Utilizing the O-antigen lipopolysaccharide biosynthesis pathway in Escherichia coli to interrogate the substrate specificities of exogenous glycosyltransferase genes in a combinatorial approach
Identifieur interne : 000167 ( Ncbi/Curation ); précédent : 000166; suivant : 000168Utilizing the O-antigen lipopolysaccharide biosynthesis pathway in Escherichia coli to interrogate the substrate specificities of exogenous glycosyltransferase genes in a combinatorial approach
Auteurs : Eric B. Johansen [États-Unis] ; Francis C. Szoka [États-Unis] ; Anthony Zaleski [États-Unis] ; Michael A. Apicella [États-Unis] ; Bradford W. Gibson [États-Unis]Source :
- Glycobiology [ 0959-6658 ] ; 2010.
Descripteurs français
- KwdFr :
- Antigènes O (), Antigènes O (biosynthèse), Antigènes O (génétique), Conformation des glucides, Escherichia coli (métabolisme), Glycosyltransferase (génétique), Glycosyltransferase (métabolisme), Haemophilus influenzae (enzymologie), Haemophilus influenzae (génétique), Spécificité du substrat, Séquence glucidique, Test ELISA, Vecteurs génétiques (génétique).
- MESH :
- biosynthèse : Antigènes O.
- enzymologie : Haemophilus influenzae.
- génétique : Antigènes O, Glycosyltransferase, Haemophilus influenzae, Vecteurs génétiques.
- métabolisme : Escherichia coli, Glycosyltransferase.
- Antigènes O, Conformation des glucides, Spécificité du substrat, Séquence glucidique, Test ELISA.
English descriptors
- KwdEn :
- Carbohydrate Conformation, Carbohydrate Sequence, Enzyme-Linked Immunosorbent Assay, Escherichia coli (metabolism), Genetic Vectors (genetics), Glycosyltransferases (genetics), Glycosyltransferases (metabolism), Haemophilus influenzae (enzymology), Haemophilus influenzae (genetics), O Antigens (biosynthesis), O Antigens (chemistry), O Antigens (genetics), Substrate Specificity.
- MESH :
- chemical , biosynthesis : O Antigens.
- chemical , chemistry : O Antigens.
- chemical , genetics : Glycosyltransferases, O Antigens.
- enzymology : Haemophilus influenzae.
- genetics : Genetic Vectors, Haemophilus influenzae.
- metabolism : Escherichia coli, Glycosyltransferases.
- Carbohydrate Conformation, Carbohydrate Sequence, Enzyme-Linked Immunosorbent Assay, Substrate Specificity.
Abstract
In previous work, our laboratory generated novel chimeric lipopolysaccharides (LPS) in
Url:
DOI: 10.1093/glycob/cwq033
PubMed: 20208062
PubMed Central: 2900885
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PMC:2900885Le document en format XML
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<sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Utilizing the O-antigen lipopolysaccharide biosynthesis pathway in <italic>Escherichia coli</italic>
to interrogate the substrate specificities of exogenous glycosyltransferase genes in a combinatorial approach</title>
<author><name sortKey="Johansen, Eric B" sort="Johansen, Eric B" uniqKey="Johansen E" first="Eric B." last="Johansen">Eric B. Johansen</name>
<affiliation wicri:level="1"><nlm:aff id="aff1"><addr-line>Department of Pharmaceutical Chemistry and Pharmaceutical Sciences</addr-line>
,<institution>University of California</institution>
,<addr-line>San Francisco, CA 94143</addr-line>
,<country>USA</country>
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<country xml:lang="fr">États-Unis</country>
<wicri:regionArea># see nlm:aff country strict</wicri:regionArea>
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<author><name sortKey="Szoka, Francis C" sort="Szoka, Francis C" uniqKey="Szoka F" first="Francis C." last="Szoka">Francis C. Szoka</name>
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,<institution>University of California</institution>
,<addr-line>San Francisco, CA 94143</addr-line>
,<country>USA</country>
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<country xml:lang="fr">États-Unis</country>
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<author><name sortKey="Zaleski, Anthony" sort="Zaleski, Anthony" uniqKey="Zaleski A" first="Anthony" last="Zaleski">Anthony Zaleski</name>
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,<institution>University of Iowa</institution>
,<addr-line>Iowa City, IA 52242</addr-line>
,<country>USA</country>
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<author><name sortKey="Apicella, Michael A" sort="Apicella, Michael A" uniqKey="Apicella M" first="Michael A." last="Apicella">Michael A. Apicella</name>
<affiliation wicri:level="1"><nlm:aff id="aff2"><addr-line>Department of Microbiology</addr-line>
,<institution>University of Iowa</institution>
,<addr-line>Iowa City, IA 52242</addr-line>
,<country>USA</country>
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<wicri:regionArea># see nlm:aff country strict</wicri:regionArea>
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<author><name sortKey="Gibson, Bradford W" sort="Gibson, Bradford W" uniqKey="Gibson B" first="Bradford W." last="Gibson">Bradford W. Gibson</name>
<affiliation wicri:level="1"><nlm:aff id="aff3"><institution>Buck Institute for Age Research</institution>
,<addr-line>8001 Redwood Blvd., Novato, CA 94945</addr-line>
,<country>USA</country>
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<country xml:lang="fr">États-Unis</country>
<wicri:regionArea># see nlm:aff country strict</wicri:regionArea>
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<series><title level="j">Glycobiology</title>
