The O-antigen flippase Wzk can substitute for MurJ in peptidoglycan synthesis in Helicobacter pylori and Escherichia coli

The peptidoglycan (PG) cell wall is an essential component of the cell envelope of most bacteria. Biogenesis of PG involves a lipid-linked disaccharide-pentapeptide intermediate called lipid II, which must be translocated across the cytoplasmic membrane after it is synthesized in the inner leaflet of this bilayer. Accordingly, it has been demonstrated that MurJ, the proposed lipid II flippase in Escherichia coli, is required for PG biogenesis, and thereby viability. In contrast, MurJ is not essential in Bacillus subtilis because this bacterium produces AmJ, an unrelated protein that is functionally redundant with MurJ. In this study, we investigated why MurJ is not essential in the prominent gastric pathogen, Helicobacter pylori. We found that in this bacterium, Wzk, the ABC (ATP-binding cassette) transporter that flips the lipid-linked O- or Lewis- antigen precursors across the inner membrane, is redundant with MurJ for cell viability. Heterologous expression of wzk in E. coli also suppresses the lethality caused by the loss of murJ. Furthermore, we show that this cross-species complementation is abolished when Wzk is inactivated by mutations that target a domain predicted to be required for ATPase activity. Our results suggest that Wzk can flip lipid II, implying that Wzk is the flippase with the most relaxed specificity for lipid-linked saccharides ever identified

Wzk can substitute for MurJ in <i>E</i>. <i>coli</i>.

<p>(A) Summary of complementation experiments. The loss of <i>murJ</i> in <i>E</i>. <i>coli</i> can only be accomplished with a plasmid encoding wild-type <i>wzk</i>. The <i>wzkE525A</i> allele, which carries a mutation in the Walker B motif that is required for ATPase activity, cannot complement a Δ<i>murJ</i> allele. (B) Growth of <i>E</i>. <i>coli</i> strain NR3647 [MG1655 Δ<i>lacIZYA</i>::<i>FRT</i> Δ<i>murJ</i>::<i>kan</i> (pIH23)] in LB broth supplemented with 40 μM IPTG as determined by OD₆₀₀. The data represents the average and standard deviation of six independent cultures. (C) <i>E</i>. <i>coli</i> strain NR2920 (MG1655 Δ<i>lacIYZA</i>::<i>FRT</i> Δ<i>murJ</i>::<i>kan</i> (pRC7KanMurJ, pIH23) expressing both <i>murJ</i> and <i>wzk</i> exhibits the rod-shape cellular morphology typical of wild-type <i>E</i>. <i>coli</i> cells (MurJ⁺ Wzk⁺ panel) under a 100X phase-contrast objective. Δ<i>murJ</i> cells complemented with <i>wzk</i> (Wzk⁺ panels) exhibit morphology characteristic of cells with PG defects: larger size, aberrant morphology, and lysis (marked with white arrow heads).</p

Generation of enzymatically inactive Wzk variant.

<p>(A) Expression of different <i>H</i>. <i>pylori</i> Wzk variants was induced by IPTG in <i>E</i>. <i>coli</i> DH5α for 4 h. Cell lysates from normalized cultures were separated on SDS-PAGE, followed by immunoblotting to detect the expression levels of Wzk mutants relative to wild type enzyme. Among the different Wzk variants, WzkE525A displayed expression levels comparable to that of wild type. (B) WzkE525A flippase activity is diminished <i>in vivo</i>. Both AcrA from <i>C</i>. <i>jejuni</i> and the accessory gene cluster required for its <i>N</i>-glycosylation were reconstituted in <i>E</i>. <i>coli</i>, with the exception of the native flippase. Flippase activity of Wzk and its E525A variant was tested by monitoring the glycosylation levels of <i>C</i>. <i>jejuni</i> AcrA via immunoblotting. Monoclonal anti-histidine was used to detect the expression of histidine-tagged AcrA (green), while the <i>C</i>. <i>jejuni</i> glycan was detected by the rabbit polyclonal anti-<i>C</i>. <i>jejuni</i> glycan (red). The glycosylated form of AcrA (G) is marked by the colocalization of both signals (yellow). Unglycosylated form of AcrA is marked U.</p

MurJ and Wzk translocate Und-PP linked saccharides across the cytoplasmic membrane.

<p>Nucleotide-linked sugars (not shown) are used to build the PG precursor lipid II and the O-antigen precursor Und-PP-O-antigen on the membrane-bound lipid carrier Und-P. The first Und-P glycosyltransferase in each respective pathway are MraY (not shown) and WecA. After each Und-PP-linked intermediate is synthesized in the inner leaflet of the cytoplasmic membrane, flippases MurJ and Wzk translocate them across the bilayer. At the periplasmic leaflet of the cytoplasmic membrane, the disaccharide-pentapeptide component of lipid II is used to build glycan chains that are crosslinked into the preexisting PG matrix, while the O polysaccharide portion of Und-PP-O-antigen is transferred onto LPS molecules by the WaaL ligase (not shown). After these steps, the lipid carrier is recycled (green dotted arrows). The O polysaccharide is composed of <i>N</i>-acetyl glucosamine (GlcNAc, grey hexagon labeled G), galactose (green squares), and fucose (orange triangles); the disaccharide in lipid II is composed of <i>N</i>-acetyl muramic acid (MurNAc, black hexagon labeled M) and GlcNAc (grey hexagon labeled G), while the pentapeptide is composed of L-Ala (light blue circle), D-Glu (green circle), <i>meso</i>-2,6-diaminopimelic acid (<i>meso</i>-A₂pm, red circle) and two D-Ala (dark blue circle) residues.</p