Figure 1.
Pathways of nucleotide-activated rhamnose biogenesis.
Schematic depiction of pathways of dTDP-rhamnose biosynthesis in Bacteria and of UDP-rhamnose biosynthesis in plants and viruses. UGD, UDP-D-glucose-4,6-dehydratase; UGER, UDP-4-keto-6-deoxy-D-glucose-3,5-epimerase/4-reductase.
Table 1.
Primers used in this study.
Figure 2.
Agl11 is a glucose-1-phosphate thymidylyltransferase.
A. Hfx. volcanii cells transformed to express CBD-Agl11 were subjected to cellulose-based chromatography. A cell extract and cellulose-bound proteins were separated on 10% SDS-PAGE and Coomassie-stained. A cellulose-bound ∼55 kDa protein band corresponding to CBD-Agl11 is observed. The positions of molecular weight markers are shown on the left. B. Cellulose-bound CBD-Agl11 or cellulose beads alone (blank) were resuspended in reaction buffer and incubated in the presence of dTTP and glucose-1-phosphate, with each substrate separately or without both substrates. Aliquots removed immediately after substrate addition and up to 40 min later were incubated with pyrophosphatase and the extent of phosphate release was measured 29]. The results represent average of triplicates ± standard deviation for one of three repeats of the experiment. C. The assay products obtained after a 5 h incubation at 42°C were separated by TLC, along with glucose-1-phosphate and dTDP-glucose standards, as described in Experimental Procedures.
Figure 3.
Agl12 functions as dTDP-D-glucose-4,6-dehydratase.
A. Hfx. volcanii cells transformed to express CBD-Agl12 were subjected to cellulose-based capture. A cell extract and cellulose-bound protein were separated on 10% SDS-PAGE and Coomassie-stained. Protein band corresponding to CBD-Agl11 (∼51 kDa) is observed. The positions of molecular weight markers are shown on the left of the gel. B. Cellulose-bound CBD-Agl12 or cellulose beads alone (blank) was resuspended in reaction buffer and incubated in the presence of dTDP-glucose or no substrate. Immediately after substrate addition and up to 40 min of incubation at 42°C, the samples were incubated with 0.1 M NaOH for 20 min at 42°C and the increase of absorbance at 320 nm was measured. C. The same assay was repeated for up to 60 min using dTDP-glucose or UDP-glucose as substrate or with no substrate. The assay results presented in B and C represent averages of triplicates ± standard deviation obtained in one of two repeats of the experiment.
Figure 4.
Agl13 and Agl14 together convert dTDP-4-keto-6-deoxy-glucose into dTDP-rhamnose.
Cellulose-bound CBD-Agl13 and CBD-Agl14 were combined with dTDP-4-keto-6-deoxy-glucose and NADPH and the soluble fraction was examined by nano- ESI/MS analysis. Peaks corresponding to dTDP-rhamnose and the sodium adduct are indicated. Inset: Purification of CBD-Agl13 and CBD-Agl14. Cell extracts and cellulose-bound protein from Hfx. volcanii cells transformed to express CBD-Agl13 (left) or CBD-Agl14 (right) were separated on 10% SDS-PAGE and Coomassie-stained. Protein bands corresponding to CBD-Agl13 and CBD-Agl14 are observed in each lane of cellulose-bound material. The positions of molecular weight markers are shown on the left of each gel.
Figure 5.
agl11 and agl13 are co-transcribed.
A. Schematic depiction of the position and orientation of agl11-agl14 in the Hfx. volcanii genome. The length of each gene is arbitrarily drawn. B. PCR amplifications were performed using a forward primer against a region within agl11 and a reverse primer against a region within agl13, together with genomic DNA or RNA isolated from Hfx. volcanii cells, cDNA prepared from the same RNA or no nucleic acid (blank) as template. The positions of Kbp markers are shown on the left.
Table 2.
Archaea where rmlABCD are clustered.