Nucleoside transport systems in Escherichia coli K12: Specificty and regulation

Two nucleoside transport systems have been verified and separated by mating and recombination experiments. The recipient strain was a mutant which is negative for transport of all nucleosides. The two systems differ in specificity and in regulation. One system transports pyrimidine and adenine in specificity and in regulation. One system transports pyrimidine and adenine nucleosides, but not guanine nucleosides. It is regulated by the cytR gene. The other system transports all nucleosides and is regulated by the cytR as well as by the deoR genes. Enzyme assays performed on whole cells of strains, able or unable to transport nucleosides, indicate that the nucleoside catabolizing enzymes are located inside the permeability barrier of the cell.     

Ferrous iron transport mutants in Escherichia coli K12

A ferrous iron transport system in Escherichia coli is described. Mutants in this transport system were isolated using the antibiotic streptonigrin. The gene locus feo (for ferrous iron transport) was mapped near pncA at 38.5 min on the genetic map of E. coli K12. The transport of ferrous iron was regulated by fur as the siderophore transport systems.     

L-arabinose transport systems in Escherichia coli K-12.

Mutations in the arabinose transport operons of Escherichia coli K-12 were isolated with the Mu lac phage by screening for cells in which beta-galactosidase is induced in the presence of L-arabinose. Standard genetic techniques were then used to isolate numerous mutations in either of the two transport systems. Complementation tests revealed only one gene, araE, in the low-affinity arabinose uptake system. P1 transduction placed araE between lysA (60.9 min) and thyA (60.5 min) and closer to lysA. The operon of the high-affinity transport system was found to contain two genes: araF, which codes for the arabinose-binding protein, and a new gene, araG. The newly identified gene, araG, was shown by two-dimensional gel electrophoresis to encode a protein which is located in the membrane. Only defects in araG could abolish uptake by the high-affinity system under the conditions we used.     

TonB-independent ferrioxamine B-mediated iron transport in Escherichia coli K12

Two variants of Escherichia coli heat-stable enterotoxin Ip, in which the amino acid residue at position 11 was substituted with lysine or arginine, were purified to near homogeneity from the culture supernatants of toxin-producing mutant strains. Neither the purified heat-stable enterotoxin Ip(Lys-11) nor the purified heat-stable enterotoxin Ip(Arg-11) showed a positive response in the suckling mouse assay or in the mouse intestinal loop assay. Furthermore, live bacteria producing these mutant heat-stable Ip enterotoxins did not cause fluid accumulation in mouse intestinal loops, in contrast to bacteria producing native heat-stable enterotoxin Ip. Nevertheless, antisera raised against both heat-stable enterotoxin Ip(Lys-11) and heat-stable enterotoxin Ip(Arg-11) neutralized the enterotoxic activity of native heat-stable enterotoxin Ip. These results demonstrate that heat-stable enterotoxin Ip(Lys-11) and heat-stable enterotoxin Ip(Arg-11) lose enterotoxicity but retain epitopes which are common to native heat-stable enterotoxin Ip.     

The alpha-methylglucoside transport in Escherichia coli K12 cells]

The transport of alpha-methylglucoside (MG) in the wild type cells of Escherichia coli K12 and the isogenic mutant strains, defective in the activity of phosphoenolpyruvate: sugar phosphotransferase system components was studied. It was shown that the enzyme IIB' in the absence of enzyme I and HPr is able to transport MG into the cells by a "facilitated" diffusion mechanism. Compounds which dissipate the energy of membrane protone potential such as NaN3, carbonylcyanide-m-chlorophenylhydrasone, dicyclohexylcarbodiimide, enhance the utilization of MG by the wild-type cells. However, the cells retaining intact enzyme IIB' but deficient in the phospho approximately HPr-generating system, were not sensitive to the action of poisons. The cells possessing the intact phospho HPr-generating system and inactive enzyme IIB' are also unaffected by the poisons. It seems that these results do not confirm the hypothesis of the direct delta mu H+ involvement in the regulation of transmembrane phosphorylation. The hypothesis is postulated that the energy metabolism inhibitors influence the phosphatase activity of factor III of the phosphotransferase system. The present data are well explained by this hypothesis.     

Multiplicity of leucine transport systems in Escherichia coli K-12

The major component of leucine uptake in Escherichia coli K-12 is a common system for l-leucine, l-isoleucine, and l-valine (LIV-I) with a Michaelis constant (K(m)) value of 0.2 muM (LIV-I system). The LIV-binding protein appears to be associated with this system. It now appears that the LIV-I transport system and LIV-binding protein also serve for the entry of l-alanine, l-threonine, and possibly l-serine. A minor component of l-leucine entry occurs by a leucine-specific system (L-system) for which a specific leucine-binding protein has been isolated. A mutant has been obtained that shows increased levels of the LIV-I transport activity and increased levels of both of the binding proteins. Another mutant has been isolated that shows only a major increase in the levels of the leucine-specific transport system and the leucine-specific binding protein. A third binding protein that binds all three branched-chain amino acids but binds isoleucine preferentially has been identified. The relationship of the binding proteins to each other and to transport activity is discussed. A second general transport system (LIV-II system) with a K(m) value of 2 muM and a relatively low V(max) can be observed in E. coli. The LIV-II system is not sensitive to osmotic shock treatment nor to growth of cells in the presence of leucine. This high K(m) system, which is specific for the branched-chain amino acids, can be observed in membrane vesicle preparations.     

