WecA is an integral membrane protein that initiates the biosynthesis of enterobacterial common antigen and O-antigen lipopolysaccharide (LPS) by catalyzing the transfer of N-acetylglucosamine (GlcNAc)-1-phosphate onto undecaprenyl phosphate (Und-P) to form Und-P-P-GlcNAc. WecA belongs to a large family of eukaryotic and prokaryotic prenyl sugar transferases. Conserved aspartic acids in putative cytoplasmic loops 2 (Asp90 and Asp91) and 3 (Asp156 and Asp159) were targeted for replacement mutagenesis with either glutamic acid or asparagine. We examined the ability of each mutant protein to complement O-antigen LPS synthesis in a wecA-deficient strain and also determined the steady-state kinetic parameters of the mutant proteins in an in vitro transfer assay. Apparent K m and V max values for UDP-GlcNAc, Mg 2؉ , and Mn 2؉ suggest that Asp156 is required for catalysis, while Asp91 appears to interact preferentially with Mg 2؉ , possibly playing a role in orienting the substrates. Topological analysis using the substituted cysteine accessibility method demonstrated the cytosolic location of Asp90, Asp91, and Asp156 and provided a more refined overall topological map of WecA. Also, we show that cells expressing a WecA derivative C terminally fused with the green fluorescent protein exhibited a punctate distribution of fluorescence on the bacterial surface, suggesting that WecA localizes to discrete regions in the bacterial plasma membrane.
Genetic evidence suggests that a family of bacterial and eukaryotic integral membrane proteins (referred to as Wzx and Rft1, respectively) mediates the transbilayer movement of isoprenoid lipid-linked glycans. Recent work in our laboratory has shown that Wzx proteins involved in O-antigen lipopolysaccharide (LPS) assembly have relaxed specificity for the carbohydrate structure of the O-antigen subunit. Furthermore, the proximal sugar bound to the isoprenoid lipid carrier, undecaprenyl-phosphate (Und-P), is the minimal structure required for translocation. In Escherichia coli K-12, N-acetylglucosamine (GlcNAc) is the proximal sugar of the O16 and enterobacterial common antigen (ECA) subunits. Both O16 and ECA systems have their respective translocases, Wzx O16 and Wzx E , and also corresponding polymerases (Wzy O16 and Wzy E ) and O-antigen chain-length regulators (Wzz O16 and Wzz E ), respectively. In this study, we show that the E. coli wzx E gene can fully complement a wzx O16 translocase deletion mutant only if the majority of the ECA gene cluster is deleted. In addition, we demonstrate that introduction of plasmids expressing either the Wzy E polymerase or the Wzz E chain-length regulator proteins drastically reduces the O16 LPS-complementing activity of Wzx E . We also show that this property is not unique to Wzx E , since Wzx O16 and Wzx O7 can cross-complement translocase defects in the O16 and O7 antigen clusters only in the absence of their corresponding Wzz and Wzy proteins. These genetic data are consistent with the notion that the translocation of O-antigen and ECA subunits across the plasma membrane and the subsequent assembly of periplasmic O-antigen and ECA Und-PP-linked polymers depend on interactions among Wzx, Wzz, and Wzy, which presumably form a multiprotein complex.
The asgA gene is required for generation of extracellular A signal, which serves as a cell-density signal for fruiting body development in Myxococcus xanthus. The AsgA protein is a histidine protein kinase and consists of a receiver domain that is conserved among response regulators of twocomponent signal transduction systems, followed by a histidine protein kinase domain that is conserved among sensor proteins of two-component systems.AsgA is thought to function in a signal transduction pathway that leads to expression of genes required for A-signal generation. A genetic suppression analysis of an asgA missense mutation was undertaken in order to identify genes that may provide information regarding the role of AsgA in A-signal generation and fruiting body formation. Twenty-two independent strains containing mutations that suppress asgA473 were isolated by selecting for production of heat-resistant spores under conditions that promote fruiting body development in wild-type cells. Ten of the 22 suppressor strains contained bypass suppressors. All the suppressor strains had direct spore counts a t least three to four times greater than the original asgA473 mutant, and three strains had viable counts that exceeded wild-type by more than one order of magnitude. Surprisingly, none of the suppressor strains produced wild-type levels of extracellular A-signal.
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