Regulation of the Peptidoglycan Polymerase Activity of PBP1b by Antagonist Actions of the Core Divisome Proteins FtsBLQ and FtsN

Abstract

Peptidoglycan (PG) is an essential constituent of the bacterial cell wall. During cell division, PG synthesis localizes at midcell under the control of a multiprotein complex, the divisome, allowing the safe formation of two new cell poles and separation of daughter cells. Genetic studies in Escherichia coli pointed out that FtsBLQ and FtsN participate in the regulation of septal PG (sPG) synthesis; however, the underlying molecular mechanisms remained largely unknown. Here we show that FtsBLQ subcomplex directly interacts with the PG synthase PBP1b and with the subcomplex FtsW-PBP3, mainly via FtsW. Strikingly, we discovered that FtsBLQ inhibits the glycosyltransferase activity of PBP1b and that this inhibition was antagonized by the PBP1b activators FtsN and LpoB. The same results were obtained in the presence of FtsW-PBP3. Moreover, using a simple thioester substrate (S2d), we showed that FtsBLQ also inhibits the transpeptidase domain of PBP3 but not of PBP1b. As the glycosyltransferase and transpeptidase activities of PBP1b are coupled and PBP3 activity requires nascent PG substrate, the results suggest that PBP1b inhibition by FtsBLQ will block sPG synthesis by these enzymes, thus maintaining cell division as repressed until the maturation of the divisome is signaled by the accumulation of FtsN, which triggers sPG synthesis and the initiation of cell constriction. These results confirm that PBP1b plays an important role in E. coli cell division and shed light on the specific role of FtsN, which seems to counterbalance the inhibitory effect of FtsBLQ to restore PBP1b activity.IMPORTANCE Bacterial cell division is governed by a multiprotein complex called divisome, which facilitates a precise cell wall synthesis at midcell and daughter cell separation. Protein-protein interactions and activity studies using different combinations of the septum synthesis core of the divisome revealed that the glycosyltransferase activity of PBP1b is repressed by FtsBLQ and that the presence of FtsN or LpoB suppresses this inhibition. Moreover, FtsBLQ also inhibits the PBP3 activity on a thioester substrate. These results provide enzymatic evidence of the regulation of the peptidoglycan synthase PBP1b and PBP3 within the divisome. The results confirm that PBP1b plays an important role in E. coli cell division and shed light on the specific role of FtsN, which functions to relieve the repression on PBP1b by FtsBLQ and to initiate septal peptidoglycan synthesis.

Keywords: FtsBLQ; FtsN; PBP1b; divisome; lipid II; peptidoglycan.

FIG 1

Purification of the FtsBLQ ternary complex and Western blot analysis. (A) SDS-PAGE analysis of HisFtsBLQ and StepFtsBLQ complexes purified on HisTrap (His) or StrepTrap (Strep), respectively. Numbers at left are molecular masses in kilodaltons. (B) Western blot analyses performed on HisFtsBLQ complex purified on HisTrap using specific antibodies against each protein and anti-His antibody for His-FtsB.

FIG 2

Protein-protein interactions using coexpression and copurification. The proteins indicated below each panel (A to K) were coexpressed in E. coli and copurified on a nickel column using one (His)-tagged protein as a bait. The eluted proteins were then labeled with fluorescent ampicillin, when PBPs were present, and analyzed by SDS-PAGE followed by fluorescence (FL) imaging and protein staining with Coomassie blue (CB). M, protein standard; EL, elution fractions from Ni affinity column; FL, fluorescently labeled PBPs; Ex, protein extracts; FT, indicates the flowthrough fractions. The bands of the proteins are indicated by arrowheads. FtsW* is a degradation product of FtsW. α-HA indicates immunoblotting (IM) analysis using antibodies against the HA epitope of FtsW_HA. Numbers at left of panels are molecular masses in kilodaltons.

