Dorothy M. Wilson

Cation specificity for sugar substrates of the melibiose carrier in Escherichia coli

A study has been made of the sugar substrate specificities and the cation specificities of the melibiose transport system of Escherichia coli. The following beta-galactosides were found to be transported: lactose, L-arabinose-beta-D-galactoside, D-fructose-beta-D-galactoside, o- and p-nitrophenyl-beta-D-galactosides. These beta-galactosides were cotransported with Na+ but not with H+. The alpha-galactosides raffinose, melibiose and p-nitrophenyl-alpha-galactoside were transported with either H+ or Na+. Of the monosaccharides tested D-galactose could use either Na+ or H+ for cotransport whereas D-fucose, L-arabinose and D-galactosamine could use only Na+. The sugar specificity requirements for H+ cotransport are therefore more exacting than those for Na+ cotransport.

The lactose carrier of Klebsiella pneumoniae M5a1; the physiology of transport and the nucleotide sequence of the lacY gene

A comparison has been made between the physiology and amino acid sequence of the lactose carriers of Klebsiella pneumoniae M5a1 and Escherichia coli K-12. The membrane transport of lactose was much weaker in Klebsiella than in E. coli. On the other hand o-nitrophenylgalactoside uptake by Klebsiella was distinctly greater than with E. coli. In spite of the differences in sugar transport between the two organisms, the amino acid sequences of the respective lactose carriers were remarkably similar (60% of the amino acids are identical).

Inhibition of growth of Escherichia coli by lactose and other galactosides.

A study has been made of the inhibition of growth caused by the addition of lactose or other galactosides to lac constitutive Escherichia coli growing in glycerol minimal medium. The effect was greater at pH 5.9 and pH 7.9 than at pH 7.0. Inhibition of growth by lactose was observed also in the case of a beta-galactosidase negative mutant. However, a lacY mutant, which has a defect in the entry of protons normally coupled with galactoside transport, showed only slight inhibition of growth on the addition of galactosides. In the case of the parental strain the addition of lactose resulted in a sharp fall in delta pH across the cell membrane and a reduction in intracellular ATP, and the recovery was slow. Under the same conditions the lacY mutant showed a smaller and only transient effect. It is postulated that the sudden entry of protons associated with lactose uptake lowers the protonmotive force, reducing the ATP levels and inhibiting growth of the cells. This hypothesis would account also for the selection of lacY mutants found when E. coli is grown in the presence of isopropyl-beta-D-thiogalactoside.

Evidence for a close association between helix IV and helix XI in the melibiose carrier of Escherichia coli

The melibiose carrier of Escherichia coli is a cation-sugar cotransport protein. Asp124 in membrane-spanning helix IV of the carrier protein was replaced with Ser, Ile or Phe by site-directed mutagenesis of a plasmid containing the melB gene. Each of these mutants failed to show membrane transport of melibiose and melibiose-positive revertants could be isolated on melibiose MacConkey indicator plates. D124F showed only one type of revertant (D124C) and D124I showed only revertants to the normal (D124). Second site revertants were not found with either of these mutants. S124, however, showed two types of second site revertants: D124S/V375A and D124S/V375G. The revertant D124S/V375A had lost melibiose/proton cotransport but showed 25% of normal melibiose (20 mM) uptake in the presence of 10 mM sodium ion at 37 degrees C. The value for the parental strain D124S was 2%. The second revertant D124S/V375G showed greater activity than S124 in the presence of 100 mM NaCl at both 20 degrees C and 37 degrees C. It was concluded that in the normal carrier protein Asp124 in helix IV is probably close to Val375 in helix XI. Since Lys377 is close to Val375, it is possible that Asp124 may interact with Lys377 to form a salt bridge.

