Molecular analysis of the gat genes from Escherichia coli and of their roles in galactitol transport and metabolism

Abstract

In enteric bacteria, the hexitol galactitol (Gat) (formerly dulcitol) is taken up through enzyme II (II Gat ) of the phosphoenolpyruvate-dependent phosphotransferase system (PTS), and accumulated as galactitol 1-phosphate (Gat1P). The gat genes involved in galactitol metabolism have been isolated from the wild-type isolate Escherichia coli EC3132 and cloned on a 7.8-kbp PstI DNA fragment. They comprise six complete open reading frames and one truncated open reading frame in the order gatYZABCDR. The genes gatABC code for the proteins GatA (150 residues) and GatB (94 residues), which correspond to the hydrophilic domains IIA Gat and IIB Gat , and GatC, which represents a membrane-bound transporter domain IIC Gat (35 kDa, 427 residues). The three polypeptides together constitute a II Gat of average size (671 residues). Gene gatD codes for a Gat1P-specific NAD-dependent dehydrogenase (38 kDa, 346 residues), gatZ codes for a protein (42 kDa, 378 residues) of unknown function, and gatY (31 kDa, 286 residues) codes for a D-tagatose-1,6-bisphosphate aldolase with similarity to other known ketose-bisphosphate aldolases. The truncated gatR gene, whose product shows similarity to the glucitol repressor GutR, closely resembles a gatR gene fragment from E. coli K-12. The gat genes map in both organisms at similar positions, in E. coli K-12, where they are transcribed counterclockwise at precisely 46.7 min or 2,173 to 2,180 kbp. The genes are expressed constitutively in both strains, probably due to a mutation(s) in gatR. Transcription initiation sites for the gatYp and the gatRp promoters were determined by primer extension analysis.In enteric bacteria, the polyhydric alcohol galactitol (Gat) (formerly dulcitol) is transported and phosphorylated through a phosphoenolpyruvate (PEP)-dependent galactitol:phosphotransferase system (PTS) (II Gat ) (9, 10). During uptake of a PTS carbohydrate, a phosphoryl group is transferred by the general PTS proteins enzyme I (EI) (gene ptsI) and histidine protein (HPr) (gene ptsH) from PEP to the substrate-specific enzymes II (EIIs) (for a review, see reference 13). The phosphoryl group is first accepted by the hydrophilic domain IIA, then by IIB. In a third step it is transferred to the substrate located in the membrane-bound transporter IIC, and the substrate phosphate is released into the cytoplasm. The three domains of an EII may be fused into a single protein, or they can be separated into two or three distinct proteins.During galactitol uptake through II Gat , galactitol 1-phosphate (Gat1P) is generated and converted by an NAD-dependent Gat1P-dehydrogenase (gene gatD) into D-tagatose 6-phosphate (Tag6P) (9,10,20). Further metabolism requires phosphofructokinase I (gene pfkA) to generate D-tagatose 1,6-bisphosphate. This kinase can be substituted for by phosphofructokinase II (gene pfkB) in strains overexpressing the enzyme, which is normally expressed at a low level in Escherichia coli K-12 (5). Finally, the bisphosphate is hydrolyzed by a ketose-bisphosphate aldolase into dihydroxyace...

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