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Abstract

Bacteria are known to tightly control the spatial distribution of certain proteins by positioning them to distinct regions of the cell, particularly the cell poles. These regions represent important organizing platforms for several processes essential for bacterial survival and reproduction. The proteins localized at the cell poles are recruited to these positions by interaction with other polar proteins or protein complexes. The α-proteobacterium Caulobacter crescentus possesses a self-organizing polymeric polar matrix constituted of the scaffolding protein PopZ. PopZ recruits to the cell poles several proteins involved in various essential processes such as chromosome segregation and the regulation of cell division. This latter process is controlled by the spatial regulator MipZ, which coordinates chromosome segregation with cell division. The two essential proteins PopZ and MipZ both physically interact with the centromere binding protein ParB, an essential element of the chrosomome segregation system of Caulobacter crescentus. The main function of the ATPase MipZ is to position the cell division apparatus by spatially restricting the localization of the key cell division protein FtsZ to midcell. MipZ accomplishes this function by interacting with chromosomal DNA and forming a shallow gradient, with a high concentration at the cell poles and a low concentration near the midcell, therefore permitting FtsZ polymerization solely at midcell. The formation of the MipZ bipolar gradient is intimately linked to the establishment of the multimeric matrix PopZ at the cell poles, which insures the anchorage of ParB-parS complexes at the cell poles. In this study, we have uncovered the inhibitory mode of action of the polar element MipZ on FtsZ polymerization and identified the interaction regions of MipZ with its three interaction partners, ParB, FtsZ and the chromosomal DNA. We found that similarly to the FtsZ assembly inhibitor from Escherichia coli MinC, MipZ is capable of inhibiting FtsZ polymerization as well as shortening FtsZ polymers into smaller oligomers. Our results show also that the inhibitory effect of MipZ on FtsZ polymerization is independent of its ability to stimulate the FtsZ GTPase activity. Mapping of the binding interfaces of MipZ revealed that the DNA- and ParB-binding regions are overlapping and mainly constituted of positively charged residues, whereas two distinct regions appear to be involved in FtsZ-binding. We also purified the polar factor PopZ from soluble fractions and provided relevant data related to its secondary structure composition and its assembly into higher-order structures. Our in vitro analysis on PopZ, revealed among others that it is mainly composed of α-helices and unstructured regions and forms relatively straight filament-like structures differing from what was previously reported. Altogether, the data obtained in this work bring more knowledge about two key elements of C. crescentus.