Cation transport in Escherichia coli. IX. Regulation of K transport

Kinetics of K exchange in the steady state and of net K uptake after osmotic upshock are reported for the four K transport systems of Escherichia coli: Kdp, TrkA, TrkD, and TrkF. Energy requirements for K exchange are reported for the Kdp and TrkA systems. For each system, kinetics of these two modes of K transport differ from those for net K uptake by K-depleted cells (Rhoads, D. B. F.B. Walters, and W. Epstein. 1976. J. Gen. Physiol. 67:325-341). The TrkA and TrkD systems are inhibited by high intracellular K, the TrkF system is stimulated by intracellular K, whereas the Kdp system is inhibited by external K when intracellular K is high. All four systems mediate net K uptake in response to osmotic upshock. Exchange by the Kdp and TrkA systems requires ATP but is not dependent on the protonmotive force. Energy requirements for the Kdp system are thus identical whether measured as net K uptake or K exchange, whereas the TrkA system differs in that it is dependent on the protonmotive force only for net K uptake. We suggest that in both the Kpd and TrkA systems formation of a phosphorylated intermediate is necessary for all K transport, although exchange transport may not consume energy. The protonmotive-force dependence of the TrkA system is interpreted as a regulatory influence, limiting this system to exchange except when the protonmotive force is high.     

Flagellar formation in Escherichia coli electron transport mutants.

Mutants of Escherichia coli lacking ubiquinone or heme have been tested for motility and found to be essentially immotile. The loss of motility is identified with the loss of flagellum synthesis.     

Cation Transport in Escherichia coli : III. Potassium fluxes in the steady-state

The present study is concerned with the measurement of the unidirectional K flux in E. coli. Methods are described by means of which a fairly dense suspension of cells may be maintained in a well defined steady-state with respect to the intracellular K concentration and the pH of the medium. The kinetics of K⁴² exchange under these conditions are consistent with the presence of a single intracellular K compartment with a unidirectional K flux of 1 pmol/(cm² sec.). This rate is independent of the extracellular K concentration over the range studied. The simultaneous rate of H secretion averages 16 pmols/(cm² sec.) indicating that in the steady-state the efflux of metabolically produced H is not linked mole for mole to K movement.     

Cation Transport in Escherichia coli : VII. Potassium requirement for phosphate uptake

When Escherichia coli K-12 is grown in media containing limiting amounts of K, growth continues normally until all the extracellular K has been consumed. Thereafter the rates of growth, glucose consumption, and oxygen consumption decrease progressively, and the cell contents of K and P fall. These changes, referred to as K limitation, are all reversed by the addition of K. By specifically altering the ionic composition of the cells it was shown that these metabolic disturbances are not due to changes in the cell content of K or Na, but are directly related to the absence of K from the extracellular medium. The cell pool of inorganic P and the uptake of PO₄ from the medium are low in K-limited cells and are immediately stimulated by the addition of K, suggesting that the primary effect of K limitation is to inhibit PO₄ uptake. All the metabolic effects of K limitation can be attributed to inhibition of PO₄ uptake. The requirement of extracellular K for PO₄ uptake may be due to a coupling between the uptake of K and PO₄.     

Genetic analysis of Escherichia coli oligopeptide transport mutants.

The composition of the outer membrane channels formed by the OmpF and OmpC porins is important in peptide permeation, and elimination of these proteins from the Escherichia coli outer membrane results in a cell in which the primary means for peptide permeation through this cell structure has been lost. E. coli peptide transport mutants which harbor defects in genes other than the ompF/ompC genes have been isolated on the basis of their resistance to toxic tripeptides. The genetic defects carried by these oligopeptide permease-negative (Opp-) strains were found to map in two distinct chromosomal locations. One opp locus was trp linked and mapped to the interval between att phi 80 and galU. Complementation studies with F'123 opp derivatives indicated that this peptide transport locus resembles that characterized in Salmonella typhimurium as a tetracistronic operon (B. G. Hogarth and C. F. Higgins, J. Bacteriol. 153:1548-1551, 1983). The second opp locus, which we have designated oppE, was mapped to the interval between dnaC and hsd at 98.5 min on the E. coli chromosome. The differences in peptide utilization, sensitivity and resistance to toxic peptides, and the L-[U-14C]alanyl-L-alanyl-L-alanine transport properties observed with these Opp-E. coli strains demonstrated that the transport systems encoded by the trp-linked opp genes and by the oppE gene(s) have different substrate preferences. Mutants harboring defects in both peptide transport loci defined in this study would not grow on nutritional peptides except for tri-L-methionine, were totally resistant to toxic peptides, and would not actively transport L-[U-14C]alanyl-L-alanyl-L-alanine.     

