Wzx proteins involved in biosynthesis of O antigen function in association with the first sugar of the O-specific lipopolysaccharide subunit

Abstract

One of the most common pathways for the export of O-specific lipopolysaccharide (LPS) across the plasma membrane requires the participation of the Wzx protein. Wzx belongs to a family of integral membrane proteins that share little conservation in their primary amino acid sequence, making it difficult to delineate functional domains. This paper reports the cloning and expression in Escherichia coli K-12 of various Wzx homologues from different bacteria as FLAG epitope-tagged protein fusions. A reconstitution system for O16 LPS synthesis was used to assess the ability of each Wzx protein to complement an E. coli K-12 Dwzx mutant. The results demonstrate that Wzx proteins from O-antigen systems that use N-acetylglucosamine or N-acetylgalactosamine for the initiation of the biosynthesis of the O repeat can fully complement the formation of O16 LPS. Partial complementation was seen with Wzx from Pseudomonas aeruginosa, a system that uses N-acetylfucosamine in the initiation reaction. In contrast, there was negligible complementation with the Wzx protein from Salmonella enterica, a system in which galactose is the initiating sugar. These results support a model whereby the first sugar of the O repeat can be recognized by the O-antigen translocation machinery.

Selective Sorting of Cargo Proteins into Bacterial Membrane Vesicles

“…First, we tested the flippase activity of porS. Flippases generally have relaxed specificities toward the translocated substrates (28,29). We confirmed a flippase function for PorS by demonstrating its ability to restore O antigen biosynthesis and Campylobacter jejuni protein N-glycosylation reconstituted in an E. coli flippase-deficient strain (the details are given in supplemental data and supplemental Fig.…”

Substrate Specificity and Membrane Topology of Escherichia coli PgpB, an Undecaprenyl Pyrophosphate Phosphatase

“…Whether C 55 -PP-linked substrates are translocated through the membrane by the same mechanism remains to be determined. It is well established that translocation of C 55 -PP-linked substrates requires specific flippases/translocators (O-antigen biosynthesis, some types of bacterial protein glycosylation) (47)(48)(49), but in the case of peptidoglycan synthesis this translocator is not known (50).…”

Distinct functional domains of the Salmonella enterica WbaP transferase that is involved in the initiation reaction for synthesis of the O antigen subunit

“…Indeed, the growth rate of E. coli DH5a (pSM13) was significantly slower (doubling time 106±3 min, n57) than that of E. coli DH5a carrying the vector control (doubling time 76±3.4 min; P,0.001, n57). We have previously demonstrated that the E. coli Wzx flippase does not recognize the product of the reaction catalysed by the Salmonella WbaP protein (Und-PP-Gal) (Feldman et al, 1999;Marolda et al, 2004). Therefore, the growth defect caused by WbaP expression in E. coli could be due to accumulation of Und-PP-Gal in the cytoplasmic side of the plasma membrane, which could presumably block the recycling of Und-P and thus compromise cell wall biosynthesis.…”

“…Protein aggregates that barely penetrate the gel matrix and anomalous migration in the gel are commonly observed with membrane proteins upon heat denaturation, particularly in proteins with high pIs (Kashino, 2003). To avoid this problem, the protein samples were incubated at 45 u C for 30 min, as we have done in previous studies with the integral membrane proteins WecA and Wzx (Amer & Valvano, 2000;Lehrer et al, 2007;Marolda et al, 2004). In this case, we also detected a polypeptide with an apparent mass of 26 kDa corresponding to WbaP M170-R354-FLAG (Fig.…”