Protection of the DNA during the exposure of Escherichia coli cells to a toxic metabolite: the role of the KefB and KefC potassium channels

The effect of the toxic metabolite methylglyoxal on the DNA of Escherichia coli cells has been investigated. Exposure of E. coli cells to methylglyoxal reduces the transformability of plasmid DNA and results in the degradation of genomic DNA. The activity of the KefB and KefC potassium channels protects E. coli cells against methylglyoxal and limits the amount of DNA damage. In mutants lacking KefB and KefC, methylglyoxal-induced DNA damage was reduced by incubation with a weak acid that lowers the pHi to the same extent as through KefB and KefC activation. This provides evidence that acidification of the cytoplasm protects E. coli DNA against methylglyoxal. By the analysis of cells lacking UvrA, we demonstrate that this repair protein is required for the degradation of the DNA upon methylglyoxal exposure. However, protection by KefB and KefC occurred independently of UvrA. Although we present evidence that exposure of E. coli cells to methylglyoxal results in DNA degradation, our results suggest this event is not essential for methylglyoxal-induced death. The implications of these findings will be discussed.     

Survival of Escherichia coli cells exposed to iodoacetate and chlorodinitrobenzene is independent of the glutathione-gated K+ efflux systems KefB and KefC.

The KefB and KefC systems of Escherichia coli cells are activated by iodoacetate (IOA) and chlorodinitrobenzene (CDNB), leading to a rapid drop in the intracellular pH. However, survival of exposure to IOA or CDNB was found to be essentially independent of KefB and KefC activation. No correlation was found between the toxicity of the compound and its ability to elicit protective acidification via activation of KefB and KefC.     

Activation of potassium channels during metabolite detoxification in Escherichia coli

In bacteria the detoxification of compounds as diverse as methylglyoxal and chlorodinitrobenzene proceeds through the formation of a glutathione adduct. In the Gram-negative bacteria, e.g. Escherichia coli, such glutathione adducts activate one, or both, of a pair of potassium efflux systems KefB and KefC. These systems share many of the properties of cation-translocating channels in eukaryotes. The activity of these systems has been found to be present in a range of Gram-negative bacteria, but not in the glutathione-deficient species of Gram-positive organisms. The conservation of the activity of these systems in a diverse range of organisms suggested a physiological role for these systems. Here we demonstrate that in E. coli cells activation of the KefB efflux system is essential for the survival of exposure to methylglyoxal. Methylglyoxal can be added to the growth medium or its synthesis can be stimulated in the cytoplasm. Under both sets of conditions survival is aided by the activity of KefB. Inhibition of KefB activity by the addition of 10 mM potassium to the growth medium stimulates methylglyoxal-induced cell death. This establishes an essential physiological function for the KefB system.     

Glutathione and the gated potassium channels of Escherichia coli

Glutathione-deficient mutants of Escherichia coli were found to require high potassium concentrations for growth, unless supplemented with glutathione. The unsupplemented mutants exhibited a rapid leak of potassium when transferred to a K+-free medium and a fast K+ turnover at the steady state of K+ accumulation, contrasting with the slow rate of the same processes in the wild-type. The steady-state level of K+ accumulation in low potassium medium increased immediately upon addition of glutathione, even in the absence of protein synthesis. K+-independent revertants were found to possess restored glutathione synthesis. Many properties of the glutathione-deficient mutants were identical with those of the potassium leaky K-B- and K-C- mutants, which, however, have a normal glutathione content. Both types of mutants differ from the wild-type in their response to thiol reagents in that no rapid loss of K+ is observed: they have, however, clear-cut differences under these circumstances. These results suggest that the products of trkB and trkC genes are essential for the formation of the potassium channel and glutathione plays an important role in the gating process.     

Identification of an ancillary protein, YabF, required for activity of the KefC glutathione-gated potassium efflux system in Escherichia coli

A new subunit, YabF, for the KefC K(+) efflux system in Escherichia coli has been identified. The subunit is required for maximum activity of KefC. Deletion of yabF reduces KefC activity 10-fold, and supply of YabF in trans restores activity. IS2 and IS10R insertions in yabF can be isolated as suppressors of KefC activity consequent upon the V427A and D264A KefC mutations.     