Sucrose transport through maltoporin mutants of Escherichia coli.

Maltoporin (LamB) and sucrose porin (ScrY) reside in the bacterial outer membrane and facilitate the passive diffusion of maltodextrins and sucrose, respectively. To gain further insight into the determinants of solute specificity, LamB mutants were designed to allow translocation of sucrose, which hardly translocates through wild-type LamB. Three LamB mutants were studied. (a) Based on sequence and structure alignment of LamB with ScrY, two LamB triple mutants were generated (R109D, Y118D,D121F; R109N,Y118D,D121F) to mimic the ScrY constriction. The crystal structure of the first of these mutants was determined to be 3.2 A and showed an increased ScrY-like cross-section except for D109 that protrudes into the channel. (b) Based on this crystal structure a double mutant was generated by truncation of the two residues that obstruct the channel most in LamB (R109A,Y118A). Analysis of liposome swelling and in vivo sugar uptake demonstrated substantial sucrose permeation through all mutants with the double alanine mutant performing best. The triple mutants did not show a well-defined binding site as indicated by sugar-induced ion current noise analysis, which can be explained by remaining steric interference as deduced from the crystal structure. Binding, however, was observed for the double mutant that had the obstructing residues truncated to alanines.     

Temperature-sensitive mutants of Escherichia coli affecting beta-galactoside transport

Six different temperature-sensitive (ts) mutants have been isolated which have parental beta-galactoside permease levels at low temperatures but have decreased permease levels when grown at high temperatures. These mutants were derived from Escherichia coli ML308 (lacI(-)Y(+)Z(+)A(+)). After N-methyl-N'-nitro-N'-nitro-soguanidine mutagenesis, ampicillin was used to select for cells unable to grow on low lactose concentrations at 42 C. Temperature-sensitive mutants were assayed for galactoside permease activity after growth in casein hydrolysate medium at 25 or 42 C by measuring both radioactive methylthio-beta-d-galactoside uptake and in vivo o-nitrophenyl-beta-d-galactoside hydrolysis. The six conditional isolates have decreased levels of galactoside permease which are correlated with decreased growth rates at elevated temperatures. The low permease levels are not due to a temperature labile lacY gene product but rather to a temperature labile synthesis rate of functional permease. Some of the mutants exhibit a ts increase in permeability as shown by the increased leakage of intracellular beta-galactosidase and by the increased rate of in vivo o-nitrophenyl-beta-d-galactoside hydrolysis via the nonpermease mediated entry mechanism. Preliminary evidence indicates that transport in general is decreased in these mutants, yet there is some specificity in the mutational lesion since glucoside transport is unaffected. All these observations suggest that these mutants have ts alterations in membrane synthesis which results in pleiotropic effects on various membrane functions.     

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.     

Defective transport in S-adenosylmethionine synthetase mutants of Escherichia coli

The transport of several metabolites is decreased in mutant strains of Escherichia coli (Met K, E4 and E40), which contain decreased levels of S-adenosylmethionine synthetase. The rates and extents of uptake for lysine, leucine, methionine, and α-methylglucoside in both whole cells and membrane vesicles isolated from these mutants are 2- to 10-fold lower than in corresponding preparations from wild-type cells, although proline uptake is normal. The addition of S-adenosylmethionine to cultures of strain E40 can partially restore the rate and extent of lysine uptake. Lysine transport is lower in mutant vesicles in the presence of either d-lactate, succinate, α-hydroxylbutyrate, or NADH even though these substrates are oxidized at rates comparable to those in wild-type vesicles. This suggests that the defect is not related to the ability of vesicles to oxidize electron donors, but is very likely related to the ability of mutant vesicles to couple respiration to lysine transport. In addition, temperature-induced efflux of α-methylglucoside phosphate and dinitrophenol-induced efflux of lysine are similar in both the mutant and wild-type membranes, indicating that the barrier properties of the membrane and the activity of the lysine carrier are normal.     

