Abstract
BackgroundAssembly of the tubulin-like GTPase, FtsZ, at the future division site initiates the process of bacterial cytokinesis. The FtsZ ring serves as a platform for assembly of the division machinery and constricts at the leading edge of the invaginating septum during cytokinesis. In vitro, FtsZ assembles in a GTP-dependent manner, forming straight filaments that curve upon GTP hydrolysis. FtsZ binds but cannot hydrolyze GTP as a monomer. Instead, the active site for GTP hydrolysis is formed at the monomer-monomer interface upon dimerization. While the dynamics of GTP hydrolysis and assembly have been extensively studied in vitro, significantly less is known about the role of GTP binding and hydrolysis in vivo. ftsZ84, a GTPase defective allele of Escherichia coli ftsZ, provides a striking example of the disconnect between in vivo and in vitro FtsZ assembly.ResultsAlthough ftsZ84 mutants are defective for FtsZ ring formation and division under nonpermissive conditions, they are near wild type for ring formation and division under permissive conditions. In vitro, however, purified FtsZ84 is defective in GTP binding, hydrolysis and assembly under standard reaction conditions. To clarify the nature of the FtsZ84 assembly defect, we isolated and characterized three intragenic suppressors of ftsZ84. All three suppressor mutations increased the apparent affinity of FtsZ84 for GTP, consistent with improved subunit-subunit interactions along the longitudinal interface. Although kinetic analysis indicates that the suppressor mutations increase the affinity of FtsZ84 for GTP, all three exhibit reduced rates of GTP hydrolysis and fail to support assembly in vitro.ConclusionTogether, our data suggest that FtsZ, and potentially other enzymes whose assembly is similarly regulated, can compensate for defects in catalysis through increases in substrate binding and subunit-subunit interactions. In addition, these results highlight the dichotomy between commonly used in vitro assembly conditions and FtsZ ring formation in the complex intracellular milieu.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-015-0544-z) contains supplementary material, which is available to authorized users.
Highlights
Of the tubulin-like GTPase, FtsZ, at the future division site initiates the process of bacterial cytokinesis
Arjes et al BMC Microbiology (2015) 15:209 from our laboratory and others indicates that FtsZ concentration is constant throughout the cell cycle and that the precise spatial and temporal regulation of bacterial cell division is governed by tightly orchestrated changes in FtsZ assembly dynamics [3, 4, 7]
Based on our finding that the FtsZ84* mutants restore GTP binding as estimated by Km, we propose the intragenic suppressor mutations increase subunit-subunit affinity and promote nucleotide binding by trapping GTP between in the active site between FtsZ subunits (Fig. 7c)
Summary
Of the tubulin-like GTPase, FtsZ, at the future division site initiates the process of bacterial cytokinesis. FtsZ assembles in a GTP-dependent manner, forming straight filaments that curve upon GTP hydrolysis. Of the tubulin-like GTPase FtsZ at the future division site is a fundamental step in bacterial cytokinesis [1, 2]. The active site for GTP hydrolysis is formed at the longitudinal interface between two FtsZ subunits where the GTPbinding pocket of one monomer contacts the T7 synergy loop of the adjacent monomer (Fig. 1a) [2, 8]. GTP binding stimulates FtsZ assembly into single stranded polymers ( known as protofilaments). Once GTP has been hydrolyzed to GDP, polymers can disassemble and exchange GDP for GTP and assemble once again [2]
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