A wild-type isolate, EC3132, of Escherichia coli, that is able to grow on sucrose was isolated and its csc genes (mnemonic for chromosomally coded sucrose genes) transferred to strains of E. coli K12. EC3132 and all sucrose-positive exconjugants and transductants invariably showed a D-serine deaminase (Dsd)-negative phenotype. The csc locus maps adjacent to dsdA, the structural gene for the D-serine deaminase, and contains an inducible regulon, controlled by a sucrose-specific repressor CscR, together with structural genes for a sucrose hydrolase (invertase) CscA, for a D-fructokinase CscK, and for a transport system CscB. Based on DNA sequencing studies, this last codes for a hydrophobic protein of 415 amino acids. CscB is closely related to the beta-galactoside transport system LacY (31.2% identical residues) and a raffinose transport system RafB (32.3% identical residues) of the enteric bacteria, both of the proton symport type. A two-dimensional model common to the three transport proteins, which is based on the integrated consensus sequence, will be discussed.

The enzymes for catabolism of the pentitols D-arabinitol (Dal) and ribitol (Rbt) and the corresponding genes from Klebsiella pneurnoniae (dal and rbt) and Escherichia coli (at/ and rtl) have been used intensively in experimental evolutionary studies. Four dal and four rbt genes from the chromosome of K. pneurnoniae 1033-5P14 were cloned and sequenced. These genes are clustered in two adjacent but divergently transcribed operons and separated by two convergently transcribed repressor genes, dalR and rbtR. Each operon encodes an NAD-dependent pentose dehydrogenase (dalD and rbtl)), an ATP-dependent pentulose kinase (dalK and rbtK) and a pentose-specif ic ion symporter (dalT and rbtl). Although the biochemical reactions which they catalyse are highly similar, the enzymes showed interesting deviations. Thus, DalR (313 aa) and RbtR (270 aa) belong to different repressor families, and DalD (455 aa) and RbtD (248 aa), which are active as a monomer or as tetramers, respectively, belong to different dehydrogenase families. Of the two kinases (19.3% identity), DalK (487 aa) belongs to the subfamily of short 0-xylulokinases and RbtK (D-ribulokinase; 535 aa) to the subfamily of long kinases. The repressor, dehydrogenase and kinase genes did not show extensive similarity beyond local motifs. This contrasts with the ion symporters (86.6 Yo identity) and their genes (8207% identity). Due to their unusually high similarity, parts of dalT and rbtT have previously been claimed erroneously to correspond to 'inverted repeats' and possible remnants of a 'metabolic transposon' comprising the dal and rbt genes. Other characteristic structures, e.g. a secondary attl site and chi-like sites, as well as the conservation of this gene group in E. coli C are also discussed.

Two new genes, dalT and rbtT, have been cloned from the dal operon for D-arabinitol and the rbt operon for ribitol uptake and degradation, respectively, in Klebsiella pneumoniae 1033-5P14, derivative KAY2026. Each gene codes for a specific transporter which, based on sequence data, belongs to a large family of carbohydrate transporters which constitutes 12 transmembrane helices. DalT and RbtT show an unusually high similarity (86.2% identical residues for totals of 425 and 427 amino acids, respectively). This allowed the construction of DalT-RbtT and RbtT-DalT crossover hybrids by using a natural restriction site overlapping Met202. This site is located within the large cytoplasmic loop which connects the putative helices 6 and 7 and in particular the amino-and the carboxy-terminal halves of the transporters. Both hybrids have close to normal transport activities but essentially the substrate specificities and kinetic properties of the amino-terminal half. This result localizes essential substrate binding and recognition sites to the amino-terminal halves of the proteins in this important class of carbohydrate transporters.Membrane transport systems from prokaryotic and eukaryotic organisms form a large superfamily of facilitators (MFS) that catalyzes uniport, symport, and antiport (11,19). Members of the MFS superfamily share conserved amino acid sequence motifs and structures. These transporters include in particular facilitators and H ϩ -symporters for pentoses, hexoses, and polyhydric alcohols. The presence of conserved sequences and structural similarities implies common mechanisms of action. Replacement by localized mutagenesis of defined amino acids which impair transport properties has been used to identify putative catalytic centers. Alternatively, hybrids have been constructed in which a part of a transporter is replaced by the corresponding part of a related transporter having, e.g., different substrate specificity. At present, active hybrids only have been obtained when small parts have been exchanged or when very closely related transporters, as often found in one eukaryotic organism, have been used, e.g., four human glucose transporters (4, 35) or the glucose and galactose transporters from yeast (23). The results from such studies with 12 transmembrane helix facilitators and H ϩ -symporters tend to localize a region involved in substrate recognition to the carboxy-terminal half or they indicate the presence of a second site in the middle part of these transporters.In this paper we describe hybrids between a transporter (DalT) for the polyhydric alcohol (pentitol) D-arabinitol and one (RbtT) for its isomer ribitol. The corresponding genes, dalT and rbtT, respectively, have been located within the dal and the rbt operons of Klebsiella pneumoniae 1033-5P14 and have been cloned from the chromosome of its derivative KAY2026. Direct (15) and indirect (9,13,21,27,30,34) evidence for the existence of a transporter for each pentitol in this organism has been given. The corresponding genes, however, have no...