Cation transport in Escherichia coli. VIII. Potassium transport mutants

Analysis of K transport mutants indicates the existence of four separate K uptake systems in Escherichia coli K-12. A high affinity system called Kdp has a Km of 2 muM, and Vmax at 37 degrees C of 150 mumol/g min. This system is repressed by growth in high concentrations of K. Two constitutive systems, TrkA and TrkD, have Km's of 1.5 and 0.5 mM and Vmax's of 550 and 40 at 37 and 30 degrees C, respectively. Mutants lacking all three of these saturable systems take up K slowly by a process, called TrkF, whose rate of transport is linearly dependent on K concentration up to 105 mM. On the whole, each of these systems appears to function as an independent path for K uptake since the kinetics of uptake when two are present is the sum of each operating alone. This is not true for strains having both the TrkD and Kdp systems, where presence of the latter results in K uptake which saturates at a K concentration well below 0.1 mM. This result indicates some interaction between these systems so that uptake now has the affinity characteristic of the Kdp system. All transport systems are able to extrude Na during K uptake. The measurements of cell Na suggest that growing cells of E. coli have very low concentrations of Na, considerably lower than indicated by earlier studies.     

Cation Transport in Escherichia coli : VI. K exchange

K influx and net K flux have been measured in suspensions of chloramphenicol-arrested Escherichia coli. The rate of K exchange in the steady state was independent of the K concentration of the medium over a 200-fold range. Under a number of experimental conditions the rate of exchange may be considerably increased or decreased without changing the cellular K content. These results show that under these conditions changes in K influx are associated with equal changes in K efflux, and suggest that the latter process is, at least in part, both carrier-mediated and tightly coupled to the influx process.     

Cation Transport in Escherichia coli : V. Regulation of cation content

Measurement of cellular K and Na concentrations in growing Escherichia coli indicates that the osmololity of the medium is a major determinant of the cell K concentration. In contrast, the cell Na concentration is independent of the medium osmolality and is largely dependent on the Na concentration of the medium. Sudden changes in the osmolality of the medium lead to rapid changes in K content. Washing the cells with solutions of lower osmolality results in a very rapid loss of K, which is greater in more dilute and in cold solutions. A sudden increase in the osmolality of the growth medium produces a rapid uptake of K by a mechanism whose rate is a saturable function of the K concentration of the medium and which appears to involve an exchange of K for cellular H.     

A K+ transport ATPase in Escherichia coli.

A K+ -stimulated ATPase in membranes of Escherichia coli has been identified as an activity of the Kdp system, and ATP-driven K+ transport system. Three characteristics support association of the ATPase with the Kdp system: (i) ATPase and Kdp transport are both repressed by growth in media containing high concentrations of K+; (ii) the ATPase and Kdp system accept only K+ as substrate, neither requires Na+ nor accepts Rb+ as a substrate; (iii) the affinity of the ATPase and that of th Kdp system for K+ is similar and is altered by mutations in the structural genes of the Kdp system. Discovery of an ATPase associated with a bacterial transport system suggests functional similarities with the ATP-driven transport systems of animal cells.     

Regulation of lysine biosynthesis in Escherichia coli K12.

A general survey of the regulation in lysine biosynthesis in Escherichia coli K12 is presented. No polygenic operon exists for the genes that code for enzymes of the lysine biosynthetic pathway. Lysyl-tRNA is not directly involved as a co-repressor in the pathway. Different regulation mechanisms must exist for the different enzymes. In the case of the last enzyme, diaminopimelate decarboxylase, its synthesis is induced in vivo by the lysine-sensitive aspartokinase under its non-inhibited allosteric conformation.     

Regulation of branched-chain amino acid transport in Escherichia coli.

The repression and derepression of leucine, isoleucine, and valine transport in Escherichia coli K-12 was examined by using strains auxotrophic for leucine, isoleucine, valine, and methionine. In experiments designed to limit each of these amino acids separately, we demonstrate that leucine limitation alone derepressed the leucine-binding protein, the high-affinity branched-chain amino acid transport system (LIV-I), and the membrane-bound, low-affinity system (LIV-II). This regulation did not seem to involve inactivation of transport components, but represented an increase in the differential rate of synthesis of transport components relative to total cellular proteins. The apparent regulation of transport by isoleucine, valine, and methionine reported elsewhere was shown to require an intact leucine, biosynthetic operon and to result from changes in the level of leucine biosynthetic enzymes. A functional leucyl-transfer ribonucleic acid synthetase was also required for repression of transport. Transport regulation was shown to be essentially independent of ilvA or its gene product, threonine deaminase. The central role of leucine or its derivatives in cellular metabolism in general is discussed.     

