Z-Ring-Associated Proteins Regulate Clustering of the Replication Terminus-Binding Protein ZapT in Caulobacter crescentus

Abstract

Regulated organization of the chromosome is essential for faithful propagation of genetic information. In the model bacterium Caulobacter crescentus, the replication terminus of the chromosome is spatially arranged in close proximity to the cytokinetic Z-ring during the cell cycle. Although the Z-ring-associated proteins ZapA and ZauP interact with the terminus recognition protein ZapT, the molecular functions of the complex that physically links the terminus and the Z-ring remain obscure. In this study, we found that the physical linkage helps to organize the terminus DNA into a clustered structure. Neither ZapA nor ZauP was required for ZapT binding to the terminus DNA, but clustering of the ZapT-DNA complexes over the Z-ring was severely compromised in cells lacking ZapA or ZauP. Biochemical characterization revealed that ZapT, ZauP, and ZapA interacted directly to form a highly ordered ternary complex. Moreover, multiple ZapT molecules were sequestered by each ZauP oligomer. Investigation of the functional structure of ZapT revealed that the C terminus of ZapT specifically interacted with ZauP and was essential for timely positioning of the Z-ring in vivo. Based on these findings, we propose that ZauP-dependent oligomerization of ZapT-DNA complexes plays a distinct role in organizing the replication terminus and the Z-ring. The C termini of ZapT homologs share similar chemical properties, implying a common mechanism for the physical linkage between the terminus and the Z-ring in bacteria. IMPORTANCE Rapidly growing bacteria experience dynamic changes in chromosome architecture during chromosome replication and segregation, reflecting the importance of mechanisms that organize the chromosome globally and locally within a cell to maintain faithful transmission of genetic material across generations. During cell division in the model bacterium Caulobacter crescentus, the replication terminus of the chromosome is physically linked to the cytokinetic Z-ring at midcell. However, the functions of this physical linkage are not fully understood. We adopted biochemical and cell-biological techniques to characterize the linkage, including the terminus-binding protein ZapT and the Z-ring-associated protein ZauP. We obtained evidence that the Z-ring organizes the chromosome terminus into a compact structure at midcell via specific interaction between ZapT and ZauP oligomers. Because these proteins are conserved in diverse Gram-negative bacteria, our findings highlight a novel and conserved role for the linker complex in regulated organization of the chromosome terminus.

Mechanisms for Chromosome Segregation in Bacteria

“…These three proteins interact to form a complex that anchors the ter region to the Z-ring ( Espéli et al, 2012 ; Männik et al, 2016 ), thus orchestrating divisome positioning with chromosome segregation. A similar coupling of the terminus and the divisome was recently also found in C. crescentus ( Ozaki et al, 2020 , 2021 ). ZapA and ZauP, the functional counterparts of ZapA and ZapB in E. coli , interact with ZapT (the MatP counterpart).…”

Engineering aSinorhizobium melilotichassis with monopartite, single replicon genome configuration

“…For most of the cell cycle, the replication terminus of the C. crescentus chromosome is colocalizing with FtsZ, which accumulates at the new cell pole in G 1 -phase cells and forms the Z-ring in the midcell region towards the end of the cell cycle [63]. Spatial coupling of both is mediated by interaction of the terminus recognition protein ZapT [63], the Z-ring associated proteins ZapA [73] and ZauP [64, 74]. A potential homolog of ZapA (SMc03976) was reported in S. meliloti [75] and we found a candidate for a ZapT homologue (SMc01787) by sequence comparisons.…”

“…(49)(50)(51)(52) In C. crescentus, tethering of terC to the new cell pole is mediated by the terminus recognition protein ZapT, the Z-ring associated proteins ZapA and ZauP that colocalize with FtsZ at the new cell pole. (53)(54)(55) It can be assumed that orthologous proteins and similar mechanisms are also involved in the spatial confinement of oriC and terC at the old and new cell poles, respectively, in S. meliloti. However, factors responsible for the spatial confinement of oriA and oriB to the subpolar region of the old pole are still enigmatic.…”

The Caulobacter crescentus DciA promotes chromosome replication through topological loading of the DnaB replicative helicase at replication forks

“…This assay was performed essentially as described previously ( 39 , 40 ). In brief, exponentially growing cells (100 μl) were fixed in ice-cold 70% ethanol, washed, and resuspended in buffer supplemented with 2 μM SYTOX Green (Thermo Fisher Scientific).…”

“…This assay was performed essentially as described previously ( 22 , 39 ). Briefly, proteins were loaded onto a Superdex 200 Increase 10/300 GL column (24 ml column volume), equilibrated with SEC buffer (25 mM Tris–HCl [pH 7.5], 300 mM sodium chloride, 20% glycerol, 0.1 mM ATP and 5 mM magnesium chloride), and fractionated at a flow rate of 0.2 ml/min.…”