Membrane topology mapping of the O-antigen flippase (Wzx), polymerase (Wzy), and ligase (WaaL) from Pseudomonas aeruginosa PAO1 reveals novel domain architectures.
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.
Islam
Salim T
ST
Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada.
Taylor
Véronique L
VL
Qi
Meng
M
Lam
Joseph S
JS
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
D001426:Bacterial Proteins; D008565:Membrane Proteins; D006602:Hexosyltransferases; C085652:O-antigen polymerase; D019729:Carbon-Oxygen Ligases; C528973:WaaL protein, Pseudomonas aeruginosa
Environ Microbiol. 2002 Dec;4(12):883-97
12534470
Mol Microbiol. 1993 Mar;7(5):725-34
7682279
J Bacteriol. 2007 Mar;189(6):2203-9
17209027
Eur J Biochem. 2003 Mar;270(5):799-813
12603313
Antimicrob Agents Chemother. 1993 Apr;37(4):715-21
8494366
Nat Methods. 2006 Apr;3(4):303-13
16554836
J Bacteriol. 2003 Oct;185(20):6112-8
14526023
J Biol Chem. 1999 Dec 3;274(49):35129-38
10574995
Trends Microbiol. 1995 May;3(5):178-85
7542987
Methods Cell Biol. 1991;34:61-75
1943817
Trends Genet. 1988 Aug;4(8):223-6
3072723
J Bacteriol. 2000 Jul;182(14):4068-76
10869087
FEMS Microbiol Lett. 2008 Oct;287(1):76-84
18707624
J Immunol. 2001 Jun 15;166(12):7410-8
11390493
Proc Natl Acad Sci U S A. 2002 Mar 5;99(5):2690-5
11867724
J Bacteriol. 2003 Apr;185(8):2503-11
12670974
EMBO J. 1982;1(8):945-51
6329717
J Bacteriol. 2005 May;187(9):3002-12
15838026
Nat Struct Mol Biol. 2008 Feb;15(2):130-8
18204465
Rev Infect Dis. 1991 Mar-Apr;13 Suppl 4:S279-84
1710816
Proc Natl Acad Sci U S A. 2006 Sep 12;103(37):13658-63
16954199
Mol Microbiol. 2007 Sep;65(5):1345-59
17697256
J Bacteriol. 2009 Nov;191(21):6618-31
19717596
Bioinformatics. 2001 Sep;17(9):849-50
11590105
Infect Immun. 1994 Mar;62(3):809-17
8112851
Carbohydr Res. 2003 Nov 14;338(23):2503-19
14670712
Nucleic Acids Res. 2005 Jul 1;33(Web Server issue):W36-8
15980489
Microbiol Rev. 1993 Mar;57(1):50-108
8096622
Infect Immun. 1989 Jun;57(6):1707-13
2498205
Virology. 2004 Apr 10;321(2):217-21
15051382
J Bacteriol. 1999 Feb;181(3):973-80
9922263
Microbes Infect. 2000 Jul;2(9):1051-60
10967285
J Mol Biol. 1999 Jan 29;285(4):1503-13
9917392
J Endotoxin Res. 2006;12(6):324-36
17254386
J Biol Chem. 2009 Mar 13;284(11):7395-403
19129185
Mol Microbiol. 1998 Jun;28(6):1211-22
9680210
FEMS Microbiol Lett. 1999 Oct 15;179(2):187-202
10518715
J Mol Biol. 2007 Nov 30;374(3):671-87
17950311
J Biol Chem. 2005 Jul 8;280(27):25936-47
15908430
Appl Environ Microbiol. 2008 Dec;74(23):7422-6
18849445
Mol Microbiol. 1995 May;16(3):565-74
7565115
Mol Microbiol. 2008 Dec;70(6):1424-40
19019161
Microbiology (Reading). 2004 Dec;150(Pt 12):4095-105
15583162
Arch Microbiol. 2005 Oct;184(1):1-10
16044265
J Mol Biol. 1988 Apr 5;200(3):501-11
3294421
Innate Immun. 2009 Oct;15(5):261-312
19710102
J Bacteriol. 1989 Sep;171(9):4609-16
2527843