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.

Enteric bacteria (Enteriobacteriaceae) carry on their single chromosome about 4000 genes that all strains have in common (referred to here as "obligatory genes"), and up to 1300 "facultative" genes that vary from strain to strain and from species to species. In closely related species, obligatory and facultative genes are orthologous genes that are found at similar loci. We have analyzed a set of facultative genes involved in the degradation of the carbohydrates galactitol, D-tagatose, D-galactosamine and N-acetyl-galactosamine in various pathogenic and non-pathogenic strains of these bacteria. The four carbohydrates are transported into the cell by phosphotransferase (PTS) uptake systems, and are metabolized by closely related or even identical catabolic enzymes via pathways that share several intermediates. In about 60% of Escherichia coli strains the genes for galactitol degradation map to a gat operon at 46.8 min. In strains of Salmonella enterica, Klebsiella pneumoniae and K. oxytoca, the corresponding gat genes, although orthologous to their E. coli counterparts, are found at 70.7 min, clustered in a regulon together with three tag genes for the degradation of D-tagatose, an isomer of D-fructose. In contrast, in all the E. coli strains tested, this chromosomal site was found to be occupied by an aga/kba gene cluster for the degradation of D-galactosamine and N-acetyl-galactosamine. The aga/kba and the tag genes were paralogous either to the gat cluster or to the fru genes for degradation of D-fructose. Finally, in more then 90% of strains of both Klebsiella species, and in about 5% of the E. coli strains, two operons were found at 46.8 min that comprise paralogous genes for catabolism of the isomers D-arabinitol (genes atl or dal) and ribitol (genes rtl or rbt). In these strains gat genes were invariably absent from this location, and they were totally absent in S. enterica. These results strongly indicate that these various gene clusters and metabolic pathways have been subject to convergent evolution among the Enterobacteriaceae. This apparently involved recent horizontal gene transfer and recombination events, as indicated by major chromosomal rearrangements found in their immediate vicinity.

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...

Mtlcomplexes by selecting for growth on this polyhydric alcohol. More than 40 different mutants were analyzed to determine their ability to grow on mannitol, as well as their ability to bind and transport free mannitol and, after restoration of the missing domain(s), their ability to phosphorylate mannitol. Four mutations were identified (E218A, E218V, H256P, and H256Y); all of these mutations are located in the highly conserved loop 5 of the IIC membrane-bound transporter, and two are located in its GIHE motif. These mutations were found to affect the various functions in different ways. Interestingly, in the presence of all II Mtl variants, whether they were in the truncated form or in the complete form, in the phosphorylated form or in the nonphosphorylated form, and in the wild-type form or in the mutated form, growth occurred on the low-affinity analogue D-arabinitol with good efficiency, while only the uncoupled mutated forms transported mannitol at a high rate.The bacterial phosphoenolpyruvate (PEP)-dependent mannitol phosphotransferase system (PTS) catalyzes the concomitant transport and phosphorylation of D-mannitol (Mtl) (14,15,46). Transfer of the phosphoryl group from PEP to the substrate is catalyzed in four reversible steps by a soluble PEP-dependent protein kinase designated enzyme I, a soluble histidine protein (HPr), and an mtlA-encoded D-mannitol-specific enzyme II (II Mtl ) which also accepts, but with a lower affinity, D-glucitol and D-arabinitol (16, 50 Mtl (amino acids 1 to 346) forms six transmembrane segments which are joined by three short periplasmic loops and by two large intracellular loops (loop 3, residues 70 to 134; loop 5, residues 185 to 273) (19,24,37,45). Loop 5, which comprises a conserved structure centered around a characteristic GIHE motif, has been postulated to be essential in substrate binding and translocation (16,49). In the enteric bacteria, the three domains are fused in a large peptide consisting of 637 amino acids and function as a dimeric complex (37). The domains must, however, be relatively autonomous because artificial splitting and fusion at the natural linkers do not grossly affect their activities. Thus, after deletion of IIA and IIB, the IIC domain alone still seems to form a functional transporter which binds and discriminates the various substrates like the intact II Mtl complex (8,22). In mutants which lack the protein kinase enzyme I and HPr, enzymes II cannot be phosphorylated and are unable to transport substrates at a rate sufficient to support growth (34). ptsHI mutants of Salmonella enterica serovar Typhimurium and Escherichia coli K-12 have been isolated which have increased rates of transport through an intact II Glc (30,40). In such mutants, uptake of free glucose occurred via facilitated diffusion, and fermentation required an ATP-dependent glucokinase. Thus, translocation and phosphorylation of the substrate can be uncoupled. Sequencing revealed that all uncoupling mutations mapped in loop 5 close to the conserved GITE motif of II Glc . All mu...