Roles of Predicted Glycosyltransferases in the Biosynthesis of the Rhizobium etli CE3 O Antigen

In Vitro Biosynthesis and Chemical Identification of UDP-N-acetyl-d-quinovosamine (UDP-d-QuiNAc)

“…For determination of neutral and amino sugars, LPS preparations were treated with 2 M trifluoroacetic acid for 2 h at 121°C. After reduction with NaBD 4 and acetylation, alditol acetate derivatives of the LPS sugars were analyzed by gas chromatography (GC) under conditions described previously (8).…”

“…This fragment was subcloned by insertion into the EcoRI and KpnI sites of plasmid pFAJ1708 (18), generating plasmid pLS31. The pLS31 plasmid was introduced into the R. etli wreQ::Tn5 mutant CE166 by a triparental mating procedure described previously (8). LPS was prepared from 0.5 ml of full-grown cultures and analyzed by SDS-PAGE with periodate-silver stain (8).…”

“…The pLS31 plasmid was introduced into the R. etli wreQ::Tn5 mutant CE166 by a triparental mating procedure described previously (8). LPS was prepared from 0.5 ml of full-grown cultures and analyzed by SDS-PAGE with periodate-silver stain (8). To determine whether QuiNAc was present, LPS was extracted from washed bacterial pellets (from 4.0 liters of culture) by the hot phenol-water method; dialyzed; treated with RNase A, DNase I, and protease K; and dialyzed again followed by lyophilization (8).…”

“…LPS was prepared from 0.5 ml of full-grown cultures and analyzed by SDS-PAGE with periodate-silver stain (8). To determine whether QuiNAc was present, LPS was extracted from washed bacterial pellets (from 4.0 liters of culture) by the hot phenol-water method; dialyzed; treated with RNase A, DNase I, and protease K; and dialyzed again followed by lyophilization (8). For determination of neutral and amino sugars, LPS preparations were treated with 2 M trifluoroacetic acid for 2 h at 121°C.…”

“…Complementation with wbpM Gene of P. aeruginosa-Site-directed mutagenesis of R. etli wreV gene was performed using the same method as described in the previous study (8). The gentamicin resistance cassette from plasmid pUCGm (19) was inserted in the R. etli CE3 wreV gene through the SalI sites, generating strain CE568.…”