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Updated: Jul 13, 2026
Biochemical and Structural Characterization of the Carbohydrate Transport Substrate-binding-protein SP0092
Published on: October 2, 2017
Biochemical characterization of MsbA from Pseudomonas aeruginosa
Hamed Ghanei1, Priyanka D Abeyrathne1, Joseph S Lam1
1Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2W1, Canada.
Abstract:
Lipopolysaccharide of Pseudomonas aeruginosa is a major constituent of the outer membrane, and it is composed of three distinct regions: lipid A, core oligosaccharide, and O antigen. Lipid A and core oligosaccharides (OS) are synthesized and assembled at the cytoplasmic side of the inner membrane and then translocated to the periplasmic side of the membrane where lipid A-core becomes the acceptor of the O antigens. Here we show that MsbA encoded by pA4997 of the P. aeruginosa genome is a member of the ABC transporter family, but this protein has distinctive features when compared with other MsbA proteins. msbA is an essential gene in this organism since mutation in this gene is lethal to the bacterium. Disruption of the chromosomal msbA was achieved only when a functional copy of the gene was provided in trans. msbA from Escherichiacoli (msbA(Ec)) could not cross complement the msbA merodiploid cells of P. aeruginosa. MsbA was expressed and purified, and the kinetic of its ATPase activity is vastly different than that of MsbA(Ec). The activity of MsbA could be selectively stimulated by different truncated versions of core OS of P. aeruginosa LPS. Specifically, phosphate substituents in the lipid A-core are important for stimulating ATPase activity of MsbA. Expression of MsbA(Ec) but not MsbA(Pa) conferred resistance to erythromycin in P. aeruginosa.
Insights
Pseudomonas aeruginosa lipopolysaccharide (LPS) transport involves the essential MsbA ABC transporter. Its ATPase activity is stimulated by core oligosaccharides, unlike E. coli MsbA.
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- Lipopolysaccharide (LPS) is a crucial component of the Pseudomonas aeruginosa outer membrane, comprising lipid A, core oligosaccharide, and O antigen.
- LPS assembly involves synthesis and translocation of lipid A-core to the periplasm before O antigen attachment.
Purpose of the Study:
- To characterize the function and properties of the MsbA ABC transporter in Pseudomonas aeruginosa.
- To investigate the distinct features of P. aeruginosa MsbA compared to other MsbA proteins, particularly from E. coli.
Main Methods:
- Genetic analysis involving complementation studies of essential msbA gene disruption.
- Protein expression, purification, and characterization of ATPase activity.
- Stimulation assays using truncated core oligosaccharides and analysis of phosphate substituent importance.
Main Results:
- P. aeruginosa msbA is essential for bacterial viability.
- P. aeruginosa MsbA exhibits unique kinetic properties and ATPase activity distinct from E. coli MsbA.
- Phosphate substituents in the lipid A-core are critical for stimulating P. aeruginosa MsbA ATPase activity.
- E. coli MsbA confers erythromycin resistance in P. aeruginosa, but P. aeruginosa MsbA does not.
Conclusions:
- P. aeruginosa MsbA is a functionally distinct ABC transporter essential for LPS biogenesis.
- Core oligosaccharide structure, specifically phosphate groups, directly influences MsbA activity.
- Differences in MsbA function may explain variations in antibiotic resistance mechanisms between P. aeruginosa and E. coli.
