Wolfgang Epstein

The roles and regulation of potassium in bacteria.

Potassium is the major intracellular cation in bacteria as well as in eucaryotic cells. Bacteria accumulate K+ by a number of different transport systems that vary in kinetics, energy coupling, and regulation. The Trk and Kdp systems of enteric organisms have been well studied and are found in many distantly related species. The Ktr system, resembling Trk in many ways, is also found in many bacteria. In most species two or more independent saturable K(+)-transport systems are present. The KefB and KefC type of system that is activated by treatment of cells with toxic electrophiles is the only specific K(+)-efflux system that has been well characterized. Pressure-activated channels of at least three types are found in bacteria; these represent nonspecific paths of efflux when turgor pressure is dangerously high. A close homolog of eucaryotic K+ channels is found in many bacteria, but its role remains obscure. K+ transporters are regulated both by ion concentrations and turgor. A very general property is activation of K+ uptake by an increase in medium osmolarity. This response is modulated by both internal and external concentrations of K+. Kdp is the only K(+)-transport system whose expression is regulated by environmental conditions. Decrease in turgor pressure and/or reduction in external K+ rapidly increase expression of Kdp. The signal created by these changes, inferred to be reduced turgor, is transmitted by the KdpD sensor kinase to the KdpE-response regulator that in turn stimulates transcription of the kdp genes. K+ acts as a cytoplasmic-signaling molecule, activating and/or inducing enzymes and transport systems that allow the cell to adapt to elevated osmolarity. The signal could be ionic strength or specifically K+. This signaling response is probably mediated by a direct sensing of internal ionic strength by each particular system and not by a component or system that coordinates this response by different systems to elevated K+.

Potassium Transport in Bacteria

Publisher Summary This chapter focuses on potassium (K + ) transport in bacteria. K + is a “compatible” cation, best tolerated of monovalent cations when present in the cell at high concentration. This tolerance allows K + to make a major contribution to regulating cell osmolality as supported by the correlation between internal K + and medium osmolality in several species. Bacteria have multiple transport systems for K + . Multiplicity of systems, each with characteristic properties, allows cells to adapt to a wider range of environments. Systems for influx are separate from systems for efflux. A single system does not appear to mediate movements in both directions. Stimulation of K + transport is an early, or the earliest, event in many bacteria when confronted with a change in medium osmolality. A scheme of osmotic adaptation for E. coli and the cyanobacteria is shown diagrammatically in the chapter. Feedback-regulated transport readily accounts for the way a cell maintains a fixed K + concentration during growth. The increase in cell volume during growth dilutes cell solutes, thereby reducing turgor pressure.

Discrimination between Rb+ and K+ by Escherichia coli.

1. The K+ requirment of Escherichia coli is only partially fulfilled by Rb+. The molar growth yield on Rb+ was about 5% of that on K+ and the growth rate in Rb+-supplemented media is lower thatn in K+ influx by any of the four K+ transport systems of E. coli. The high-affinity Kdp system (Km = 2 micron) is poorly traced by 86Rb+. It discriminates against a 86Rb+ tracer at least 1000-fold. The two moderate affinity systems, the high-rate TrkA system (Km = 1.5 mM) and the moderate rate TrkD system (Km = 0.5 mM), discriminate against a 86Rb+ tracer by approximately 10-fold and 25-fold, respectively. 86Rb+ is preferred by the low-rate TrkF system and overestimates its K+ influx by 40%.

Chapter 9 The Kdp System: A Bacterial K+ Transport ATPase

Publisher Summary Escherichia coli essentially have two transport systems that are capable of accumulating K + in order to achieve high cellular concentrations. These two systems generally exhibit certain common features. When E. coli is grown in customary media containing excess K + , only the constitutive Trk system, which has a modest affinity for K + ( K m = 1.5 m M ) is present. When the Trk system is not able to satisfy their K + needs, the cells derepress a second system, commonly called Kdp. This system has a very high affinity for K + ( K m = 2 μ M ), which allows it to scavenge traces of K+ in the medium. This chapter focuses on Kdp system and describes its structure, function, and regulation. Kdp appears to have a reaction cycle involving an acyl phosphate intermediate similar to that of the vertebrate transport ATPases. The regulation of Kdp largely reflects the needs of its bacterial home. Its transport activity is feedback regulated by the turgor pressure and osmotic pressure difference exerted across the cell membrane. Turgor pressure is also implicated as the force that is responsible for derepressing the Kdp system when the cells require K + .

Isolation and characterization of specialized transducing bacteriophages for the recA gene of Escherichia coli.

Abstract Lysogens of Escherichia coli have been isolated in which λc1857 is integrated near the recA locus in a gene required for sorbitol utilization (srl). These secondary-site lysogens produce lysates containing plaque-forming specialized transducing phages which carry part of the srl locus and the recA gene. Genetic evidence, DNA heteroduplex examination, and restriction enzyme analysis demonstrate that the bacterial DNA substitution occurs in the b2 region of the transducing λ. One such recA transducing phage, designated λprecA, contains approximately 3% less DNA than the parental λ.

