A thermosensitive defect in the ATP binding pocket of FtsA can be suppressed by allosteric changes in the dimer interface

Abstract

In Escherichia coli, initial assembly of the Z ring for cell division requires FtsZ plus the essential Z ring-associated proteins FtsA and ZipA. Thermosensitive mutations in ftsA, such as ftsA27, map in or near its ATP binding pocket and result in cell division arrest at non-permissive temperatures. We found that purified wild-type FtsA bound and hydrolysed ATP, whereas FtsA27 was defective in both activities. FtsA27 was also less able to localize to the Z ring in vivo. To investigate the role of ATP transactions in FtsA function in vivo, we isolated intragenic suppressors of ftsA27. Suppressor lesions in the ATP site restored the ability of FtsA27 to compete with ZipA at the Z ring, and enhanced ATP binding and hydrolysis in vitro. Notably, suppressors outside of the ATP binding site, including some mapping to the FtsA-FtsA subunit interface, also enhanced ATP transactions and exhibited gain of function phenotypes in vivo. These results suggest that allosteric effects, including changes in oligomeric state, may influence the ability of FtsA to bind and/or hydrolyse ATP.

FIG. 1. Thermosensitive mutations of ftsA map to the ATP-binding site

A structure of FtsA from T. maritima with a magnified region highlighting locations of lesions that render E. coli FtsA thermosensitive highlighted in yellow (S195P, encoded by ftsA27 and the focus of this study) or purple (all other known lesions). Water molecules are denoted by red dots.

FIG. 2. Intragenic suppressors of ftsA27 restore thermoresistance and map to different regions of FtsA

A) Serial dilution growth assay of WT E. coli and cells expressing chromosomal ftsA27 or an intragenic suppressor of ftsA27 spotted on plates incubated at the indicated temperatures. B) Locations of the intragenic suppressor mutations on the dimer structure of T. maritima FtsA.

FIG. 3. Resistance of ftsA27 and ftsA27 suppressors to overproduced ZipA

A–C) IFM images showing localization of FtsA or FtsZ in cells with WT chromosomal ftsA (A) ftsA27 (encoding S195P) (B) or the suppressor encoding G50E S195P (C), containing zipA expressed from a sodium salicylate-inducible plasmid at three concentrations of inducer. Scale bars are 2 µm. D) Serial dilution growth assay of WT and ftsA27 cells containing zipA expressed from a sodium salicylate-inducible plasmid, under the indicated inducer concentrations. E) Growth assay performed as described in B, with ftsA27 suppressor cells. Growth assays from different plates are shown as composite images.

FIG. 4. Some mutations that suppress ftsA27 (S195P) also suppress R300E toxicity and loss of function

Flag-ftsA alleles indicated were expressed from a plasmid with the IPTG-inducible trc promoter in WM1115 (ftsA12 (ts)) and grown in serial dilution spots at the indicated temperature with indicated amounts of IPTG. Growth assays from different plates are shown as composite images.

FIG. 5. Some mutations that suppress ftsA27 allow bypass of zipA and inhibit formation of cellular FtsAΔMTS polymers

A) Serial dilution growth assay of indicated flag-ftsA alleles expressed from the IPTG inducible weak trc promoter on plasmids (uninduced) in a W3110 zipA1 (ts) background at indicated temperatures. Growth assays from different plates are shown as composite images. B) Presence or absence of cellular FtsA polymers after overproduction of several selected FtsA derivatives deleted for their C-terminal membrane targeting sequences. Overproduction of the FLAG-FtsA variants from the stronger trc promoter on pDSW208 was induced for 1 h with 0.1 mM IPTG; cells were then stained for FtsA and imaged by IFM. Scale bar = 2 µm.

FIG. 6. Purified FtsA, FtsA27 and its suppressors display levels of ATPase activity that roughly correlate with their in vivo activities

A) Rates of WT FtsA ATPase activity in 1 mM ATP with indicated amounts of FtsA protein. B) Rates of ATPase activity with 0.25 µM of indicated FtsA protein variant.

FIG. 7. Rates of FtsA ATPase activity depend on type and amount of Mg⁺⁺ ions as well as temperature

A) Rates of ATPase activity with WT FtsA in the presence of indicated metal ions. B) Rates of ATPase activity with WT FtsA and FtsA27 at the indicated concentrations of MgCl₂. C) Rates of ATPase activity with the indicated FtsA protein at indicated temperatures. All reactions contained 0.25 µM protein.