title: 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.
authors.affiliation: Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada.
authors.forename: Salim T
authors.initials: ST
authors.lastname: Islam
authors.forename: Véronique L
authors.initials: VL
authors.lastname: Taylor
authors.forename: Meng
authors.initials: M
authors.lastname: Qi
authors.forename: Joseph S
authors.initials: JS
authors.lastname: Lam
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019729:Carbon-Oxygen Ligases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006602:Hexosyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011550:Pseudomonas aeruginosa / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D008565:Membrane Proteins; D006602:Hexosyltransferases; C085652:O-antigen polymerase; D019729:Carbon-Oxygen Ligases; C528973:WaaL protein, Pseudomonas aeruginosa
references.citation: Environ Microbiol. 2002 Dec;4(12):883-97
references.pmid: 12534470
references.citation: Mol Microbiol. 1993 Mar;7(5):725-34
references.pmid: 7682279
references.citation: J Bacteriol. 2007 Mar;189(6):2203-9
references.pmid: 17209027
references.citation: Eur J Biochem. 2003 Mar;270(5):799-813
references.pmid: 12603313
references.citation: Antimicrob Agents Chemother. 1993 Apr;37(4):715-21
references.pmid: 8494366
references.citation: Nat Methods. 2006 Apr;3(4):303-13
references.pmid: 16554836
references.citation: J Bacteriol. 2003 Oct;185(20):6112-8
references.pmid: 14526023
references.citation: J Biol Chem. 1999 Dec 3;274(49):35129-38
references.pmid: 10574995
references.citation: Trends Microbiol. 1995 May;3(5):178-85
references.pmid: 7542987
references.citation: Methods Cell Biol. 1991;34:61-75
references.pmid: 1943817
references.citation: Trends Genet. 1988 Aug;4(8):223-6
references.pmid: 3072723
references.citation: J Bacteriol. 2000 Jul;182(14):4068-76
references.pmid: 10869087
references.citation: FEMS Microbiol Lett. 2008 Oct;287(1):76-84
references.pmid: 18707624
references.citation: J Immunol. 2001 Jun 15;166(12):7410-8
references.pmid: 11390493
references.citation: Proc Natl Acad Sci U S A. 2002 Mar 5;99(5):2690-5
references.pmid: 11867724
references.citation: J Bacteriol. 2003 Apr;185(8):2503-11
references.pmid: 12670974
references.citation: EMBO J. 1982;1(8):945-51
references.pmid: 6329717
references.citation: J Bacteriol. 2005 May;187(9):3002-12
references.pmid: 15838026
references.citation: Nat Struct Mol Biol. 2008 Feb;15(2):130-8
references.pmid: 18204465
references.citation: Rev Infect Dis. 1991 Mar-Apr;13 Suppl 4:S279-84
references.pmid: 1710816
references.citation: Proc Natl Acad Sci U S A. 2006 Sep 12;103(37):13658-63
references.pmid: 16954199
references.citation: Mol Microbiol. 2007 Sep;65(5):1345-59
references.pmid: 17697256
references.citation: J Bacteriol. 2009 Nov;191(21):6618-31
references.pmid: 19717596
references.citation: Bioinformatics. 2001 Sep;17(9):849-50
references.pmid: 11590105
references.citation: Infect Immun. 1994 Mar;62(3):809-17
references.pmid: 8112851
references.citation: Carbohydr Res. 2003 Nov 14;338(23):2503-19
references.pmid: 14670712
references.citation: Nucleic Acids Res. 2005 Jul 1;33(Web Server issue):W36-8
references.pmid: 15980489
references.citation: Microbiol Rev. 1993 Mar;57(1):50-108
references.pmid: 8096622
references.citation: Infect Immun. 1989 Jun;57(6):1707-13
references.pmid: 2498205
references.citation: Virology. 2004 Apr 10;321(2):217-21
references.pmid: 15051382
references.citation: J Bacteriol. 1999 Feb;181(3):973-80
references.pmid: 9922263
references.citation: Microbes Infect. 2000 Jul;2(9):1051-60
references.pmid: 10967285
references.citation: J Mol Biol. 1999 Jan 29;285(4):1503-13
references.pmid: 9917392
references.citation: J Endotoxin Res. 2006;12(6):324-36
references.pmid: 17254386
references.citation: J Biol Chem. 2009 Mar 13;284(11):7395-403
references.pmid: 19129185
references.citation: Mol Microbiol. 1998 Jun;28(6):1211-22
references.pmid: 9680210
references.citation: FEMS Microbiol Lett. 1999 Oct 15;179(2):187-202
references.pmid: 10518715
references.citation: J Mol Biol. 2007 Nov 30;374(3):671-87
references.pmid: 17950311
references.citation: J Biol Chem. 2005 Jul 8;280(27):25936-47
references.pmid: 15908430
references.citation: Appl Environ Microbiol. 2008 Dec;74(23):7422-6
references.pmid: 18849445
references.citation: Mol Microbiol. 1995 May;16(3):565-74
references.pmid: 7565115
references.citation: Mol Microbiol. 2008 Dec;70(6):1424-40
references.pmid: 19019161
references.citation: Microbiology (Reading). 2004 Dec;150(Pt 12):4095-105
references.pmid: 15583162
references.citation: Arch Microbiol. 2005 Oct;184(1):1-10
references.pmid: 16044265
references.citation: J Mol Biol. 1988 Apr 5;200(3):501-11
references.pmid: 3294421
references.citation: Innate Immun. 2009 Oct;15(5):261-312
references.pmid: 19710102
references.citation: J Bacteriol. 1989 Sep;171(9):4609-16
references.pmid: 2527843