Probing the catalytic activity of a cell division-specific transpeptidase in vivo with beta-lactams

Abstract

Penicillin-binding protein 3 (PBP3; also called FtsI) is a transpeptidase that catalyzes cross-linking of the peptidoglycan cell wall in the division septum of Escherichia coli. To determine whether the catalytic activity of PBP3 is activated during division, we assayed acylation of PBP3 with three beta-lactams (cephalexin, aztreonam, and piperacillin) in growing cells. Acylation of PBP3 with cephalexin, but not aztreonam or piperacillin, appeared to be stimulated by cell division. Specifically, cephalexin acylated PBP3 about 50% faster in a population of dividing cells than in a population of filamentous cells in which division was inhibited by inactivation or depletion of FtsZ, FtsA, FtsQ, FtsW, or FtsN. However, in a simpler in vitro system using isolated membranes, acylation with cephalexin was not impaired by depletion of FtsW or FtsN. A conflicting previous report that the ftsA3(Ts) allele interferes with acylation of PBP3 was found to be due to the presence of a thermolabile PBP3 in the strain used in that study. The new findings presented here are discussed in light of the hypothesis that the catalytic activity of PBP3 is stimulated by interaction(s) with other division proteins. We suggest that there might be allosteric activation of substrate binding.

FIG. 1.

Recruitment of proteins to the septal ring of E. coli. The first event is polymerization of FtsZ into the FtsZ ring. Then FtsA and ZipA, which bind directly to the FtsZ ring, localize independently of each other. The remaining proteins localize subsequently in the order FtsK, FtsQ, FtsL and YgbQ (codependently), FtsW, PBP3 (FtsI), and FtsN.

FIG. 2.

Effect of FtsW depletion on acylation of PBP3 with β-lactams. (A) Fluorescence image of a representative gel. Cells were grown in the presence of cephalexin for 30 min (cephalexin concentrations [in micrograms per milliliter] are shown above the lanes), and then residual unacylated PBPs were detected by counterlabeling with Bocillin FL, a fluorescent penicillin derivative. The cells were grown in the presence of arabinose or glucose. Arabinose induces ftsW; these cells exhibited normal morphology. Glucose represses ftsW; these cells were filamentous. (B) Fraction of PBP3 acylated with cephalexin, piperacillin, or aztreonam. Antibiotic concentrations are in micrograms per milliliter. The percentages of PBP3 bound are the means ± standard deviations (error bars) from four to six experiments with cephalexin, two experiments with piperacillin, and three experiments with aztreonam. (C) Time course of acylation of PBP3 with cephalexin in an FtsW depletion strain growing in the presence of arabinose (black squares) or glucose (white squares). Growing cells (or filaments) were exposed to 5 μg of cephalexin per ml for the time indicated, and then residual unacylated PBP3 was detected by counterlabeling with Bocillin FL. Data points are the means ± standard deviations (error bars) of two (arabinose) or three (glucose) experiments, each done in triplicate.

FIG. 3.

Amounts of PBP3 in FtsQ, FtsW, and FtsN depletion strains. Gels used for experiments reported in Table 2 were analyzed by Western blotting with a chemiluminescent substrate and exposure to film. Only the portion of the blot with PBP3 is shown.

FIG. 4.

Time course of acylation of PBP3 with Bocillin FL in membranes isolated from an FtsW depletion strain (A) or an FtsN depletion strain (B). (A) Bocillin FL was used at 20, 10, and 5 μM (squares, circles, and triangles, respectively). Cells were grown with arabinose (ftsW induced) (black symbols) or glucose (ftsW repressed) (white symbols) prior to membrane isolation. Data points are the means of two experiments. For clarity, standard deviations are not shown but were mostly within 10% of the mean. (B) Bocillin FL was used at 20 and 8 μM (squares and circles, respectively). Data points are the means of three experiments, and standard deviations were mostly within 10% of the mean. Fluorescence is shown in 10⁵ units.

FIG. 5.

Half-lives of wild-type PBP3 (white squares) and mutant PBP3 (ftsI321) (black squares) at 42°C. A wild-type strain, DHB4, harboring pDSW478 or pDSW514 was induced to overexpress the respective ftsI alleles. The membranes were isolated and incubated at 42°C for the time indicated. Residual active PBP3 was detected by counterlabeling with Bocillin FL at 30°C. Data are graphed as the means ± standard deviations (error bars) from three experiments. Each line is a best-fit line determined by linear regression.