FIG 3

Effect of FtsBLQ and/or FtsN and LpoB on the activity of PBP1b in the presence and absence of FtsW/PBP3. The GTase activity of PBP1b with dansyl-lipid II is measured using a continuous fluorescence assay (the concentrations of PBP1b and other proteins and detergent concentrations were optimized for each experiment). Upon PG polymerization, the fluorescence decreases over time. Values are the mean ± SD from three experiments or a representative of three assays. (A) Inhibition of lipid II (LII) polymerization by PBP1b (1b) using variable concentrations of FtsBLQ (BLQ) subcomplex. (B and C) FtsN or LpoB, respectively, suppresses the inhibitory effect of FtsBLQ on PBP1b. (D and E) FtsN or LpoB, respectively, suppresses the inhibitory effect of FtsBLQ on PBP1b in the presence of FtsW/PBP3 (1bW3). (F and G) Activation of PBP1b by FtsN or LpoB, respectively, in the presence of FtsW/PBP3.

FIG 4

Inhibition of PBP1b GTase activity by FtsBLQ. TLC analysis of the reaction products formed by PBP1b from radioactive lipid II substrate. The addition of FtsBLQ in the PBP1b reaction mixture inhibits the polymerization of lipid II into PG polymer.

FIG 5

Effect of FtsBLQ subunits and mutants and FtsN mutants on the activity of PBP1b. The GTase activity of PBP1b was measured by continuous fluorescence assay (the concentration of PBP1b and other proteins and detergent concentration were optimized for each experiment). (A and B) Comparison of the effects of FtsBLQ with the subunit FtsQ or FtsBL, respectively, on the activity of PBP1b. (C) Comparison of the effect of FtsBLQ containing the mutation FtsL^D93A and control complex on the activity of PBP1b. (D) Comparison of the effects of the FtsN^W83L and FtsN^Y85W mutants and wild-type FtsN on the activity of PBP1b. (E) Comparison of the effects of FtsN and FtsN^W83L mutant on PBP1b inhibited by FtsBLQ. (F) Effect of FtsBLQ containing the mutation FtsB^D59H on the activity of PBP1b.

FIG 6

Phenotype of E. coli cells (C43 DE3) overexpressing FtsBLQ complex or mutants. FtsBLQ (WT) induces extensive cell filamentation after induction, while the cells overproducing FtsBLQ complexes with a mutation in FtsB or FtsL exhibit comparable cell length before and after induction. The average cell length (L) ± SD (µm) and the number of cells measured (n) are shown below the corresponding representative image. The average cell width in all cases was ∼1.3 µm. Bar, 5 μm.

FIG 7

Effect of FtsBLQ and subunits on the hydrolysis of S2d by PBP3 and PBP1b TPase domain. (A) Inhibition of PBP3 activity by FtsBLQ. (B) FtsBLQ does not inhibit the hydrolysis of S2d by PBP1b TPase domain. (C) Hydrolysis of S2d by PBP3 is inhibited by FtsQ but not by FtsBL. Abs 330 nm, absorbance at 330 nm.

FIG 8

Schematic representation of the linear recruitment pathway of the divisome and a regulation model of the septal synthase subcomplex (FtsW-PBP3-PBP1b) by FtsBLQ, FtsN, and the lipoprotein LpoB. (A) Linear recruitment pathway of the divisome with the septal peptidoglycan (sPG) synthesis core shown in a frame; protein-protein interactions are shown with arrows. (B) Topology of the synthase subcomplex and the regulatory proteins (FtsBLQ, FtsN, and LpoB) in the cell envelope. FtsBLQ subcomplex inhibits the GTase activity of PBP1b (via FtsL) and the TPase domain of PBP3 (via FtsQ). Interaction of FtsN via the essential region (^EFtsN) and of LpoB with PBP1b suppresses the inhibition by FtsBLQ, activating PBP1b and probably the whole synthase subcomplex. Interaction of FtsN with FtsA and LpoB with the outer membrane allows the coordination of sPG synthesis with cytoplasmic, inner (IM), and outer membrane (OM) events. CCD, constriction control domain of FtsL.