Transport properties of Asp-51 → Glu and Asp-120 → Glu mutants of the melibiose carrier of Escherichia coli

Asp-51-->Glu and Asp-120-->Glu mutants of the melibiose carrier of Escherichia coli were investigated for their cation/sugar cotransport properties. The carrier containing Glu-51 showed proton/melibiose cotransport but was extremely defective in Na+ or Li+ stimulation of sugar accumulation. On the other hand, the carrier containing Glu-120 had lost the ability to couple protons with melibiose uptake while retaining considerable Na+ or Li+ cotransport with melibiose (40-fold accumulation versus 90-fold for the wild type in the presence of Na+). It is concluded that both Asp-51 and Asp-120 are important for cation recognition.

Reconstitution of the melibiose carrier of Escherichia coli.

Different conditions were studied for optimal solubilization and reconstitution of the melibiose carrier of Escherichia coli. Several alpha- and beta-galactosides, known to be substrates for the melibiose carrier, were found to inhibit [3H]-melibiose uptake by proteoliposomes. In the presence of 10 mM Na+ the Km for melibiose counterflow was 0.42 mM. Melibiose and raffinose were good substrates for counterflow, while thiomethyl-beta-galactoside and p-nitrophenyl-alpha-galactoside were accumulated very poorly. Although the latter two sugars are known to be substrates for the carrier, they showed a very rapid rate of passive diffusion across the liposome membrane. The proton ionophore carbonylcyanidechlorophenylhydrazone had no effect on uptake, suggesting that a proton motive force is not essential for the counterflow phenomenon.

[30] Methods for the study of the melibiose carrier of Escherichia coli

Publisher Summary This chapter describes methods used for measuring the transport of both the sugar and cation substrates. Some of these methods such as the preparation of radioactive melibiose and the construction of the Li+-selective electrode were devised specifically for studies of the melibiose transport system. The melibiose carrier of Escherichia coli is a representative of the class of carriers that require a cation for cotransport. An unusual feature of this transport system is that it is able to utilize Na + , Li + , or H + as the coupling cation depending on the conditions. Another interesting property of the carrier is that different sugar substrates show different specificities for the cation requirement. A variety of strains of E. coli has been isolated for use in experiments on the melibiose carrier. It is essential to start with a strain from which the lactose carrier (lac Y) has been deleted, as the lac carrier also is capable of transporting melibiose. The growth of normal E. coli cells on melibiose minimal medium is strongly inhibited by 10 mM Li + and it is therefore possible to isolate Li + -resistant mutants. This organism is dependent on Na + or Li + for growth on melibiose as it has lost the ability to transport the disaccharide with protons.

Control of lactose transport in Escherichia coli

It is essential for optimal growth rate on a carbon source such as lactose that cells carefully regulate the transport and the first step in metabolism. Inducibility as a mode of regulation of catabolic pathways is physiologically economical since specific proteins are produced only when needed by the cell. In the case of the lac operon of Escherichiu coli, lOOO-fold variation in the levels of transport and fl-galactosidase is possible. It is interesting to note that lactose itself does not induce this operon but is converted by fl-galactosidase into another disaccharide, alIolactose, which is the true physiological inducer [ 11. Normal induction requires the proper relationship between the levels of P-galactosidase and transport. According to Mieschendahl et al. [2] the absence of P-galactosidase (lac2) leads to partial (10%) constitutivity of lactose transport and the transacetylase. They propose that this phenomenon is due to the accumulation of an endogenous inducer which is normally destroyed by /3-galactosidase in the uninduced cell. An interesting type of imbalance of an opposite nature is seen in cells which possess an abnormally high level of /3-galactosidase compared with the level of transport. When a la& gene is introduced (via an episome) into a cell with low levels of lac expression the cell becomes lactose negative. The lactose negative phenotype may be due to the failure of the cell to accumulate sufficiently high levels of allolactose [3] or to the destruction of an endogenous inducer [2]. An important further control is that exerted by glucose. Early studies of Monod [4] indicated that when cells of E. coli were placed in a medium con-