Biochemical and genetic characterization of the pathways for trehalose metabolism in Propionibacterium freudenreichii, and their role in stress response

Abstract

Propionibacterium freudenreichii accumulates high levels of trehalose, especially in response to stress. The pathways for trehalose metabolism were characterized, and their roles in response to osmotic, oxidative and acid stress were studied. Two pathways were identified: the trehalose-6-phosphate synthase/phosphatase (OtsA-OtsB) pathway, and the trehalose synthase (TreS) pathway. The former was used for trehalose synthesis, whereas the latter is proposed to operate in trehalose degradation. The activities of OtsA, OtsB and TreS were detected in cell extracts; the corresponding genes were identified, and the recombinant proteins were characterized in detail. In crude extracts of P. freudenreichii, OtsA was specific for ADP-glucose, in contrast to the pure recombinant OtsA, which used UDP-, GDP-and TDP-glucose, in addition to ADP-glucose. Moreover, the substrate specificity of OtsA in cell extracts was lost during purification, and the recombinant OtsA became specific to ADP-glucose upon incubation with a dialysed cell extract. The level of OtsA was enhanced (approximately twofold) by osmotic, oxidative and acid stress, whereas the level of TreS remained constant, or it decreased, under identical stress conditions. Therefore, the OtsA-OtsB pathway plays an important role in the synthesis of trehalose in response to stress. It is most likely that trehalose degradation proceeds via TreS to yield maltose, which is subsequently catabolized via amylomaltase activity. Hydrolytic activities that are potentially involved in trehalose degradation (trehalase, trehalose phosphorylase, trehalose-6-phosphate phosphorylase and trehalose-6-phosphate hydrolase) were not present. The role of trehalose as a common response to three distinct stresses is discussed.

A Unique Combination of Genetic Systems for the Synthesis of Trehalose in Rubrobacter xylanophilus : Properties of a Rare Actinobacterial TreT

“…Cells were grown in Thermus medium without or with 2.5% NaCl at 60 or 67°C in a medium containing 1.0 g/liter tryptone and 1.0 g/liter yeast extract (10). The cultures were also grown in a medium containing 2.0 g/liter maltose as a single carbon source, 1 g/liter NH 4 Cl, and 0.25 g/liter yeast extract. Chromosomal DNA was isolated as previously described (32).…”

“…R. xylanophilus cells were recovered by centrifugation; extracts were obtained using a French pressure cell and dialyzed (20 mM Tris-HCl, pH 7.5). To provide evidence of the activities of the TreP, Tps/Tpp, TreT, TreS, and TreY/TreZ pathways in the cell extracts, the assays were performed as previously described, with slight modifications (4,7,29,37). The mixtures contained 15 g of protein of R. xylanophilus extract (1 g/l) and the appropriate substrates in a final volume of 50 l. The reactions were performed at 60°C for 30 min in 25 mM bis-Tris propane buffer at pH 7.0 with 4 mM MgCl 2 and then stopped on ice.…”

“…In many organisms osmoregulated trehalose synthesis appears to involve Tps and Tpp; however, the accumulation of trehalose during osmotic adjustment in Corynebacterium glutamicum, which also possess TreS, is linked to the TreY/TreZ pathway (38). Propionibacterium freudenreichii, on the other hand, depends on the Tps/Tpp pathway for the synthesis of trehalose in response to osmotic, oxidative, and acid stresses, while TreS is involved in trehalose degradation (4).…”

Dairy Propionibacteria: Less Conventional Probiotics to Improve the Human and Animal Health

“…As described in paragraphs above, propionibacteria synthesize trehalose as a reserve compound and as a stress-response metabolite [52][53][54][55].…”

Molecular and Physiological Role of the Trehalose-Hydrolyzing α-Glucosidase from Thermus thermophilus HB27

“…These enzymes may also play important roles in the regulation of the level of trehalose in a cell. Trehalose can also be converted into maltose by trehalose synthase (TreS; EC 5.4.99.16) and then metabolized via amylomaltase, maltose/maltodextrin phosphorylase, or other ␣-glucosidases with ␣-1,4-linkage hydrolytic activity (7,14,18). Although T. thermophilus HB27 lacks the genetic machinery for trehalose biosynthesis, this disaccharide is taken up from the medium (2, 24) but does not serve as a compatible solute for strain HB27, unlike the majority of the strains of this species.…”