MurJ and a novel lipid II flippase are required for cell wall biogenesis inBacillus subtilis

Abstract

Bacterial surface polysaccharides are synthesized from lipid-linked precursors at the inner surface of the cytoplasmic membrane before being translocated across the bilayer for envelope assembly. Transport of the cell wall precursor lipid II in Escherichia coli requires the broadly conserved and essential multidrug/oligosaccharidyl-lipid/polysaccharide (MOP) exporter superfamily member MurJ. Here, we show that Bacillus subtilis cells lacking all 10 MOP superfamily members are viable with only minor morphological defects, arguing for the existence of an alternate lipid II flippase. To identify this factor, we screened for synthetic lethal partners of MOP family members using transposon sequencing. We discovered that an uncharacterized gene amj (alternate to MurJ; ydaH) and B. subtilis MurJ (murJ Bs ; formerly ytgP) are a synthetic lethal pair. Cells defective for both Amj and MurJ Bs exhibit cell shape defects and lyse. Furthermore, expression of Amj or MurJ Bs in E. coli supports lipid II flipping and viability in the absence of E. coli MurJ. Amj is present in a subset of gram-negative and gram-positive bacteria and is the founding member of a novel family of flippases. Finally, we show that Amj is expressed under the control of the cell envelope stress-response transcription factor σ M and cells lacking MurJ Bs increase amj transcription. These findings raise the possibility that antagonists of the canonical MurJ flippase trigger expression of an alternate translocase that can resist inhibition.T he bacterial cell wall or peptidoglycan (PG) is composed of glycan strands cross-linked together by short peptides. This 3D meshwork protects the cell from osmotic lysis and determines shape, and its assembly is the target of some of our most successful antibiotics. Cell wall synthesis begins in the cytoplasm, where a set of highly conserved enzymes catalyze the formation of the lipid-linked precursor lipid II, which is composed of undecaprenyl-pyrophosphate (UndPP) linked to N-acetylglucosamine-N-acetylmuramic acid pentapeptide. Lipid II is synthesized on the inner face of the cytoplasmic membrane (1). The molecule is then translocated to the outer face of the membrane, where the disaccharide-peptide monomer is incorporated into the existing PG by cell wall synthetic machineries composed of penicillin-binding proteins and additional factors (2). The enzymes that transport lipid II across the membrane have been the subject of extensive research and speculation (3-6). Recent work in Escherichia coli has provided strong evidence that the polytopic membrane protein MurJ is required for lipid II transport across the membrane, and is likely to be a lipid II flippase (6). MurJ is a member of the multidrug/oligosaccharidyl-lipid/polysaccharide (MOP) exporter superfamily (7). It is broadly conserved among Eubacteria and essential for viability in many organisms. Intriguingly, work in the model gram-positive bacterium Bacillus subtilis indicates that cells lacking four MOP superfamily members similar to MurJ are viable and have...

Depletion of Undecaprenyl Pyrophosphate Phosphatases Disrupts Cell Envelope Biogenesis in Bacillus subtilis

“…Chromosomal and plasmid DNA transformation was performed as described previously (32). Markerless in-frame deletion mutants were constructed from BKE strains as described previously (5). Briefly, BKE strains were acquired from the Bacillus Genetics Stock Center, chromosomal DNA was extracted, and the mutation, containing an erm cassette, was transformed into our wild-type (WT) strain 168.…”

“…The identity of the lipid II flippase required for the export of this essential precursor from the cytoplasmic to the external face of the membrane has been controversial. Recent results provide strong support for a role of MurJ as the lipid II flippase (4) and have further shown that this enzyme is redundant in function with a stressregulated protein named alternate to MurJ (Amj) (5). The recycling of the UPP carrier to Und-P also involves redundant enzymes, perhaps to allow the conversion to occur on either the inner or outer face of the membrane.…”

Cell-wall recycling and synthesis in Escherichia coli and Pseudomonas aeruginosa – their role in the development of resistance

“…Lipid II is flipped over into the periplasmic space using a 'flippase' enzyme [37]. The identity of 'flippase' remains ambiguous and is the subject of current research [38][39][40]. In E. coli, FtsW and MurJ have been independently shown to be the proteins responsible for translocation of the lipid-linked muropeptides from the cytoplasm into the periplasmic space [41][42][43][44].…”

The Blueprint of a Minimal Cell: MiniBacillus

“…Most of the enzymes participating in the pathways mentioned above are essential. MurJ is an exception, as B. subtilis also possesses the weakly expressed functional paralog Amj (142).…”