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

M. Soledad Saldías, Kinnari Patel, Cristina L. Marolda, Mauricio Bittner, Inés Contreras, Miguel A. Valvano

Resultado de la investigación: Article

44 Citas (Scopus)

Resumen

WbaP is a membrane enzyme that initiates O antigen synthesis in Salmonella enterica by catalysing the transfer of galactose 1-phosphate (Gal-1-P) onto undecaprenyl phosphate (Und-P). WbaP possesses at least three predicted structural domains: an N-terminal region containing four transmembrane helices, a large central periplasmic loop, and a C-terminal domain containing the last transmembrane helix and a large cytoplasmic tail. In this work, we investigated the contribution of each region to WbaP function by constructing a series of mutant WbaP proteins and using them to complement O antigen synthesis in ΔwbaP mutants of S. enterica serovars Typhi and Typhimurium. Truncated forms of WbaP lacking the periplasmic loop exhibited altered chain-length distributions in O antigen polymerization, suggesting that this central domain is involved in modulating the chain-length distribution of the O polysaccharide. The N-terminal and periplasmic domains were dispensable for complementation of O antigen synthesis in vivo, suggesting that the C-terminal domain carries the sugar-phosphate transferase activity. However, despite the fact that they complemented the synthesis of O antigen in the ΔwbaP mutant in vivo, membrane extracts containing WbaP derivatives without the N-terminal domain failed to transfer radioactive Gal from UDP-Gal into a lipid-rich fraction. These results suggest that the N-terminal region of WbaP, which contains four transmembrane domains, is essential for the insertion or stability of the protein in the bacterial membrane. We propose that the domain structure of WbaP enables this protein not only to function in the transfer of Gal-1-P to Und-P but also to establish critical interactions with additional proteins required for the correct assembly of O antigen in S. enterica.

Idioma originalEnglish
Páginas (desde-hasta)440-453
Número de páginas14
PublicaciónMicrobiology
Volumen154
N.º2
DOI
EstadoPublished - feb 2008

Huella dactilar

O Antigens
Salmonella enterica
Transferases
Membranes
Sugar Phosphates
Salmonella typhi
Uridine Diphosphate
Bacterial Proteins
Protein Stability
Mutant Proteins
Polymerization
Polysaccharides
Proteins
Lipids
Enzymes

ASJC Scopus subject areas

  • Microbiology

Citar esto

Saldías, M. Soledad ; Patel, Kinnari ; Marolda, Cristina L. ; Bittner, Mauricio ; Contreras, Inés ; Valvano, Miguel A. / Distinct functional domains of the Salmonella enterica WbaP transferase that is involved in the initiation reaction for synthesis of the O antigen subunit. En: Microbiology. 2008 ; Vol. 154, N.º 2. pp. 440-453.
@article{60d39d406e434e1b817225c15b49d10d,
title = "Distinct functional domains of the Salmonella enterica WbaP transferase that is involved in the initiation reaction for synthesis of the O antigen subunit",
abstract = "WbaP is a membrane enzyme that initiates O antigen synthesis in Salmonella enterica by catalysing the transfer of galactose 1-phosphate (Gal-1-P) onto undecaprenyl phosphate (Und-P). WbaP possesses at least three predicted structural domains: an N-terminal region containing four transmembrane helices, a large central periplasmic loop, and a C-terminal domain containing the last transmembrane helix and a large cytoplasmic tail. In this work, we investigated the contribution of each region to WbaP function by constructing a series of mutant WbaP proteins and using them to complement O antigen synthesis in ΔwbaP mutants of S. enterica serovars Typhi and Typhimurium. Truncated forms of WbaP lacking the periplasmic loop exhibited altered chain-length distributions in O antigen polymerization, suggesting that this central domain is involved in modulating the chain-length distribution of the O polysaccharide. The N-terminal and periplasmic domains were dispensable for complementation of O antigen synthesis in vivo, suggesting that the C-terminal domain carries the sugar-phosphate transferase activity. However, despite the fact that they complemented the synthesis of O antigen in the ΔwbaP mutant in vivo, membrane extracts containing WbaP derivatives without the N-terminal domain failed to transfer radioactive Gal from UDP-Gal into a lipid-rich fraction. These results suggest that the N-terminal region of WbaP, which contains four transmembrane domains, is essential for the insertion or stability of the protein in the bacterial membrane. We propose that the domain structure of WbaP enables this protein not only to function in the transfer of Gal-1-P to Und-P but also to establish critical interactions with additional proteins required for the correct assembly of O antigen in S. enterica.",
author = "Sald{\'i}as, {M. Soledad} and Kinnari Patel and Marolda, {Cristina L.} and Mauricio Bittner and In{\'e}s Contreras and Valvano, {Miguel A.}",
year = "2008",
month = "2",
doi = "10.1099/mic.0.2007/013136-0",
language = "English",
volume = "154",
pages = "440--453",
journal = "Microbiology (United Kingdom)",
issn = "1350-0872",
publisher = "Society for General Microbiology",
number = "2",

}

Distinct functional domains of the Salmonella enterica WbaP transferase that is involved in the initiation reaction for synthesis of the O antigen subunit. / Saldías, M. Soledad; Patel, Kinnari; Marolda, Cristina L.; Bittner, Mauricio; Contreras, Inés; Valvano, Miguel A.