Assembly of the Kdp complex, the multi-subunit K+-transport ATPase of Escherichia coli.

Kdp, the high affinity ATP-driven K+-transport system of Escherichia coli, is a complex of the membrane-bound subunits KdpA, KdpB, KdpC and the small peptide KdpF. The assembly of this complex was studied by the analysis of mutants that expressed two of the three large subunits and inserted them into the cytoplasmic membrane. In the strains that do not express KdpC or KdpA the other two subunits did not copurify on dye-ligand affinity columns after solubilization with non-ionic detergent. In the mutant lacking KdpB the other two subunits copurified under the same conditions. It is concluded that KdpC forms strong interactions with the KdpA subunit, serving to assemble and stabilise the Kdp complex. A structure in which KdpC could be one of the connecting links between the energy-delivering subunit KdpB and the K+-transporting subunit KdpA is suggested by these data.

Modulation of KdpD phosphatase implicated in the physiological expression of the Kdp ATPase of Escherichia coli

The KdpD sensor kinase and the KdpE response regulator control the expression of the kdpFABC operon, encoding the KdpFABC high‐affinity K+ transport system of Escherichia coli. Low turgor pressure has been postulated to be the environmental stimulus to express KdpFABC. KdpD has autokinase, phosphotransferase and, like many sensor kinases, response regulator (phospho‐KdpE) specific phosphatase activity. To determine which of these activities are altered in response to the environmental stimulus, we isolated and analysed six kdpD mutants that cause constitutive expression of KdpFABC. In three of the mutants, phosphatase activity was undetectable and, in two, phosphatase was reduced. Kinase activity was unaffected in four of the mutants, but elevated in one. In one mutant, a pseudorevertant of a kdpD null mutation, kinase and phosphatase were both reduced to 20% of the wild‐type level. These findings suggest that initiation of signal transduction by KdpD is mediated by the inhibition of the phospho‐KdpE‐specific phosphatase activity of KdpD, leading to an accumulation of phospho‐KdpE, which in turn activates the expression of the KdpFABC system. The data also suggest that levels of activity in vitro may differ from what occurs in vivo, because in vitro conditions cannot replicate those in vivo.

Roles of the trkB and trkC gene products of Escherichia coli in K+ transport

Mutations at the trkB and trkC loci of Escherichia coli produce an abnormal efflux of K+. The mutations are partially dominant in diploids and revert frequently by what appears to be intragenic suppression to the null state. The mutations can be reverted by insertion of Tn10 into the mutated gene, and spontaneous revertants are fully recessive to the mutant allele in diploids. K+ efflux produced by NEM* and by DNP* persists in strains with presumed null mutations at either locus, indicating neither gene product is the primary target for the effect of these inhibitors on K+ efflux. The results are consistent with the view that trkB and trkC encode independent systems for K+ efflux. Mutations at these loci alter regulation of the process so that K+ efflux occurs inappropriately. A second mutation to the null state abolishes this abnormal K+ efflux. These genes may encode K+/H+ antiporters, an activity postulated to mediate K+ efflux and demonstrated to exist in E. coli and other bacteria.

Kdp-ATPase of Escherichia coli

Kdp-ATPase consists of three large protein subunits each having a distinct function. The 72-kD KdpB energy-coupling sub-unit is homologous to other P-type ATPases, and is the site of acylphosphorylati

[51] Genetics of Kdp, the K+-transport ATPase of Escherichia coli

Publisher Summary This chapter describes the application of a variety of widely used techniques of bacterial genetics to the study of Kdp. Analogous genetic methods are already available for lower eukaryotes, such as yeast, but at present only a few are readily applied to higher eukaryotes. The rapid progress of genetics gives promise that the ease and extent of genetic dissection in bacteria and yeast will soon be possible in higher eukaryotes. Kdp is a complex of three-membrane proteins that form a K + -transport ATPase in Escherichia coli . Kdp belongs to the E l -E 2 class of transport ATPases, as shown by the formation of an acyl phosphate intermediate, and by homology to the Ca 2+ -ATPase and the Na + , K + -ATPase of animal cells, and to H + -ATPases of yeast and fungi. Genetic analysis has been important from the outset in the identification and characterization of the Kdp transport ATPase, in contrast to eukaryotic transport ATPases, where genetics has only recently added to the extensive information obtained by biochemical techniques. To analyze the kdp genotype of a strain and to construct strains with particular kdp genotypes for mapping, complementation analysis, or cloning, it is useful to be able to move kdp mutations from one strain to another. A convenient way to move them is cotransduction by bacteriophage P1 with a nearby marker. Three useful markers close to kdp are nagA, gltA, and nadA.