Uridine and cytidine transport in Escherichia coli B and transport-deficient mutants.

Three mutants of Escherichia coli B which are defective in components of the transport system for uridine and uracil were isolated and utilized to study the mechanism of uridine transport. Mutant U- was isolated from a culture resistant to 77 micronM 5-fluorouracil. Mutant U-UR-, isolated from a culture of mutant U-, is resistant to 770 micronM 5-fluorouracil and 750 micronM adenosine. Mutant NUC- is resistant to 80 micronM showdomycin and has been reported previously. The characteristics of uridine transport by E. coli B and the mutants provide data supporting the following conclusions. The transport of adenosine, deoxyadenosine, guanosine, deoxyguanosine, adenine, or guanine by mutant U- and mutant U-UR- is identical with that in the parental strain. Uridine is transported by E. coli B as intact uridine. In addition, extracellular uridine is also rapidly cleaved to uracil and the ribose moiety. The latter is transported into the cells, whereas uracil appears in the medium and is transported by a separate uracil transport system. The entry of the ribose moiety of uridine is fast relative to the uracil and uridine transport processes. The Km values and the inhibitory effects of heterologous nucleosides for the transport of uridine and the ribose moiety of uridine are similar. Studies of cytidine uptake in the parental and mutant strains provide evidence that cytidine is transported by two independent systems, one of which is the same as that involved in the transport of intact uridine. Uridine inhibits but is not transported by the other system for cytidine transport. Evidence for the above conclusions was based on comparisons of the characteristics of [2-14C]uridine, [U-14C]uridine, and [2-14C]cytidine transport using E. coli B and the three transport mutants under conditions which measure initial rates. The nature of the inhibitory effects of heterologous nucleosides on the uridine transport processes and identification of extracellular components from radioactive uridine provides supportive data for the conclusions.     

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.     

Peptidase-deficient mutants of Escherichia coli.

Mutant derivatives of Escherichia coli K-12 deficient in several peptidases have been obtained. Mutants lacking a naphthylamidase, peptidase N, were isolated by screening for colonies unable to hydrolyze L-alanine beta-naphthylamide. Other mutants were isolated using positive selections for resistance to valine peptides. Mutants lacking peptidase A, a broad-specificity aminopeptidase, were obtained by selection for resistance to L-valyl-L-leucine amide. Mutants lacking a dipeptidase, peptidase D, were isolated from a pepN pepA strain by selection for resistance to L-valyl-glycine. Starting with a pepN pepA pepD strain, selection for resistance to L-valyl-glycyl-glycine or several other valine peptides produced mutants deficient in another aminopeptidase, peptidase B. Mutants resistant to L-valyl-L-proline lack peptidase Q, an activity capable of rapid hydrolysis of X-proline dipeptides. Using these selection procedures, a strain (CM89) lacking five different peptidases has been isolated. Although still sensitive to valine, this strain is resistant to a variety of valine di- and tripeptides. The ability of this strain to use peptides as sources of amino acids is much more restricted than that of wild-type E. coli strains. Strains containing only one of the five peptidases missing in CM89 have been constructed by transduction. The peptide utilization profiles of these strains show that each of the five peptidases can function during growth in the catabolism of peptides.     

icdB mutants of Escherichia coli.

icdB mutations map at 16 min, lead to the specific loss of citrate synthase, and are complemented by a prophage containing a gltA+ gene. Thus, they are allelic with gltA.     

Metabolite transport in mutants of Escherichia coli K12 defective in electron transport and coupled phosphorylation.

