Table 1.
Primers used in this study.
Fig 1.
The cholesterol glycolipids of Borrelia burgdorferi and predicted reaction.
A. Chemical structure of cholesteryl-β-D-galacto-pyranoside (CGal); and cholesteryl 6-O-acyl-β-D-galactopyranoside (ACGal) B. Predicted reaction showing UDP-alpha-galactose as the sugar donor and cholesterol as the acceptor molecule to yield cholesteryl β-D-galactopyranoside (CGal) which possess a galactosyl residue with its anomeric bond in the β-configuration.
Fig 2.
Multiple sequence alignment of the BB0572 sequence from Borrelia burgdorferi against Lyme disease and relapsing fever causing spirochetes.
Red box indicates the active motifs of the GT2 glycosyltransferase enzyme. A star indicates positions which have a single, fully conserved residue. A period (.) indicates conservation between groups of weakly similar properties.
Fig 3.
Verification of expression of bb0572 in E coli.
Coomassie stained SDS-PAGE gel (A) and anti-His tag Western blot (B) of whole cell lysate from uninduced (lanes (2, 4 and 6) and IPTG induced (lanes 3, 5 and 7) cultures of E. coli expressing the WT bb0572 (lane 2 and 3), mutated bb0572 ΔCFF/DGD/I (lane 4 and 5) or possessing the empty pET28a vector (lane 6 and 7). Lane 1(ladder).
Fig 4.
Cell density and kinetics of bb0572 expression in Borrelia burgdorferi cultured in vitro.
Bb cells at early exponential phase (1x106) were cultured and harvested at 6, 12, 18, 24, 30 and 36 h to determine expression of bb0572. A. Cell densities were monitored by dark-field microscopy every six hours, corresponding to cell harvest for RNA extraction for expression analysis. B. Gene expression of bb0572 was normalized with the flaB as the house keeping gene. Fold change represents change in expression of bb0572 between 6 to 36 h relative to time zero (baseline). No significant differences in expression were noted during selected time intervals. To ensure adequate number of cells, culture with cell density of (1 x108 cells/ml) was used in cell-free assays (black arrow).
Fig 5.
TLC of lipids from cell free assays for the enzymatic incorporation of [26- 14C] cholesterol into cholesteryl-β-D-galacto-pyranoside (CGal). A.
The formation of cholesteryl-β-D-galacto-pyranoside (CGal) (arrow) in whole cell lysates of Bb and recombinant E. coli expressing WT bb0572 (lane 1 and 2). As expected, CGal was not formed by the whole cell lysate of E. coli expressing mutant bb0572 ΔCFF/I/DGD (lane 3). Lane 4 is the empty expression vector control, and lane 6 and 7 are from boiled whole cell lysates of Bb and recombinant E. coli expressing WT bb0572, respectively. Note lane 5 was not used. B. Substrate controls: Bb and E. coli WT bb0572 whole cell lysates with UDP-Gal (lane 1 and 2); GDP-Man (lanes 3 and 4), or cholesterol oxidase treated [26- 14C] cholesterol with UDP-Gal (lane 5 and 6) as substrate. CHO, cholesterol; ACGal, cholesteryl 6-O-acyl-β-D-galactopyranoside and MGal, monogalactosyl diacylglycerol. Ec WT (Escherichia coli wild type), Ec MT (E.coli mutant—bb0572 ΔCFF/DGD/I) and Ec VC- E.coli vector control).