Cation Transport in Escherichia coli : IV. Kinetics of net K uptake

The resuspension of K-poor, Na-rich stationary phase E. coli in fresh medium at pH 7.0 results in a rapid uptake of K and extrusion of Na by the cells. In all experiments net K uptake exceeded net Na extrusion. An investigation of the uptake of glucose, PO₄, and Mg and the secretion of H by these cells indicates that the excess K uptake is not balanced by the simultaneous uptake of anions but must be accompanied by the extrusion of cations from the cell. The kinetics of net K uptake are consistent with the existence of two parallel influx processes. The first is rapid, of brief duration, and accounts for approximately 60 per cent of the total net K uptake. This process is a function of the extracellular K concentration, is inhibited in acid media, and appears to be a 1 for 1 exchange of extracellular K for intracellular H. The second influx process has a half-time of approximately 12 minutes, and is not affected by acid media. This process is a function of the intracellular Na concentration, is dependent upon the presence of K in the medium, and may be ascribed to a 1 for 1 exchange of extracellular K for intracellular Na.     

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.     

Regulation of the L-arabinose transport operons in Escherichia coli

l-Arabinose is transported into Escherichia coli via two independent transport systems, a system possessing relatively low affinity for arabinose, the araE system, and a system of higher affinity for arabinose, the araFG system. In the work reported here we demonstrate that insertion of the Mu-lac bacteriophage isolated by Casadaban &; Cohen (1979) permits a reliable measurement of the expression of these two operons. Using appropriate Mu-lac insertion strains we found that both of the arabinose transport operons can be induced approximately 150-fold by the presence of arabinose, and that induction of both transport operons requires CRP (cyclic AMP receptor protein), but that their catabolite sensitivities differ from one another. In addition, we show that the araC⁺ allele is dominant to the C^c allele in the control of the transport operons, just as is found in the araBAD operon.     

Amino-sugar transport systems of Escherichia coli K12

Glucosamine, mannose and 2-deoxyglucose enter Escherichia coli by the phosphotransferase system coded for by the gene ptsM. The glucosamine- and mannose-negative, deoxyglucose-resistant phenotype of ptsM mutants can be suppressed by a mutation mapping near ptsG that allows constitutive expression of the glucose phosphotransferase coded for by the gene ptsG. N-Acetylglucosamine enters E. coli by two distinct phosphotransferase systems (White, 1970). One of these is the PtsM system, the other is coded for by a gene which maps near the nagA,B genes at about min 15 on the E. coli chromosome. We propose that this gene be designated ptsN. Strains with either of these components of the phosphotransferase system will utilize N-acetylglucosamine as sole carbon source.     

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.     

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⁺.     

Regulation of aromatic amino acid transport systems in Escherichia coli K-12.

The regulation of the aromatic amino acid transport systems was investigated. The common (general) aromatic transport system and the tyrosine-specific transport system were found to be subject to repression control, thus confirming earlier reports. In addition, tryosine- and tryptophan-specific transport were found to be enhanced by growth of cells with phenylalanine. The repression and enhancement of the transport systems was abolished in a strain carrying an amber mutation in the regulator gene tyrR. This indicates that the tyrR gene product, which was previously shown to be involved in regulation of aromatic biosynthetic enzymes, is also involved in the regulation of the aromatic amino acid transport systems.     

Pantothenate transport in Escherichia coli.

The function of the stable 6S RNA of Escherichia coli is not known. Recently, it was proposed that the 6S RNA is a component of a bacterial signal recognition particle required for protein secretion. To test this proposal, we isolated a mutant that lacks the 6S RNA. Studies of the mutant show that the 6S RNA is not essential for growth or for protein secretion. The gene for the 6S RNA (ssr) maps near serA at 63 min on the E. coli genetic map.