(3–6). MurJ is a polytopic IM protein and a mem-
ber of the MOP (multidrug/oligo-saccharidyl-
lipid/polysaccharide) exporter superfamily (7).
It is essential in Escherichia coli. Cells depleted
of MurJ fail to complete PG biogenesis, accu-
mulate PG precursors, and lyse (4, 6). A three-
dimensional structural model and corresponding
transmembrane topology of MurJ are similar
to those of MOP exporters of amphipathic drugs
and undecaprenyl-PP–linked oligosaccharides (8).
Furthermore, a hydrophilic central cavity in MurJ
is essential for function. Fts W and its paralog
RodA are polytopic IM proteins that belong to
the SEDS (shape, elongation, division, and spor-
ulation) superfamily and are required for PG syn-
thesis during division (Fts W) or elongation (RodA)
(3, 5, 9). Support for SEDS proteins functioning as
flippases is based on in vitro studies in which lipid
II flippase activity was detected for purified Fts W
incorporated into liposomes (10).
The identity of the lipid II flippase has been
sought after for decades (2). Determining which
proteins flip lipid II in vivo requires a sensitive
method to detect lipid II flippase activity and a
method to connect this activity to a specific protein within the cell. When added to E. coli, the
protein toxin colicin M (ColM) is translocated into
the periplasm, where it cleaves lipid II (Fig. 1A)
(11, 12). We therefore reasoned that ColM could
be used in an assay to detect freshly flipped lipid II.
To evaluate this possibility, cells were metabolically
labeled with [3H]-mDAP, an amino acid unique to
the PG peptide, and either left untreated or incubated with purified ColM. Cells were then extracted
with hot water followed by butanol to separate
water-soluble PG intermediates and ColM-derived
products from lipid-linked PG precursors. High-performance liquid chromatography (HPLC) analysis of the water-soluble extract revealed a new
peak in the ColM-treated samples (Fig. 1B and
fig. S1), and its appearance correlated with the
loss of radiolabel in the butanol extract (Fig. 1C).
Moreover, increasing the cellular lipid II concentration by overproducing the lipid-carrier synthase UppS (2) enhanced the production of the
ColM-specific peak (fig. S2). The ColM-specific
product was identified as PP-Mpep4-G (figs. S3 and
S4), which presumably results from the processing of the ColM product, PP-Mpep5-G, by a carboxy-peptidase (Fig. 1A). Because carboxypeptidases
function only in the periplasm (1), this result
confirms that ColM acts on flipped lipid II.
To test whether MurJ flips lipid II, a method
to rapidly and specifically inactivate it was needed.
A collection of 39 functional single-Cys MurJ variants modifiable by the Cys-reacting molecule
MTSES (2-sulfonatoethyl methanethiosulfonate)
was previously used to determine the membrane
topology of MurJ (8). We asked whether any of
these mutant proteins were rendered nonfunctional by derivitization with MTSES. Treatment
of Cys-free MurJ (MurJWT) cells with MTSES had
no effect on growth, but the addition of MTSES to
cells producing derivatives with Cys substitutions
Fig. 1. In vivo assay for lipid
II flippase activity. (A) PG
precursor synthesis starts
with the conversion of UDP-N-acetylglucosamine (UDP-G)
to UDP-N-acetylmuramic acid
(UDP-M), followed by the
addition of amino acids
(represented by colored
spheres) to UDP-M to form
the pentapeptide (pep5) stem
L-Ala-γ-D-Glu-m-DAP-D-Ala-D-Ala). The [3H]-mDAP label
is indicated by the red star.
The UDP sugars are transferred
to undecaprenol-P (Und-P)
in the IM to form lipid II, which
is flipped across the IM to
expose the disaccharide-pep5
(Mpep5-G) for polymerization
and cross-linking into PG (not shown in the figure). Exogenous ColM binds to FhuA and is translocated
across the OM, presumably through a porin. In the periplasm, ColM cleaves lipid II into undecaprenol
(Und-OH) and soluble PP-Mpep5-G, which is further processed by carboxypeptidases (CPase) to produce
PP-Mpep4-G. (B and C) Cells of DmurJ DlysA strains producing FLAG-MurJ lacking endogenous Cys residues, referred
to as MurJWT (NR2592) or its derivative MurJA29C (NR2593), were labeled with [3H]-mDAP. cps, counts per second.
After 15 min, ColM and/or MTSES were added as indicated, and growth was continued for 10 min. Samples were then
withdrawn and either extracted with hot water alone or sequentially with water, then butanol. Hot-water extracts were subjected to HPLC and radiodetection to quantify
the labeled ColM product (B); scintillation counting was used to quantify label in the lipid (butanol) fraction (C). See figs. S1 to S4 for experimental details and peak
identification. Shown are the mean T SD from three experiments. P value determined with Student’s t test. N.S., not significant.
Fig. 2. MTSES specifically
inhibits the function of MurJA29C.
(A) Structural model of MurJ (8).
Sensitivity to MTSES is limited to
specific residues within the MurJ
cavity: residues 29 (green) in
transmembrane domain (TMD) 1;
49 (red) in TMD 2; and 263
(orange), 269 (blue), and 273
(magenta) in TMD 8. (B) Effect of
MTSES on the growth of haploid
cells producing MurJWT (left) or
MurJA29C in glucose M63 medium.
OD600, optical density at 600 nm.
Lysis of MurJA29C cells is suppressed by the presence of a wild-type murJ allele (right). Arrows
indicate the time of MTSES addition; solid symbols, no MTSES;
open symbols, MTSES-treated. Data represent mean T SD from three experiments. See fig. S5 for the
MTSES sensitivity of other variants.