Mutations which uncouple transport and phosphorylation in the D-mannitol phosphotransferase system of Escherichia coli K-12 and Klebsiella pneumoniae 1033-5P14

Abstract

Mutants of Escherichia coli K-12 were isolated which lack the normal phosphotransferase system-dependent catabolic pathway for D-mannitol (Mtl). In some mutants the pts genes for the general proteins enzyme I and histidine protein of the phosphoenolpyruvate-dependent carbohydrate phosphotransferase systems were deleted. Other mutants expressed truncated mannitol-specific enzymes II (II(Mtl)) which lacked the IIA(Mtl) or IIBA(Mtl) domain(s), and the mtlA genes originated either from E. coli K-12 or from Klebsiella pneumoniae 1033-5P14. The dalD gene from Klebsiella oxytoca M5a1 was cloned on single-copy plasmids and transformed into the strains described above. This gene encodes an NAD-dependent D-arabinitol dehydrogenase (DalD) which converts D-arabinitol into D-xylulose and also converts D-mannitol into D-fructose. The different strains were used to isolate mutations which allow efficient transport of mannitol through the nonphosphorylated II(Mtl) complexes 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.

FIG. 1.

Deletions of the II^Mtl complex of E. coli K-12. The locations of domains A, B, and C of II^Mtl are indicated together with the locations of the essential residues E218, H256, C384, and H554 and the locations of the approximate borders between the domains. The longest deletions in the four deletion classes isolated previously are L611 to K637 in class 1, Q520 to K637 in class 2, M393 to K637 in class 3, and L161 to K637 in class 4. Representative deletions for each class are Δ69 (ΔL632-K637) and Δ65 (ΔE618-K637) for class 1, Δ130 (ΔV532-K637) for class 2, Δ137 (ΔE511-K637) for class 3, and ΔSnaBI (ΔV377-K637) and Δ21 (ΔE346-K637) for class 4. In K. pneumoniae, ΔKp (ΔG361-K635) was used as a representative of class 4 and corresponds to the allele present in pSOL110 (Table 1).

FIG. 2.

Intracellular d-[¹⁴C]mannitol in cell extracts from wild-type and uncoupled mutant strains. Cells of strain LGS323 [ΔmtlA55 Δ(gut-rec)63] (lane 1) and cells of transformants of this strain with the wild-type allele of mtlA on pGJ9 (lanes 2 and 3) or with the uncoupled allele on pGJ9 H256P (lane 4) were incubated for 5 min with 280 μM d-[¹⁴C]mannitol at 25°C, washed, and extracted as described in Material and Methods. Portions (5 μl) of the supernatants were applied to Nono-Sil G plates and developed in 1-butanol-ethanol-water (3:1:1). In lane 3, 1.6 × 10⁴cpm of d-[¹⁴C]mannitol was added to each of the cell extracts before application to the gel plates. Free d-mannitol and several metabolic derivatives of mannitol 1-phosphate in the wild type were visualized by radioautography. WT, wild type.