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Abstract

In most bacteria, cell division requires assembly of FtsZ, the tubulin homologue, into a ring-like structure, the so-called Z-ring. The Z-ring acts as a scaffold for the cell division machinery and marks the future division site. To precisely localize the Z-ring, bacteria have evolved different regulatory mechanisms. In the model organism Caulobacter crescentus, Z-ring positioning depends on a P-loop ATPase, MipZ. MipZ forms bipolar gradients within the cell and acts as an inhibitor of FtsZ polymerization, thereby restricting assembly of the Z-ring to the midcell region. Gradient formation is driven by the alternation of MipZ between a monomeric and dimeric state with distinct interaction patterns and diffusion rates. This alternation results in a dynamic localization cycle, in which MipZ continuously oscillates between non-specific chromosomal DNA and the polarly localized ParB protein. In this study, we investigated the function of MipZ by mapping its interaction interfaces with FtsZ, ParB and DNA. We systematically exchanged surface-exposed residues using alanine-scanning mutagenesis. Analyzing the subcellular distribution of the mutant proteins as well as their ability to support division site placement, we identified four clusters of residues that are important for MipZ activity. Two of them are likely responsible for contacting FtsZ and chromosomal DNA, respectively, whereas the other two appear to be involved in the interaction with ParB. Notably, the DNA-binding and FtsZ-binding interfaces of MipZ comprise residues from both monomeric subunits and are located on opposite sides of the dimer. This result is consistent with the previous finding that the regulatory effect of MipZ is specific for its dimeric form and that only the dimeric form contacts DNA and FtsZ. We also found that the DNA-binding region mainly consists of positively charged arginine and lysine residues. In vivo and in vitro studies showed that mutation of these residues impairs the DNA-binding activity of MipZ to different extents; moreover, mutation of R194 and R198 abolished the MipZ-DNA interaction. These results provide the first detailed analysis of the interaction determinants of MipZ and deepen our knowledge of the molecular mechanism underlying the function of this intriguing cell division regulator.