Regulation Of Catalytic Efficiency And Expression Of Trehalose-6-phosphate Synthase In Arthrobacter At Low Temperature

The enzyme OtsA (trehalose-6-phosphate synthase) is ubiquitous in both prokaryotic and eukaryotic organisms, where it plays a critical role in stress resistance and glucose metabolism. Studies in OtsA and the related regulations are very popular in plants and microorganisms areas. To understand the function and regulations of OtsA of microorganisms in stress, we chose two strains of Arthrobacter:Arthrobacter sp.Cjts and Arthrobacter strain A3 to analyze the mechanisms of catalytic efficiency and expressing regulations of OtsA in cold stress.The conclusions were obtained through cloning of the otsA genes, expressing and purifying the recombinant proteins, testing of the enzyme activities and analyzing of the protein-protein interations.(1)The catalytic efficiency of OtsA from Arthrobacter sp. Cjts is higher than that from Escherichia coli at low temperatures, which results from the high affinity of the enzyme for UDP-glucose at low temperatures.(2) The N-loop sequence is one of the important parts of catalytic center of OtsA. Exchang of N-loop sequence from Arthrobacter OtsA to Escherichia coli OtsA has increased the affinity for UDP-glucose, which suggests the sequence has a large effect on OtsA affinity for UDP-Glc. Sequence analysis indicated that the flexibility of the N-loop may be related to the catalytic efficiency of OtsA at low temperatures.(3) The change of trehalose level is mainly controlled by OtsA through immunoblot analyses and has no relations to other trehalose pathways in Arthrobacter strain A3. The same conclusion was obtained through comparison of changes of trehalose levels in wild type stain, otsA overexpressing strain and otsA knock-out strain.(4) The OtsA level decreases under cold stress and this decrease is attributed to repression of its synthesis through otsA promoter region analysis. Increasing the intracellular trehalose level of Arthrobacter could repress the otsA promoter at optimal growth temperature, and at the beginning of the cold shock the trehalose levels of Arthrobacter cells were tested increased. Therefore, the accumulation of trehalose is one of the factors which repress OtsA expression. (5) The elongation factor Tu (EF-Tu) could bind to OtsA directly and cold enhanced this binding action. EF-Tu is a genetic stabilizer for OtsA expression during cold shock with the presence of OtsA, which suggests the binding of EF-Tu and OtsA may help to stabilize expression of OtsA in the cold.Above all, this study indicates the mechanism of high catalytic efficiency of Arthrobacter OtsA at low temperatures and the factors that regulate the expression of OtsA in the cold. Therefore, it has influence on the future stress resistance studies of Arthrobacter and other microoganisms.