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

Significance: The bacterial envelope is composed of a diverse set of polysaccharides. Virtually all of these polymers are synthesized from lipid-linked precursors that are flipped across the cytoplasmic membrane by ATP-binding cassette transporters or multidrug/oligosaccharidyl-lipid/polysaccharide (MOP) exporter superfamily members. Transport of the cell wall precursor lipid II requires the MOP family member MurJ inEscherichia coli. Here, we provide evidence for a novel lipid II flippase inBacillus subtiliscalled alternate to MurJ (Amj), which bears no similarity to either family of transporters.amjis up-regulated in the absence ofB. subtilisMurJ (MurJ_Bs) via an envelope stress-response pathway, suggesting this novel flippase may serve as a defense mechanism against naturally occurring MurJ antagonists.

Summary: 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.

Summary: spxB‐encoded pyruvate oxidase is a major virulence factor of Streptococcus pneumoniae. During aerobic growth, SpxB synthesizes H₂O₂ and acetyl phosphate, which play roles in metabolism, signalling, and oxidative stress. We report here the first cis‐ and trans‐acting regulatory elements for spxB transcription. These elements were identified in a genetic screen for spontaneous mutations that caused colonies of strain D39 to change from a semitransparent to an opaque appearance. Six of the seven opaque colonies recovered (frequency ≈ 3 × 10⁻⁵) were impaired for SpxB function or expression. Two mutations changed amino acids in SpxB likely required for cofactor or subunit binding. One mutation defined a cis‐acting adjacent direct repeat required for optimal spxB transcription. The other three spontaneous mutations created the same frameshift near the start of the trans‐acting spxR regulatory gene. The SpxR protein contains helix–turn–helix, CBS and HotDog domains implicated in binding DNA, adenosyl compounds, and CoA‐containing compounds respectively, and suggest that SpxR positively regulates spxB transcription in response to energy and metabolic state. Microarray analyses unexpectedly demonstrated that SpxR also positively regulates the strH exoglycosidase gene, which, like spxB, has been implicated in colonization. Finally, SpxR is required for full virulence in a murine model of infection.