Characterization of Galacturonosyl Transferase Genes rgtA, rgtB, rgtC, rgtD, and rgtE Responsible for Lipopolysaccharide Synthesis in Nitrogen-fixing Endosymbiont Rhizobium leguminosarum
| Autor: | Elmar L. Kannenberg, Russell W. Carlson, Dusty B. Brown, L. Scott Forsberg |
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| Rok vydání: | 2012 |
| Předmět: |
biology
Mutant Mutagenesis (molecular biology technique) Cell Biology medicine.disease_cause Biochemistry Molecular biology Rhizobium leguminosarum Cell membrane Lipid A chemistry.chemical_compound medicine.anatomical_structure chemistry Glycosyltransferase medicine biology.protein Transferase Glycosyl Molecular Biology |
| Zdroj: | Journal of Biological Chemistry. 287:935-949 |
| ISSN: | 0021-9258 |
| DOI: | 10.1074/jbc.m111.311571 |
| Popis: | Rhizobium lipopolysaccharide (LPS) contains four terminally linked galacturonic acid (GalA) residues; one attached to the lipid A and three attached to the core oligosaccharide moiety. Attachment of the GalA residues requires the lipid donor dodecaprenyl-phosphate GalA (Dod-P-GalA), which is synthesized by the GalA transferase RgtE reported here. The galacturonosyl transferases RgtA, -B, and -C utilize Dod-P-GalA to attach GalAs on the LPS core region, and RgtD attaches GalA to the lipid A 4′ position. As reported here, the functions of the rgtD and rgtE genes were determined via insertion mutagenesis and structural characterization of the mutant lipid A. The rgtE− mutant lacked Dod-P-GalA as determined by mass spectrometry of total lipid extracts and the inability of rgtE− mutant membranes to provide the substrate for heterologously expressed RgtA activity. In addition, we created single mutations in each of the rgtA, -B, -C, -D, and -E genes to study the biological function of the GalA residues. The structures of the core oligosaccharide region from each of the rgt mutants were elucidated by glycosyl linkage analysis. Each mutant was assayed for its sensitivity to sodium deoxycholate and to the antimicrobial cationic peptide, polymyxin B (PmxB). The rgt mutants were more sensitive than the parent strain to deoxycholate by varying degrees. However, the rgtA, -B, and -C mutants were more resistant to PmxB, whereas the rgtD and E mutants were less resistant in comparison to the parent strain. |
| Databáze: | OpenAIRE |
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