Beta-methylgalactoside Transport System Research Articles

Sequence of the mglB gene from Escherichia coli K12: comparison of wild-type and mutant galactose chemoreceptors.

The mglB gene of Escherichia coli codes for a galactose-binding protein (GBP) that serves both as the galactose chemoreceptor and as the recognition component of the beta-methylgalactoside transport system. The mglB551 mutation eliminates the chemotactic function of GBP without altering its transport or substrate-binding properties. To investigate the interaction between GBP and Trg, the chemotactic signal transducer for galactose, we sequenced the mglB genes from wild-type and mglB551 mutant strains. The mutation causes the replacement of Gly74 of GBP by Asp. This residue is located in alpha-Helix III at the tip of the P domain in the GBP tertiary structure farthest removed from the substrate-binding cleft between the P and Q domains. We conclude that Helix III must be part of, or at least adjacent to, the recognition site for Trg. Our sequence also included part of the mglA gene, which is immediately distal to mglB. The amino acid sequence deduced for the beginning of the MglA protein showed homology with a family of polypeptides that contain an ATP-binding site and are components of binding-protein-dependent transport systems.

Identification of the mglA gene product in the beta-methylgalactoside transport system of Escherichia coli using plasmid DNA deletions generated in vitro.

Three genes (mglA, mglB, and mglC) required for active transport of substrate by the methylgalactoside permease were identified in a hybrid ColE1-DNA plasmid isolated from a clone (pLC3-14) of the Clarke-Carbon bank of Escherichia coli genes. A 4.6-kilobase DNA fragment obtained from pLC3-14 was cloned into the plasmid vector pBR322. The presence of the three mgl genes in the resultant plasmid, pMG3, was verified by genetic complementation and biochemical analysis of mgl mutants transformed with pMG3 DNA. Derivatives of pMG3 containing deletions in each mgl gene were constructed; restriction endonuclease mapping and functional analysis of these plasmids allowed us to physically locate the mgl genes within the inserted plasmid DNA and also to identify a heretofore unknown protein component of the transport system. Expression of these plasmids in vivo resulted in the specific synthesis of three major proteins of apparent molecular weight of 19,000, 36,000, and 52,000. The 36,000-dalton protein is the galactose-binding protein previously identified as the mglB product. The 19,000-dalton protein maybe the product of mglD, a regulatory gene mapping outside of the mgl gene cluster. The 52,000-dalton protein is a new permease component which we have identified here as the mglA product based on the observation that pMG6, a plasmid with a 0.6-kilobase mglA deletion, failed to encode for this protein but produced a truncated polypeptide showing a reduction in molecular weight comparable to the extent of the deletion. In bacteria bearing an mglA+, B-, C+ plasmid (Pmg4), the 52,000-dalton protein is located to a large extent (73%) in the membrane fraction.

Chemotaxis of Salmonella typhimurium to Amino Acids and Some Sugars

Patterns of chemotaxis by Salmonella typhimurium strain LT-2 to l-amino acids and to several sugars were quantitated by the Adler capillary procedure. Competition experiments indicated that LT-2 possesses three predominant receptors, or interacting sets of receptors, for amino acids. These were termed the aspartate, serine, and alanine classes, respectively. Studies with strains carrying point and deletion mutations affecting components of the phosphoenolpyruvate: glycose phosphotransferase system (PTS) made unlikely a role in primary reception of d-glucose by the three soluble PTS components, namely HPr, enzyme I, and factor III. A ptsG mutant defective in membrane-bound enzyme IIB' of the high-affinity glucose transport system was shown to exhibit normal chemotaxis providing pleiotropic effects of the mutation were eliminated by its genotypic combination with other pts mutations or, phenotypically, by addition of cyclic AMP and substrate. A correlation was demonstrated between chemotaxis to glucose and activity of the low-affinity glucose transport complex, membrane-bound enzymes IIB:IIA, and an enzyme IIB:IIA mutant was shown to have a preponderant defect in chemotaxis to glucose and mannose. Of four systems capable of galactose transport, only the beta-methylgalactoside transport system was implicated in chemotaxis to galactose. Some properties of a mutant possibly defective in processing of signals for chemotaxis to sugars is described.

Properties of the entry and exit reactions of the beta-methyl galactoside transport system in Escherichia coli.

