Lok‐To Sham

Building the cell wall is flipping difficult The cell wall of bacteria is constructed from a polysaccharide called peptidoglycan (PG). It forms a matrix that surrounds cells and is essential for the integrity of the cytoplasmic membrane. Many of our most successful antibiotics target PG synthesis. The synthetic pathway involves the assembly of sugar building blocks on a lipid carrier at the inner face of the cytoplasmic membrane. The reactions that produce this so-called lipid II precursor and the enzymes that catalyze them have been known for decades. However, the identity of the flippase enzyme that “flips” lipid II in the membrane to expose the sugar building blocks on the cell surface for polymerization has remained highly controversial. Sham et al. now show that the essential protein MurJ is the long sought-after flippase responsible for the translocation of lipid-linked cell wall precursors across the bacterial cytoplasmic membrane (see the Perspective by Young). The work completes the cell wall biogenesis pathway and defines the function of an attractive target for new antibiotics. Science , this issue p. 220 ; see also p. 139

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 (murJBs; formerly ytgP) are a synthetic lethal pair. Cells defective for both Amj and MurJBs exhibit cell shape defects and lyse. Furthermore, expression of Amj or MurJBs 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 MurJBs 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.

The peptidoglycan (PG) sacculus, a meshwork of polysaccharide strands cross-linked by short peptides, protects bacterial cells against osmotic lysis. To enlarge this covalently closed macromolecule, PG hydrolases must break peptide cross-links in the meshwork to allow insertion of new glycan strands between the existing ones. In the rod-shaped bacterium Bacillus subtilis, cell wall elongation requires two redundant endopeptidases, CwlO and LytE. However, it is not known how these potentially autolytic enzymes are regulated to prevent lethal breaches in the cell wall. Here, we show that the ATP-binding cassette transporter-like FtsEX complex is required for CwlO activity. In Escherichia coli, FtsEX is thought to harness ATP hydrolysis to activate unrelated PG hydrolases during cell division. Consistent with this regulatory scheme, B. subtilis FtsE mutants that are unable to bind or hydrolyse ATP cannot activate CwlO. Finally, we show that in cells depleted of both CwlO and LytE, the PG synthetic machinery continues moving circumferentially until cell lysis, suggesting that cross-link cleavage is not required for glycan strand polymerization. Overall, our data support a model in which the FtsEX complex is a remarkably flexible regulatory module capable of controlling a diverse set of PG hydrolases during growth and division in different organisms.

The connection between peptidoglycan remodeling and cell division is poorly understood in ellipsoid-shaped ovococcus bacteria, such as the human respiratory pathogen Streptococcus pneumoniae. In S. pneumoniae, peptidoglycan homeostasis and stress are regulated by the WalRK (VicRK) two-component regulatory system, which positively regulates expression of the essential PcsB cysteine- and histidine-dependent aminohydrolases/peptidases (CHAP)-domain protein. CHAP-domain proteins usually act as peptidoglycan hydrolases, but purified PcsB lacks detectable enzymatic activity. To explore the functions of PcsB, its subcellular localization was determined. Fractionation experiments showed that cell-bound PcsB was located through hydrophobic interactions on the external membrane surface of pneumococcal cells. Immunofluorescent microscopy localized PcsB mainly to the septa and equators of dividing cells. Chemical cross-linking combined with immunoprecipitation showed that PcsB interacts with the cell division complex formed by membrane-bound FtsX(Spn) and cytoplasmic FtsE(Spn) ATPase, which structurally resemble an ABC transporter. Far Western blotting showed that this interaction was likely through the large extracellular loop of FtsX(Spn) and the amino terminal coiled-coil domain of PcsB. Unlike in Bacillus subtilis and Escherichia coli, we show that FtsX(Spn) and FtsE(Spn) are essential in S. pneumoniae. Consistent with an interaction between PcsB and FtsX(Spn), cells depleted of PcsB or FtsX(Spn) had strikingly similar defects in cell division, and depletion of FtsX(Spn) caused mislocalization of PcsB but not the FtsZ(Spn) early-division protein. A model is presented in which the interaction of the FtsEX(Spn) complex with PcsB activates its peptidoglycan hydrolysis activity and couples peptidoglycan remodeling to pneumococcal cell division.

In most well-studied rod-shaped bacteria, peptidoglycan is primarily crosslinked by penicillin-binding proteins (PBPs). However, in mycobacteria, crosslinks formed by L,D-transpeptidases (LDTs) are highly abundant. To elucidate the role of these unusual crosslinks, we characterized Mycobacterium smegmatis cells lacking all LDTs. We find that crosslinks generate by LDTs are required for rod shape maintenance specifically at sites of aging cell wall, a byproduct of polar elongation. Asymmetric polar growth leads to a non-uniform distribution of these two types of crosslinks in a single cell. Consequently, in the absence of LDT-mediated crosslinks, PBP-catalyzed crosslinks become more important. Because of this, Mycobacterium tuberculosis (Mtb) is more rapidly killed using a combination of drugs capable of PBP- and LDT- inhibition. Thus, knowledge about the spatial and genetic relationship between drug targets can be exploited to more effectively treat this pathogen.