<idno type="ISSN">0959-6658</idno>
<idno type="eISSN">1460-2423</idno>
<imprint><date when="2010">2010</date>
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</fileDesc>
<profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Carbohydrate Conformation</term>
<term>Carbohydrate Sequence</term>
<term>Enzyme-Linked Immunosorbent Assay</term>
<term>Escherichia coli (metabolism)</term>
<term>Genetic Vectors (genetics)</term>
<term>Glycosyltransferases (genetics)</term>
<term>Glycosyltransferases (metabolism)</term>
<term>Haemophilus influenzae (enzymology)</term>
<term>Haemophilus influenzae (genetics)</term>
<term>O Antigens (biosynthesis)</term>
<term>O Antigens (chemistry)</term>
<term>O Antigens (genetics)</term>
<term>Substrate Specificity</term>
</keywords>
<keywords scheme="KwdFr" xml:lang="fr"><term>Antigènes O ()</term>
<term>Antigènes O (biosynthèse)</term>
<term>Antigènes O (génétique)</term>
<term>Conformation des glucides</term>
<term>Escherichia coli (métabolisme)</term>
<term>Glycosyltransferase (génétique)</term>
<term>Glycosyltransferase (métabolisme)</term>
<term>Haemophilus influenzae (enzymologie)</term>
<term>Haemophilus influenzae (génétique)</term>
<term>Spécificité du substrat</term>
<term>Séquence glucidique</term>
<term>Test ELISA</term>
<term>Vecteurs génétiques (génétique)</term>
</keywords>
<keywords scheme="MESH" type="chemical" qualifier="biosynthesis" xml:lang="en"><term>O Antigens</term>
</keywords>
<keywords scheme="MESH" type="chemical" qualifier="chemistry" xml:lang="en"><term>O Antigens</term>
</keywords>
<keywords scheme="MESH" type="chemical" qualifier="genetics" xml:lang="en"><term>Glycosyltransferases</term>
<term>O Antigens</term>
</keywords>
<keywords scheme="MESH" qualifier="biosynthèse" xml:lang="fr"><term>Antigènes O</term>
</keywords>
<keywords scheme="MESH" qualifier="enzymologie" xml:lang="fr"><term>Haemophilus influenzae</term>
</keywords>
<keywords scheme="MESH" qualifier="enzymology" xml:lang="en"><term>Haemophilus influenzae</term>
</keywords>
<keywords scheme="MESH" qualifier="genetics" xml:lang="en"><term>Genetic Vectors</term>
<term>Haemophilus influenzae</term>
</keywords>
<keywords scheme="MESH" qualifier="génétique" xml:lang="fr"><term>Antigènes O</term>
<term>Glycosyltransferase</term>
<term>Haemophilus influenzae</term>
<term>Vecteurs génétiques</term>
</keywords>
<keywords scheme="MESH" qualifier="metabolism" xml:lang="en"><term>Escherichia coli</term>
<term>Glycosyltransferases</term>
</keywords>
<keywords scheme="MESH" qualifier="métabolisme" xml:lang="fr"><term>Escherichia coli</term>
<term>Glycosyltransferase</term>
</keywords>
<keywords scheme="MESH" xml:lang="en"><term>Carbohydrate Conformation</term>
<term>Carbohydrate Sequence</term>
<term>Enzyme-Linked Immunosorbent Assay</term>
<term>Substrate Specificity</term>
</keywords>
<keywords scheme="MESH" xml:lang="fr"><term>Antigènes O</term>
<term>Conformation des glucides</term>
<term>Spécificité du substrat</term>
<term>Séquence glucidique</term>
<term>Test ELISA</term>
</keywords>
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<front><div type="abstract" xml:lang="en"><p>In previous work, our laboratory generated novel chimeric lipopolysaccharides (LPS) in <italic>Escherichia coli</italic>
transformed with a plasmid containing exogenous lipooligosaccharide synthesis genes (<italic>lsg</italic>
) from <italic>Haemophilus influenzae.</italic>
Analysis of these novel oligosaccharide-LPS chimeras allowed characterization of the carbohydrate structures generated by several putative glycosyltransferase genes within the <italic>lsg</italic>
locus. Here, we adapted this strategy to construct a modular approach to study the synthetic properties of individual glycosyltransferases expressed alone and in combinations. To this end, a set of expression vectors containing one to four putative glycosyltransferase genes from the <italic>lsg</italic>
locus, <italic>lsgC-F</italic>
, were transformed into <italic>E. coli</italic>
K12 (XL-1) which is defective in LPS O-antigen biosynthesis<italic>.</italic>
This strategy relied on the inclusion of the <italic>H. influenzae</italic>
gene product <italic>lsgG</italic>
in every plasmid construct, which partially rescues the <italic>E. coli</italic>
LPS biosynthesis defect by priming uridine diphosphate-undecaprenyl in the WecA-dependent O-antigen synthetic pathway with <italic>N</italic>
-acetyl-glucosamine (GlcNAc). This GlcNAc-undecaprenyl then served as an acceptor substrate for further carbohydrate extension by transformed glycosyltransferases. The resultant LPS-linked chimeric glycans were isolated from their <italic>E. coli</italic>
constructs and characterized by mass spectrometry, methylation analysis and enzyme-linked immunosorbent assays. These structural data allowed the specificity of various glycosyltransferases to be unambiguously assigned to individual genes. LsgF was found to transfer a galactose (Gal) to terminal GlcNAc. LsgE was found to transfer GlcNAc to Gal-GlcNAc, and both LsgF and LsgD were found to transfer Gal to GlcNAc-Gal-GlcNAc but with differing linkage specificities. This method can be generalized and readily adapted to study the substrate specificity of other putative or uncharacterized glycosyltransferases.</p>
</div>
</front>
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