Dataset Information

Membrane topology mapping of the O-antigen flippase (Wzx), polymerase (Wzy), and ligase (WaaL) from Pseudomonas aeruginosa PAO1 reveals novel domain architectures.

ABSTRACT: Biosynthesis of B-band lipopolysaccharide (LPS) in Pseudomonas aeruginosa follows the Wzy-dependent pathway, requiring the integral inner membrane proteins Wzx (O-antigen [O-Ag] flippase), Wzy (O-Ag polymerase), and WaaL (O-Ag ligase). For an important first step in deciphering the mechanisms of LPS assembly, we set out to map the membrane topology of these proteins. Random and targeted 3'wzx, wzy, and waaL truncations were fused to a phoA-lacZalpha dual reporter capable of displaying both alkaline phosphatase and beta-galactosidase activity. The results from truncation fusion expression and the corresponding differential enzyme activity ratios allowed for the assignment of specific regions of the proteins to cytoplasmic, transmembrane (TM), or periplasmic loci. Protein orientation in the inner membrane was confirmed via C-terminal fusion to green fluorescent protein. Our data revealed unique TM domain properties in these proteins, particularly for Wzx, indicating the potential for a charged pore. Novel periplasmic and cytoplasmic loop domains were also uncovered, with the latter in Wzy and WaaL revealing tracts consistent with potential Walker A/B motifs.

SUBMITTER: Islam ST

PROVIDER: S-EPMC2932511 | biostudies-other | 2010 Aug

REPOSITORIES: biostudies-other

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Membrane topology mapping of the O-antigen flippase (Wzx), polymerase (Wzy), and ligase (WaaL) from Pseudomonas aeruginosa PAO1 reveals novel domain architectures.

Islam Salim T ST Taylor Véronique L VL Qi Meng M Lam Joseph S JS

mBio 20100824 3

Biosynthesis of B-band lipopolysaccharide (LPS) in Pseudomonas aeruginosa follows the Wzy-dependent pathway, requiring the integral inner membrane proteins Wzx (O-antigen [O-Ag] flippase), Wzy (O-Ag polymerase), and WaaL (O-Ag ligase). For an important first step in deciphering the mechanisms of LPS assembly, we set out to map the membrane topology of these proteins. Random and targeted 3'wzx, wzy, and waaL truncations were fused to a phoA-lacZalpha dual reporter capable of displaying both alkal ...[more]

PMID: 20824106

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Project description:The P. aeruginosa reference strain PAO1 has been used to delineate much of the physiology, metabolism, and fundamental biology of the species. The wild-type parent of PAO1 was lost, and PAO1 carries a regulatory mutation introduced for positive genetic selection that affects antibiotic resistance, virulence, quorum sensing, and other traits. The mutation is a loss-of-function change in an oxidoreductase gene (mexS), which constitutively activates a stress response controlled by a positive regulator (MexT). Fitness defects associated with the constitutive response have led to the inadvertent selection of mexT-minus suppressor mutations, creating genetic heterogeneity in PAO1 sublines studied in different laboratories. To help circumvent complications due to the mexS-minus phenotypes, we created a wild-type version of PAO1 (called LPAO) by "reverting" its mexS to the functional allele likely to have been in its parent. Phenotypic analysis revealed that the mexS-minus allele in PAO1 makes growth sensitive to salt (NaCl) and is lethal when combined with mutations inactivating the major sodium antiporter (ShaABCDEF). The salt sensitivity of PAO1 may underlie some complex mexS-minus phenotypes and help explain the selection of mexT-minus suppressor mutations. To facilitate genetic comparisons of PAO1, LPAO, and other P. aeruginosa strains, we developed a transformation procedure to transfer selectable alleles, such as transposon insertion alleles, between strains. Overall, the study helps explain phenotypic heterogeneity of PAO1-derived strains and provides resources to help recognize and eliminate difficulties due to it. IMPORTANCE The P. aeruginosa reference strain PAO1 carries a regulatory mutation that may affect processes characterized in it. To eliminate complications due to the mutation, we constructed a version of the missing wild-type parent strain and developed methods to transfer mutations between PAO1 and the new strain. The methods are likely to be applicable to other isolates of P. aeruginosa as well.

Dataset Information

Membrane topology mapping of the O-antigen flippase (Wzx), polymerase (Wzy), and ligase (WaaL) from Pseudomonas aeruginosa PAO1 reveals novel domain architectures.

Publications

Membrane topology mapping of the O-antigen flippase (Wzx), polymerase (Wzy), and ligase (WaaL) from Pseudomonas aeruginosa PAO1 reveals novel domain architectures.

Similar Datasets

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