En: Microbiology, Vol. 154, N.º 2, 02.2008, p. 440-453.

Resultado de la investigación: Article

TY - JOUR

T1 - Distinct functional domains of the Salmonella enterica WbaP transferase that is involved in the initiation reaction for synthesis of the O antigen subunit

AU - Saldías, M. Soledad

AU - Patel, Kinnari

AU - Marolda, Cristina L.

AU - Bittner, Mauricio

AU - Contreras, Inés

AU - Valvano, Miguel A.

PY - 2008/2

Y1 - 2008/2

N2 - WbaP is a membrane enzyme that initiates O antigen synthesis in Salmonella enterica by catalysing the transfer of galactose 1-phosphate (Gal-1-P) onto undecaprenyl phosphate (Und-P). WbaP possesses at least three predicted structural domains: an N-terminal region containing four transmembrane helices, a large central periplasmic loop, and a C-terminal domain containing the last transmembrane helix and a large cytoplasmic tail. In this work, we investigated the contribution of each region to WbaP function by constructing a series of mutant WbaP proteins and using them to complement O antigen synthesis in ΔwbaP mutants of S. enterica serovars Typhi and Typhimurium. Truncated forms of WbaP lacking the periplasmic loop exhibited altered chain-length distributions in O antigen polymerization, suggesting that this central domain is involved in modulating the chain-length distribution of the O polysaccharide. The N-terminal and periplasmic domains were dispensable for complementation of O antigen synthesis in vivo, suggesting that the C-terminal domain carries the sugar-phosphate transferase activity. However, despite the fact that they complemented the synthesis of O antigen in the ΔwbaP mutant in vivo, membrane extracts containing WbaP derivatives without the N-terminal domain failed to transfer radioactive Gal from UDP-Gal into a lipid-rich fraction. These results suggest that the N-terminal region of WbaP, which contains four transmembrane domains, is essential for the insertion or stability of the protein in the bacterial membrane. We propose that the domain structure of WbaP enables this protein not only to function in the transfer of Gal-1-P to Und-P but also to establish critical interactions with additional proteins required for the correct assembly of O antigen in S. enterica.

AB - WbaP is a membrane enzyme that initiates O antigen synthesis in Salmonella enterica by catalysing the transfer of galactose 1-phosphate (Gal-1-P) onto undecaprenyl phosphate (Und-P). WbaP possesses at least three predicted structural domains: an N-terminal region containing four transmembrane helices, a large central periplasmic loop, and a C-terminal domain containing the last transmembrane helix and a large cytoplasmic tail. In this work, we investigated the contribution of each region to WbaP function by constructing a series of mutant WbaP proteins and using them to complement O antigen synthesis in ΔwbaP mutants of S. enterica serovars Typhi and Typhimurium. Truncated forms of WbaP lacking the periplasmic loop exhibited altered chain-length distributions in O antigen polymerization, suggesting that this central domain is involved in modulating the chain-length distribution of the O polysaccharide. The N-terminal and periplasmic domains were dispensable for complementation of O antigen synthesis in vivo, suggesting that the C-terminal domain carries the sugar-phosphate transferase activity. However, despite the fact that they complemented the synthesis of O antigen in the ΔwbaP mutant in vivo, membrane extracts containing WbaP derivatives without the N-terminal domain failed to transfer radioactive Gal from UDP-Gal into a lipid-rich fraction. These results suggest that the N-terminal region of WbaP, which contains four transmembrane domains, is essential for the insertion or stability of the protein in the bacterial membrane. We propose that the domain structure of WbaP enables this protein not only to function in the transfer of Gal-1-P to Und-P but also to establish critical interactions with additional proteins required for the correct assembly of O antigen in S. enterica.

UR - http://www.scopus.com/inward/record.url?scp=38849111460&partnerID=8YFLogxK

U2 - 10.1099/mic.0.2007/013136-0

DO - 10.1099/mic.0.2007/013136-0

M3 - Article

VL - 154

SP - 440

EP - 453

JO - Microbiology (United Kingdom)

JF - Microbiology (United Kingdom)

SN - 1350-0872

IS - 2

ER -