Protein II influences ferrichrome-iron transport in Escherichia coli K12.

Ferrichrome-promoted iron uptake in Escherichia coli K12 is strictly dependent upon the tonA gene product, a 'minor' outer membrane protein. By selection for mutants of E. coli resistant to phages which require 'major' outer membrane proteins as receptors, strains with pronounced protein deficiencies were constructed. Such strains were tested for anomalous behaviour of ferrichrome transport. No significant differences in iron uptake were detected in E. coli K12 strains with markedly reduced amounts of protein I. However, a reduction in the initial velocity (up to 40%) was observed in E. coli deficient in outer membrane protein II. This difference was only evident when cells were grown under iron-starvation conditions; it was abolished when cells were grown in rich medium. Kinetic parameters for ferrichrome transport were determined for maximum velocity but for Km; double reciprocal plots showed a biphasic nature, probably attributable to a limited number of outer membrane binding sites and to the multi-component nature of the ferrichrome-iron transport system.     

Escherichia coli K12 arabinose-binding protein mutants with altered transport properties.

The arabinose-binding protein (ABP) of Escherichia coli binds L-arabinose in the periplasm and delivers it to a cytoplasmic membrane complex consisting of the AraG and AraH proteins, for uptake into the cell. To study the interaction between the soluble and membrane components of this periplasmic transport system, regions of the ABP surface containing the opening of the arabinose-binding cleft were subjected to site-directed mutagenesis. Thirty-eight ABP variants containing one to three amino acid substitutions were recovered. ABP variants were expressed with wild-type AraG and AraH from a plasmid, in a strain lacking the chromosomal araFGH operon, and the whole cell uptake parameters, Ven (maximum initial velocity of arabinose entry) and K(en) (concentration of arabinose yielding half-maximal entry) were determined. Twenty-four mutants had normal Ven values, 3 mutants had Ven and K(en) values twice wild type, and 11 mutants had Ven and K(en) values 20-50% of wild type. Binding proteins that had altered uptake properties were each expressed, processed, and localized to the periplasm at levels equivalent to wild type. The mutant binding proteins behaved the same as wild type during purification, and each had a Kd (dissociation constant for bound arabinose) comparable to that of wild-type ABP. Mutations that resulted in altered uptake identified nine amino acids surrounding the arabinose-binding cleft, all of which are charged in the wild-type protein, and all of whose side chains project outward from the cleft. The evidence suggests that this surface of the binding protein and these nine charged loci play a major role in ABP interactions with the membrane complex.     

Analysis of regulatory mechanisms controlling the activity of the hexitol transport systems in Escherichia coli K12.

Summary The transport systems (enzymeII-complexes of the PEP-dependent sugar: phosphotransferase system) coded for the mtl and in the gut (srl) operon of E. coli K12 have been shown to be the pacemaker enzymes in the catabolism of the two hexitols D-mannitol and D-glucitol, respecitively. As for other pacemaker enzymes their activity is regulated in a complex way: (i) via competitive inhibition by analogues. (ii) via non-competitive (feedback) inhibition by the simultaneous, rapid uptake of a number of structurally related or non-related carbohydrates, regardless if these are transported by group translocation, active transport or facilitated diffusion. This type of inhibition is strongly reinforced, if the inhibitory carbohydrates are converted efficiently into hexose-phosphates at the same time. Among these, predominantly D-fructose-6-P seems to act as a feedback inhibitor for the hexitol specific enzymeII-complexes; (iii) inhibition of hexitol-phosphate accumulation by rapid exchange with and/or chase out of the cell by D-glucose-6-P. The influence of additional parameters (PEP level, PHPr level) and indications for the existence of further mechanisms controlling the activity of hexitol and other carbohydrate transport systems will be discussed, as will be the part the inhibitory mechanisms described above play in the phenomena of transient repression and inducer exclusion.     

Enzymatic degradation and transport of endothiopeptides into Escherichia coli K12 mutant strains

Transport of three synthesized endothiopeptides: AlaPsi[CSNH]Ala, AlaPsi[CSNH]Leu, and AlaPsi[CSNH]Phe, into Escherichia coli K12 mutant strains and enzymatic degradation studies were carried out. These compounds, well transported by permeases, but significantly more resistant to enzymatic cleavage than the corresponding natural peptides, seem to be useful as enzyme inhibitor carriers.