Cloning and Characterization of the Phosphatidylserine Synthase Gene of Agrobacterium sp. Strain ATCC 31749 and Effect of Its Inactivation on Production of High-Molecular-Mass (1→3)-β-d-Glucan (Curdlan)

摘要

Genes involved in the production of the extracellular (1→3)-β-glucan, curdlan, by Agrobacterium sp. strain ATCC 31749 were described previously (Stasinopoulos et al., Glycobiology 9:31-41, 1999). To identify additional curdlan-related genes whose protein products occur in the cell envelope, the transposon TnphoA was used as a specific genetic probe. One mutant was unable to produce high-molecular-mass curdlan when a previously uncharacterized gene, pssAG, encoding a 30-kDa, membrane-associated phosphatidylserine synthase was disrupted. The membranes of the mutant lacked phosphatidylethanolamine (PE), whereas the phosphatidylcholine (PC) content was unchanged and that of both phosphatidylglycerol and cardiolipin was increased. In the mutant, the continued appearance of PC revealed that its production by this Agrobacterium strain is not solely dependent on PE in a pathway controlled by the PssAG protein at its first step. Moreover, PC can be produced in a medium lacking choline. When the pssAG::TnphoA mutation was complemented by the intact pssAG gene, both the curdlan deficiency and the phospholipid profile were restored to wild-type, demonstrating a functional relationship between these two characteristics. The effect of the changed phospholipid profile could occur through an alteration in the overall charge distribution on the membrane or a specific requirement for PE for the folding into or maintenance of an active conformation of any or all of the structural proteins involved in curdlan production or transport.