The Km, Vmax, and Ki of the entry reaction were determined for three substrates of the beta-methyl galactoside transport system: D-galactose, D-glycerol-beta-D-galactoside, and beta-methyl-D-galactoside. Although the data for D-galactose and D-glycerol-beta-D-galactoside followed simple Michaelis-Menten kinetics, the results for beta-methyl-D-galactoside deviated from Michaelis-Menten kinetics in that the Ki for beta-methyl-D-galactoside inhibition of both of the other two substrates was 10-fold greater than the Km for beta-methyl-D-galactoside entry. Furthermore, two partial mgl- strains retain 56% of the parental level of the beta-methyl-D-galactoside entry reaction, but only 12% of the parental level of transport of the other two substrates. The exit reaction of beta-methyl-D-galactoside was shown to be first order. It was stimulated sixfold when the cells were provided with an energy source. This stimulation required adenosine 5'-triphosphate or a related compound. The exit reaction was not altered by mutations in any of the three cistrons which inactivate the beta-methyl-D-galactoside entry reaction, was not increased by growth in the presence of inducers of the entry reaction, and was not repressed by growth on glucose. The striking differences between the entry and exit reactions suggest that they either use different carriers or that none of the three cistrons which are currently known to code for components of the beta-methyl galactoside transport system code for its membrane carrier.

Roles of individual mgl gene products in the beta-methylgalactoside transport system of Escherichia coli K12.

Previous findings showed that galactose-binding protein defective mutants (mgl B-,A+,C+) of Escherichia coli K12 are still capable of growth on methyl-beta-D-galactopyranoside, while mgl A- and mgl C- mutants are not. When assayed by previous methods, none of these mutants exhibited methylgalactoside transport system activity. In this study, we present a modified assay developed for measuring low levels of transport. Using this assay, we found that mgl B-,A+,C+ mutants defective in galactose-binding protein accumulate methyl-beta-D-galactopyranoside up to six times the concentration gradient while mgl A- and mgl C- mutants failed to accumulate this substrate. Similar results were obtained using D-glyceryl-beta-D-galactopyranoside, another substrate of the methylgalactoside transport system. In contrast, all sugars tested which are not substrates of this system were transported equally by all mgl- mutants. The kinetic parameters of transport in mgl B- mutants were compared to those of the isogenic mgl+ strain which accumulates methyl-beta-D-galactopyranoside against a 10,000-fold concentration gradient. The apparent Km of methyl-beta-D-galactopyranoside influx was 1,000 times greater in mgl B- than in mgl+ strains. In contrast, there was no significant difference between these strains in either the Vmax of substrate influx or the rate of substrate exit. D-Galactose competitively inhibited methyl-beta-D-galactopyranoside influx into both mgl B- and mgl+ strains; the Ki of inhibition in mgl B- cells was 2,000-fold greater than that in mgl+ cells.

The regulation of the beta-methylgalactoside transport system and of the galactose binding protein of Escherichia coli K12.

In the present publication the synthesis of the galactose binding protein and the β-methyl-galactoside transport system in Escherichia coli K12 has been shown to be coregulated. With the use of mutants of different regulatory behavior with respect to the galactose operon and the phosphoenolpyruvate-dependent phosphotransferase system, the following information about the regulation of the simultaneous synthesis of the galactose binding protein and the β-methyl-galactoside transport system was obtained. 1 Fucose acts as inducer for both the galactose binding protein and the transport system. 2 GalK mutants, endogenously induced with respect to the galactose operon, also show endogenous induction of the transport system. 3 The induction by fucose as well as endogenous induction is inhibited by 5 mM methyl-1-thio-β-D-galactopyranoside. 4 GalR+, galR and galRs mutants can show full inducibility, excluding the function of galR in the regulation of the β-methylgalactoside transport system. 5 Deletions of the entire galactose operon exhibit high yet inducible transport activities excluding the existence of a regulator gene near or in the galactose operon. 6 The existence of a regulator gene mglR which is different from galR, melR lacI, araC or pts could be demonstrated. The mutation mglR was found to be located between the origin of two Hfr strains, AB313 and KL16, i.e. between 56 and 74 min on the linkage map of E. coli. 7 Mutants defective in enzyme I activity of the phosphoenolpyruvate-dependent phosphotransferase system exhibit unimpaired β-methylgalactoside transport activity excluding any function of the β-methylgalactoside permease as a specific enzyme II of the phosphotransferase system.