 50 μM final concentration. Swarmer cell isolation, electroporations, biparental matings, and bacteriophage ϕCr30-mediated generalized transductions were performed as previously described (26,59). Plasmids for β-galactosidase assays were introduced into C. crescentus by electroporation.…”

“…CtrA directly activates promoters of many cell cycle-regulated transcripts (14,16), for example, those encoding flagellar and pilus subunits, as well as an inhibitor of capsulation (HvyA) in G1phase (20). Mutations that perturb cell cycle progression, including those that reduce CtrA activity or abundance, typically cause a reduction in the number of G1 cells in the population and therefore also curb the overall motility of the population on motility (soft agar) plates (21,23,26,27).…”