<i>Bacillus subtilis</i> MurJ and Amj Lipid II flippases are not essential for growth

Identification of the protein that mediates transbilayer transport of the undecaprenyl-pyrophosphate (Und-PP) linked peptidoglycan precursor Lipid II has long been a subject of investigation. Candidates belonging to both the MOP (multidrug/oligosaccharidyl-lipid/polysaccharide) and SEDS (shape, elongation, division and sporulation) families of transmembrane proteins have been proposed, exhibiting characteristics consistent with mediating this process, including genetic essentiality and biochemical activity. While MOP family proteins including MurJ are widely considered to be the primary Lipid II transporter, questions still remain including a role for the SEDS proteins in this process. We and others previously showed that a <i>Bacillus subtilis</i> strain lacking all four MurJ homologs is viable, thereby implicating a separate mode of Lipid II transport across the membrane. However, a subsequent report of synthetic essentiality between <i>B. subtilis</i> MurJ and the flippase Amj suggested that they are necessary and sufficient. Here, we show that this effect is alleviated by excess synthesis of the enzyme responsible for Und-PP production. Thus, the inviability of a <i>murJ-amj</i> double mutant strain is not due to the essentiality of these enzymes for flipping Lipid II but is instead most likely a consequence of a reduction of free Und-PP levels. This result is consistent with a non-MOP-dependent pathway for Lipid II transport across the cytoplasmic membrane to enable cell wall peptidoglycan synthesis.IMPORTANCEThe assembly of peptidoglycan (PG), the typically essential polymer that provides structural integrity to bacterial cells, begins with the synthesis of the Lipid II monomer in the cytoplasm and along the cytoplasmic face of the inner membrane. Lipid II is then translocated across the membrane to the extracellular site of polymerization. The mechanistic basis for this process remains unclear, with genetic and/or biochemical evidence pointing to two different families of conserved membrane proteins. Here, we present genetic evidence that only one of these two families is essential in <i>Bacillus subtilis</i>.