In vivo characterization of Escherichia coli ftsZ mutants: effects on Z-ring structure and function

Abstract

We have characterized the in vivo phenotypes of 17 mutations of Escherichia coli ftsZ. In particular, we determined whether these mutations can complement a null ftsZ phenotype, and we demonstrated that two noncomplementing mutations show partial dominant-negative behavior. We performed immunofluorescence microscopy to determine whether these mutants could assemble into normal or abnormal structures in vivo. The mutants separated into four classes-those that complemented the null and formed normal FtsZ rings, those that complemented the null but formed aberrant FtsZ structures, those that formed aberrant FtsZ structures and did not complement, and those that were unable to form any FtsZ structures. We did not find any mutations that produced nonfunctional Z rings of normal appearance. Surprisingly, some mutants that produced extensively spiraled Z-ring structures divided and grew with a normal doubling time. The analysis was carried out using a complementation system based on an ftsZ deletion strain, a temperature-sensitive rescue plasmid, and a complementation vector that placed mutated ftsZ alleles under the control of the pBAD promoter, which offered several advantages over previous systems.

FIG. 1.

Comparison of wild-type FtsZ induction with rescue of a null ftsZ allele. The left axis (% FtsZ) indicates the level of FtsZ in the cells grown at 30°C, while the right axis (% Comp.) indicates the level of complementation in cells grown at 42°C. The FtsZ measured includes that expressed from both pKD3 and pJSB100. At 100% complementation, there were as many colonies on the induction plate as on the repression control. Arabinose levels (% Ara.) are given on the horizontal axis; ∗ indicates addition of 0.2% glucose in lieu of arabinose to repress plasmid-borne FtsZ production. The error bars indicate standard errors of the mean.

FIG. 2.

Complementation curves for a number of ftsZ mutants. Complementation curves are shown as in Fig. 1. Three noncomplementing mutants (ftsZ-D96A, ftsZ-D212A, and ftsZ-E250K/D253K) are at the bottom of the graph. wt, wild type.

FIG. 3.

Abnormal colony morphology in partially dominant-negative ftsZ mutants. (A) Colonies at approximately normal magnification (bar = 1 cm). The colonies in the top row were grown on 0.005% arabinose, which is insufficient to complement or cause dominant-negative effects. The colonies in the bottom row were grown on 0.5% arabinose, sufficient to cause dominant-negative effects. All plates were grown for 24 h at 30°C, except JSN101 at 0.5% arabinose, which was grown for 48 h due to its extremely low rate of growth. (B) JSN115 colonies at greater magnification (bar = 1 mm). Note the “fried-egg” appearance of the colonies and the normal sectors in some colonies.

FIG. 4.

Expression of mutated ftsZ alleles. JSN mutant strains were grown at 30°C and lysed, and Western blotting was performed using anti-FtsZ antibodies and secondary antibodies conjugated to horseradish peroxidase. A, 0.05% arabinose was added to the culture to induce FtsZ expression; G, 0.2% glucose was added to repress FtsZ expression; ∗, JSN2 without a pJSB plasmid.

FIG. 5.

Pseudochemostat growth of various strains. (A) Growth curves of five strains (measured as the OD of the culture multiplied by the dilution factor due to pseudochemostat dilutions). (B) Average cell lengths of the strains in panel A. Each average is for 30 to 60 cells; error bars are not given due to the lack of normal distribution of cell lengths. wt, wild type.

FIG. 6.

FtsZ structures in complementing strains. All panels show IF microscopy of FtsZ. All strains were grown under complementing conditions at 42°C with 0.05% arabinose to mid-log phase (see Materials and Methods). The arrowhead in panel D points to a spiral that resolves as a double ring. Magnification, ×2,550.

FIG. 7.

FtsZ structures in noncomplementing strains. All panels except C show IF microscopy of FtsZ; panel C shows a differential interference contrast image. All strains were grown at 42°C with 0.05% arabinose to mid-log phase (see Materials and Methods) except panels B to D, where the cells were grown under pseudochemostat conditions (see the text) for the indicated times. Bars = 5 μm. (Panels B and D are at a slightly different magnification.)

FIG. 8.

Locations of mutations that cause aberrant or no Z-ring formation. Mutations in regular type complement the null allele at 42°C, and boldface mutants do not complement. The underlined mutants formed spirals, the italicized mutants did not form Z rings, and the boxed mutants formed more aberrant FtsZ structures.