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

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.

Sequence of the gat operon for galactitol utilization from a wild-type strain EC3132 of Escherichia coli.

The sequence of the gat operon for galactitol (Gat) utilization from a wild-type isolate of Escherichia coli, strain EC3132, is presented. The operon comprises 7 open reading frames (ORFs) called gatYZABCDR. The genes are transcribed from a promoter located upstream of gatY. Genes gatABC encode the substrate-specific domains IIA, IIB and IIC of a galactitol-specific Enzyme II (EIIGat) of the phospho enol pyruvate-dependent carbohydrate:phosphotransferase system (PTS); gatD encodes an NAD-dependent Gat 1-phosphate dehydrogenase; and gatY an enzyme which hydrolyses tagatose 1,6-bisphosphate; gene gatZ is required in a cell to show a Gat+ phenotype, but its physiological function has not yet been identified; gatR encodes a repressor for the gat operon. All genes are highly similar to the gat genes from E. coli K-12; in this organism they map at 46.70 min of the gene map, equivalent to about 2180-2186 kbp.

Cloning of the Escherichia coli sor genes for L-sorbose transport and metabolism and physical mapping of the genes near metH and iclR.

The sor genes for L-sorbose (Sor) degradation of Escherichia coli EC3132, a wild-type strain, have been cloned on a 10.8-kbp fragment together with parts of the metH gene. The genes were mapped by restriction analysis, by deletion mapping, and by insertion mutagenesis with Tn1725. Seven sor genes with their corresponding gene products have been identified. They form an operon (gene order sorCpCDFBAME) inducible by L-sorbose, and their products have the following functions: SorC (36 kDa), regulatory protein with repressor-activator functions; SorD (29 kDa), D-glucitol-6-phosphate dehydrogenase; SorF and SorB (14 and 19 kDa, respectively), and SorA and SorM (27 and 29 kDa, respectively), two soluble and two membrane-bound proteins, respectively, of an L-sorbose phosphotransferase transport system; SorE (45 kDa), sorbose-1-phosphate reductase. The sor operon from E. coli EC3132 thus is identical to the operon from Klebsiella pneumoniae KAY2026. On the basis of restriction mapping followed by Southern hybridization experiments, the sor genes were mapped at 91.2 min on the chromosome, 3.3 kbp downstream of the metH-iclR gene cluster, and shown to be transcribed in a counterclockwise direction. The chromosomal map of the Sor+ strain EC3132 differs from that of the Sor- strain K-12 in approximately 8.6 kbp.