De novo synthesis of bacterial glycogen: Agrobacterium tumefaciens glycogen synthase is involved in glucan initiation and elongation

Abstract

Evidence is presented indicating that initiation of glycogen synthesis in Agrobacterium tumefaciens does not require the presence of alpha(1,4)-linked glucans. Crude cell extracts incubated with ADP-glucose (Glc) were able to form alpha(1,4)-linked glucans despite the fact that cells used for extract preparation displayed a genotype that prevented synthesis of Glc-containing sugar nucleotides and thus preformation of alpha(1,4)-linked glucans and that the defined growth medium used contained glycerol as carbon source. A. tumefaciens glycogen synthase (GS) purified to homogeneity from the above-mentioned cells was able to build its own primer by transferring Glc residues from ADP-Glc to an amino acid(s) in the same protein. Primed GS then became the substrate for further GS-catalyzed glucan elongation. It was concluded that, contrary to what happens in mammalian and yeast cells in which two different proteins are required for linear alpha(1,4)-linked glucan formation (glycogenin for initiation and GS for further elongation), in A. tumefaciens and probably in all other bacteria, the same protein is involved in both glycogen initiation and elongation.

Fig. 1.

SDS/PAGE of A. tumefaciens GS-reaction products. Purified GS was incubated with ADP-[¹⁴C]Glc under unprimed initiation reaction conditions, and 10% TCA-insoluble material was submitted to 10% SDS/PAGE. (A) Coomassie brilliant blue staining. (B) Autoradiography of the gel shown in A.(C) Reactions were carried out as described for A and stopped by heating tubes at 65°C for 15 min, the contents were treated with α-amylase, and precipitates insoluble in 10% TCA were submitted to 10% SDS/PAGE. Lane 1, enzymatically treated; lane 2, untreated control. The arrow indicates GS migrating position. (D and E) Reactions were carried out and stopped as described for C and submitted to proteinase K treatment, and precipitates insoluble in 10% TCA were run on 10% SDS/PAGE. (D) Coomassie brilliant blue staining. (E) Autoradiography of the gel shown in D. Lanes 1, enzymatically treated samples; lanes 2, untreated controls. For further details see Materials and Methods.

Fig. 2.

GS gel filtration. Purified GS was submitted to BioSil SEC 250 gel chromatography. The arrows indicate the elution volumes of 66- and 50-kDa protein standards. Fractions were submitted to 10% SDS/PAGE and stained with Coomassie brilliant blue. The same fractions were used as enzyme source in unprimed initiation reactions. TCA precipitates were submitted to 10% SDS/PAGE followed by autoradiography. For further details see Materials and Methods.

Fig. 3.

GS-linked glucans are intermediates in the synthesis of large polymers. Purified GS was incubated under unprimed initiation conditions for 15 or 30 min with 3 μM ADP-[¹⁴C]Glc (lanes 1 and 2, respectively) or for only 15 min, after which unlabeled ADP-Glc toa5mM final concentration was added, and the sample was incubated further for 15 min (lanes 3). Material insoluble in 10% TCA was submitted to 10% SDS/PAGE followed by autoradiography (A) or staining with Coomassie brilliant blue (B). For further details see Materials and Methods.