Molecular analysis of the ggtBCD gene cluster of Synechocystis sp. strain PCC6803 encoding subunits of an ABC transporter for osmoprotective compounds

Abstract.

Genes encoding a substrate-binding protein (ggtB) and two integral membrane proteins (ggtC and ggtD) of the binding-protein-dependent ABC transporter for glucosylglycerol were identified in the genome of Synechocystis sp. strain PCC6803. These genes are clustered on the chromosome about 220 kb away from the previously identified ggtA gene, which encodes the ATP-binding protein of this transport system. The deduced amino acid sequences show significant similarities to corresponding subunits of ABC transporters mediating uptake of maltose and other di- and oligosaccharides in bacteria and archaea. Mutants were constructed by inserting an aphII gene cassette into the coding region of the ggtB, ggtC and ggtD genes. These mutants lost the ability to take up glucosylglycerol, sucrose and trehalose, proving that these compounds are transported by the same system. A truncated ggtB gene lacking the putative signal-peptide-encoding sequence was expressed in Escherichia coli yielding a histidine-tagged soluble protein. The recombinant GgtB protein bound glucosylglycerol with a K _D of 0.45 µM and exhibited a somewhat lower affinity towards sucrose and a substantially lower affinity towards trehalose. Transcript analysis by RT-PCR indicated that the genes of the ggtBCD gene cluster form an operon. The transcript level estimated by RNA slot blot analysis using a ggtC-specific probe was very low in cells grown in basal medium but increased significantly after a salt shock.