Precise transcription timing by a second-messenger drives a bacterial G1/S cell cycle transition

Abstract

29Bacteria adapt their growth rate to their metabolic status and environmental 30 conditions by modulating the length of their quiescent G1 period. But the molecular 31 mechanisms controlling G1 length and exit from G1 are poorly understood. Here we 32 identify a key role for the second messenger c-di-GMP, and demonstrate that a gradual 33 increase in c-di-GMP concentration determines precise gene expression during G1/S in 34 Caulobacter crescentus. We show that c-di-GMP strongly stimulates the kinase ShkA, 35activates the TacA transcription factor, and initiates a G1/S-specific transcription 36 program leading to cell morphogenesis and S-phase entry. C-di-GMP activates ShkA by 37 binding to its central pseudo-receiver domain uncovering this wide-spread domain as a 38 novel signal input module of bacterial kinases. Activation of the ShkA-dependent 39 genetic program also causes c-di-GMP to reach peak levels, which triggers S-phase 40 entry and, in parallel, promotes proteolysis of ShkA and TacA. Thus, a gradual 41 increase of c-di-GMP results in a precisely tuned ShkA-TacA activity window enabling 42 G1/S specific gene expression before cells commit to replication initiation. By defining 43 a regulatory mechanism for G1/S control, this study contributes to understanding 44 bacterial growth control at the molecular level. cell division (D or G2) 1 . Since chromosome replication and cell division (C and D 49 periods) remain constant over a wide range of growth rates 2,3 , the step committing 50 cells to initiate chromosome replication largely determines bacterial proliferation rates. 51Bacteria like Escherichia coli or Bacillus subtilis can increase their growth rate by 52 bypassing the B period and by initiating replication multiple times per division cycle 2 . 53In contrast, Caulobacter crescentus strictly separates its cell cycle stages. An 54 asymmetric division generates a sessile stalked (ST) cell, which directly re-enters S-55 phase, and a motile swarmer (SW) cell that remains in G1 for a variable time 56 depending on nutrient availability 4,5 . Coincident with G1/S transition, motile SW cells 57 undergo morphogenesis to gain sessility ( Fig. 1a). But what determines the length of 58 G1 in this organism has remained unclear. 59In C. crescentus, replication initiation is regulated by the cell cycle kinase CckA. 60CckA is a bifunctional enzyme that acts as a kinase for the replication initiation 61 inhibitor CtrA in G1, but switches to being a phosphatase in S phase, resulting in the 62 inactivation of CtrA and clearance of the replication block 6 (Fig. 1a). The CckA switch 63 is governed by two response regulators, DivK and PleD. While DivK controls CckA 64 activity through protein-protein interactions 7-9 , PleD is a diguanylate cyclase, which is 65 responsible for the characteristic oscillation of c-di-GMP during the cell cycle 10 . The 66 concentration of c-di-GMP, below the detection limit in G1, increases during G1/S to 67 reach peak levels at the onset of S-phase 11 where it allosterically stimulates Cc...

Hybrid histidine kinase activation by cyclic di-GMP-mediated domain liberation

“…First, we determined the kinetics of auto-phosphorylation separately using phosphotransfer deficient variants that had the phospho-acceptor D430 mutated (ShkAD/A) or the Rec2 domain deleted (ShkAΔRec2). As found previously 9,14 and consistent with the proposed regulatory model, ShkAΔRec2 is constitutively active (Figs. 5a, b), since it lacks the obstructing Rec2 domain.…”

“…The same kind of interaction occurs in the CckA/c-di-GMP complex, where one of the guanines binds to the β-sheet edge of the CA domain 13 . In the accompanying study 9 , Y338 was identified as a c-di-GMP binding residue in a targeted alanine scan.…”

“…In addition to Y338A, several other mutants (R324A, I340A, S347A, and Q351A) showed severely impaired activity in vivo 9 . Since all these residues contribute to c-di-GMP binding (Fig.…”

“…In vitro experiments presented in this study were carried out using protein expressed from a pET-28a-shkA plasmid. The pET-28a-shkA vector carrying the gene coding for full-length, wild-type ShkA (Uniprot: Q9ABT2) was the same as used in the preceding study 9 . Most point mutants and wild-type variants were generated from this vector following the Q5 Site-Directed Mutagenesis Kit protocol (E0554, New England Biolabs) and primers 1-8 (Sup.…”

“…GMP mediated cross-linking of the DHp and CA domain was identified as the molecular mechanism of the switch 13 . Most recently it was found that ShkA, the other major kinase involved in the regulation of the C. crescentus cell cycle progression and morphogenesis 14 , is also controlled by c-di-GMP 9 . However, in contrast to CckA, which upon binding of c-di-GMP switches into phosphatase mode, the ligand is required for ShkA kinase activity suggesting a distinct mechanism of allosteric control.…”