1. The uptakes of Pi and serine by whole cells of mutant strains of Escherichia coli K12, grown under both aerobic and anaerobic conditions, were studied. 2. Uptake by aerobic cells was low in a ubiquinone-less mutant but normal in two mutant strains unable to couple phosphorylation to electron transport. 3. One of these uncoupled strains, carrying the unc-405 allele, does not form a membrane-bound Mg2+-stimulated adenosine triphosphatase aggregate, and it is concluded that the Mg2+-stimulated adenosine triphosphatase does not serve a structural role in the aerobic active transport of Pi or serine. 4. The other uncoupled strain, in which aerobic uptake is unaffected, carries a mutation in the uncB gene, thus distinguishing this gene from the etc gene, previously shown to be concerned with the coupling of electron transport to active transport. 5. The uptakes of Pi and serine by anaerobic cells were normal in the ubiquinone-less mutant, but defective in both the uncoupled strains. 6. The uptake of Pi and serine by anaerobic cells of the uncB mutant could be increased by the addition of fumarate to the uptake medium. The unc-405 mutant, however, required the addition of fumarate for growth and for uptake. 7. The uncB mutant, unlike the unc-405 mutant, is able to grow anaerobically in a minimal medium with glucose as sole source of carbon. Similarly a strain carrying a mutation in the frd gene, which is the structural gene for the enzyme fumarate reductase, is able to grow anaerobically in a glucose-minimal medium. However, a mutant strain carrying mutations in both the uncB and frd genes resembles the unc-405 mutant in not being able to grow under these conditions.     

Escherichia coli recBC deletion mutants.

Mutants of Escherichia coli with deletions of the recB and recC genes were obtained by two methods using transposable DNA elements. The phenotypes of these mutants are similar to those of mutants with recBC point mutations. These results indicate that the RecBC gene products, exonuclease V, is not essential for the growth of E. coli but is important for DNA repair and recombination.     

Galactoside transport dissociated from proton movement in mutants of Escherichia coli

Two mutants of Escherichia coli have been described in which the transport of β-galactosides is partly uncoupled from the metabolic reactions which drive active transport. It is shown that the effective inflow of H⁺, caused by the addition of β-galactoside, is much less in these mutants than in the parental strains, and it is concluded that β-galactoside transport is partly uncoupled from H⁺ transport.     

Isolation of vitamin B 12 transport mutants of Escherichia coli

Escherichia coli KBT001, a methionine-vitamin B(12) auxotroph, was found to require a minimum of 20 molecules of vitamin B(12) (CN-B(12)) per cell for aerobic growth in the absence of methionine. After mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine and penicillin selection, two kinds of B(12) transport mutant were isolated from this strain. Mutants of class I, such as KBT069, were defective in the initial rapid binding of CN-B(12) to the cell and were unable to grow in the absence of methionine even with CN-B(12) concentrations as high as 100 ng/ml. The class II mutants possessed intact initial phases of CN-B(12) uptake but were defective in the secondary energy-dependent phase. These strains were also unable to convert the CN-B(12) taken up into other cobalamins. In the absence of methionine, some of these strains (e.g., KBT103) were able to grow on media containing 1 ng of CN-B(12)/ml, whereas others (e.g., KBT041) were unable to grow with any of the CN-B(12) concentrations used. Osmotic shock treatment did not affect the initial rate of uptake of CN-B(12) but gave a substantial decrease in the secondary rate. Trace amounts of B(12)-binding macromolecules were released from the cells by the osmotic shock, but only from strains such as KBT001 and KBT041 which possessed an active initial phase of CN-B(12) uptake. These results are interpreted as being consistent with the view that the initial CN-B(12) binding site which functions in this transport system is probably bound to the cell membrane.     

Cation/proton antiport systems in Escherichia coli.

Three distinct systems which function as proton/cation antiports have been identified in $\text{E.}\text{coli}$ by the ability of the ions to dissipate the ΔpH component of the protonmotive force in everted vesicles. System I exchanges H⁺ for K⁺, Rb⁺ or Na⁺; System II has Na⁺ and Li⁺ as substrates; and System III catalyzes proton exchange for Ca²⁺, Mn²⁺ or Sr²⁺.