Charge requirements of lipid II flippase activity in Escherichia coli

Abstract

Peptidoglycan (PG) is an extracytoplasmic glycopeptide matrix essential for the integrity of the envelope of most bacteria. The PG building block is a disaccharide-pentapeptide that is synthesized as a lipid-linked precursor called lipid II. The translocation of the amphipathic lipid II across the cytoplasmic membrane is required for subsequent incorporation of the disaccharide-pentapeptide into PG. In Escherichia coli, the essential inner membrane protein MurJ is the lipid II flippase. Previous studies showed that 8 charged residues in the central cavity region of MurJ are crucial for function. Here, we completed the functional analysis of all 57 charged residues in MurJ and demonstrated that the respective positive or negative charge of the 8 aforementioned residues is required for proper MurJ function. Loss of the negative charge in one of these residues, D39, causes a severe defect in MurJ biogenesis; by engineering an intragenic suppressor mutation that restores MurJ biogenesis, we found that this charge is also essential for MurJ function. Because of the low level of homology between MurJ and putative orthologs from Gram-positive bacteria, we explored the conservation of these 8 charged residues in YtgP, a homolog from Streptococcus pyogenes. We found that only 3 positive charges are similarly positioned and essential in YtgP; YtgP possesses additional charged residues within its predicted cavity that are essential for function and conserved among Gram-positive bacteria. From these data, we hypothesize that some charged residues in the cavity region of MurJ homologs are required for interaction with lipid II and/or energy coupling during transport.

FIG 1

Specific charges in the cavity region of MurJ are required for function. (A) Structural model of MurJ (14) showing charged residues in the cavity region that are critical for function. Shown are the front view from the membrane plane with approximate membrane boundaries marked (left) and the top view from the periplasm (right). Relevant positively (blue) and negatively (red) charged side chains are rendered as spheres. (B) Summary of the effect on MurJ function conferred by Ala and Cys substitutions (14) and substitutions to the opposite (R/K to E or D/E to R/K) or conserved (R to K or D/E to E/D) charge at each residue (left column) shown in panel A. Phenotypes are given as total loss of function (TLOF) if the allele does not complement a ΔmurJ chromosomal deletion, partial loss of function (PLOF) if the allele complements a ΔmurJ chromosomal deletion but confers sensitivity to low-osmolarity conditions, and functional (F) if the allele does not confer any mutant phenotype. Phenotypes were assessed as described in Materials and Methods. Data for Cys substitutions are described by Butler et al. (14); data for changes to Ala and the opposite and conserved charge are described in Results and Tables S1 and S2 in the supplemental material. ND, not done.

FIG 2

Detection of FLAG-MurJ variants with substitutions at charged residues crucial for MurJ function. Samples prepared from overnight cultures of merodiploid (murJ⁺) or haploid (ΔmurJ) strains producing FLAG-MurJ variants bearing substitutions to Ala (A), an opposite charge (B), or a conserved charge (C) were subjected to anti-FLAG immunoblotting and compared to the wild-type (WT) parent. Data are representative of at least three independent experiments. Anti-LptB immunoblotting was performed to control for sample loading.

FIG 3

Biogenesis of MurJ requires the presence of a negatively charged residue in loop 1. Protein levels of FLAG-MurJ variants in overnight cultures of merodiploid (murJ⁺) or haploid (ΔmurJ) strains were compared via anti-FLAG immunoblotting. The presence of a negative charge at position 39 in loop 1 is required for proper biogenesis of MurJ (compare WT and D39E to D39N and D39A). The reduction in MurJ levels caused by the D39A substitution is suppressed by the introduction of a negative charge at a different position in loop 1 (compare D39A to A37D/D39A). Data are representative of at least three independent experiments. Anti-LptB immunoblotting was performed to control for sample loading.

FIG 4

Charged residues within the cavity region are required for YtgP function. (A) Alignment of the first 12 transmembrane domains of YtgP (green) and MurJ (gray) structure models showing the side chains (stick representation) of functionally important residues in MurJ (orange sticks, black labels) and YtgP (blue sticks, blue labels). The aligned structures are shown as viewed from the membrane plane (top) and from the periplasm (bottom). Refer to Fig. S2 in the supplemental material and Materials and Methods for alignment details. (B and C) Anti-His immunoblots of nonfunctional (B) and functional (C) His-YtgP variants show relative levels that are similar to those of the wild-type (WT) parent in all cases except for the R29A variant, which is present at lower levels. Data are representative of at least three independent experiments. Anti-LptB immunoblotting was performed to control for sample loading.

FIG 5

M171R substitution causes defects in MurJ biogenesis. Anti-FLAG immunoblot of samples prepared from overnight cultures of merodiploid (murJ⁺) or haploid (ΔmurJ) strains reveals that the M171R substitution is responsible for the reduced levels of functional M171R and R24A/M171R FLAG-MurJ variants. Data are representative of at least three independent experiments. Anti-LptB immunoblotting was performed to control for sample loading.