3. Flipping over the Cytoplasmic Membrane
For decades, the mechanism behind the flipping and recycling of Lipid-II across the cytoplasmic membrane remained unknown. However, two recent publications shed light on this mystery through the simultaneous discovery of two protein families capable of recycling UndP
[19,20][19][20].
3.1. The Lipid-II Flippases
Lipid-II flippases, such as MurJ, are responsible for flipping Lipid-II to the outer leaf of the cytoplasmic membrane (
Figure 1)
[18]. This protein requires proton motive force (PMF) in order to drive conformational changes for the flip of Lipid-II
[50,51][37][38]. In
E. coli, MurJ is the sole Lipid-II flippase. It is essential, and the depletion of this protein results in the inhibition of PG biosynthesis and accumulation of lipid-linked PG precursors
[18]. By contrast,
B. subtilis has four MurJ homologs. One of them (SpoVB) is required for PG synthesis during sporulation
[52][39].
E. coli MurJ has been shown to be able to complement the sporulation defect of a
spoVB mutant. In turn, both SpoVB and YtgP, another MurJ from
B. subtilis, were shown to complement the growth defect of an
E. coli strain depleted of
murJ, thus confirming their status as Lipid-II flippases. Unexpectedly, a
B. subtilis strain lacking these four MurJ homologs did not exhibit a growth defect, suggesting the existence of an alternative Lipid-II flippase family. Indeed, an additional Lipid-II flippase, Amj (alternate to MurJ), was discovered in
B. subtilis a few years ago
[53][40]. Cells lacking both Amj and the four MurJ homologs exhibited cell shape defects and lysis. Furthermore, the expression of
amj or
murJ from
B. subtilis in an
E. coli murJ mutant restored Lipid-II flipping and, consequently, the viability of this mutant. In addition, the SEDS protein, FtsW
[27][41], an essential protein carrying GTase activity required for glycan strand polymerization during septum formation in cell division, has also been shown to transport Lipid-II in vitro
[54][42]. It may thus be an alternative Lipid-II flippase, but this assumption is controversial. This proposition was based on an in vitro FtsW reconstitution assay that reported both a Lipid-II flippase activity and an ability of FtsW to translocate various phospholipids
[54][42]. However, no FtsW-dependent flippase activity could be detected when MurJ was incorporated into liposomes. In addition, no genetic evidence supports the argument that FtsW flips Lipid-II. In fact, it was shown that FtsW activity and Lipid-II synthesis are required for the recruitment of MurJ to the mid-cell in
E. coli [55][43]. The mid-cell is the place where septal PG is synthesized and thus where the Lipid-II is flipped during cell division. This result strongly suggests that MurJ and FtsW work together in vivo for the flipping of Lipid-II.
3.2. The UndP Flippases
The step following the flipping of Lipid-II to the outer face of the cytoplasmic membrane, where the muropeptide is polymerized and crosslinked to the existing PG meshwork, is the dephosphorylation of UndPP by UndPP membrane phosphatases (BacA, YbjG, PgpB and LpxT in
E. coli [56][44] or UppP and BcrC in
B. subtilis [57][45]) for its recycling (
Figure 1). The resulting UndP is flipped back to the inner side of the cytoplasmic membrane to be recycled for the production of novel Lipid-II. For years, the transporters involved in this recycling were unidentified. It was only very recently that, using genetic screens in
B. subtilis,
Staphylococcus aureus [19] and
Vibrio cholerae [20], two broadly conserved families of flippases were shown to be responsible for UndP transport across the membrane. Genetic, cytological and syntenic analyses support the idea that these two UndP transporter families (corresponding to UptA and PopT proteins) are indeed UndP flippases.