Opening of potassium channels in Escherichia coli membranes by thiol reagents and recovery of potassium tightness

The retention of high potassium levels in Escherichia coli is not dependent on intact energy metabolism, since without the presence of a carbon source or in the presence of energy inhibitors significant K+ gradients can be maintained. In contrast, with 0.5 mM N-ethylmaleimide, K+ depletion is immediate and complete. As a final result, intracellular K+ is approximately three times more concentrated than the K+ in the medium. Increase of K+ in the medium is immediately followed by K+ uptake whereas in the unpoisoned state only an increase in the osmotic pressure of the medium would result in an increase of the K+ pool. The intracellular K+ undergoes continuous turnover in the poisoned cells whereas in intact cells turnover is strictly dependent on the presence of a metabolizable carbon source. After removal of the thiol reagent the cell recovers its capacity to concentrate potassium. The recovery process is inhibited by energy inhibitors or by incubation at low temperature but not by chloramphenicol. It is only slightly slowed down by carbon or sulfur starvation. The leak provoked by N-ethylmaleimide is similar in wild-type E. coli cells when a derepressed kdp uptake system working in the micromolar range of the K+ concentration is responsible for the intracellular pool of K+ and when, in a medium millimolar K+ concentration range, the trkA and trkD systems are predominant.     

Unilateral exposure of Shaker B potassium channels to hyperosmolar solutions.

This study tests the hypothesis that ion channels will be affected differently by external (extracellular) versus internal (cytoplasmic) exposure to hyperosmolar media. We looked first for effects on inactivation kinetics in wild-type Shaker B potassium channels. Although external hyperosmolar exposure did not alter the inactivation rate, internal exposure slowed both onset and recovery from fast inactivation. Differential effects on activation kinetics were then characterized by using a noninactivating Shaker B mutant. External hyperosmolar exposure slowed the late rising phase of macroscopic current without affecting the initial delay or early rising phase kinetics. By contrast, internal exposure slowed the initial steps in channel activation with only minimal changes in the later part of the rising phase. Neither external nor internal hyperosmolar exposure affected tail current rates in these noninactivating channels. Additionally, suppression of peak macroscopic current was approximately twofold smaller during external, as compared with internal, hyperosmolar exposure. Single-channel currents, observed under identical experimental conditions, showed a differential suppression equivalent to that seen in macroscopic currents. Apparently, during unilateral hyperosmolar exposure, changes in macroscopic peak current arise primarily from changes in single-channel conductance rather than from changes in equilibrium channel gating. We conclude that unilateral hyperosmolar exposure can provide information concerning the potential structural localization of functional components within ion-channel molecules.     

The role of potassium transport in the generation of a pH gradient in Escherichia coli.

The role of K+ transport in the generation of a pH gradient in Escherichia coli has been investigated. In K+-depleted cells, net K+ uptake dissipated delta psi (membrane potential) and led to an increase in delta pH (pH gradient). The magnitude of the delta pH formed bore a simple relationship to the net K+ uptake and was substantially independent of the respiratory rate. In K+-replete cells, generation of a pH gradient was again K+-dependent, although no net uptake of this cation occurred. The results are discussed in terms of K+ cycling, and it is suggested that delta pH is in part a function of the rate of cycling and independent of the respiratory rate.     

The effects of N-ethylmaleimide on active amino acid transport in Escherichia coli.

N-Ethylmaleimide (MalNEt) binds covalently and without specificity to accessible sulfhydryl residues in proteins. In some cases specificity has been imposed on this reaction by manipulating reaction conditions, yielding information concerning both enzyme mechanism and the identity of specific proteins (for example C.F. Fox and E.P. Kennedy (1965) Proc. Natl. Acad. Sci. u.s. 54, 891-899) and R.E. McCarty and J. Fagan (1973) Biochemistry 12, 1503-1507). We have examined the effects of MalNEt on the active accumulation of nine amino acids by Escherichia coli strains ML 308-225 and DL 54. Whole cells have been used in order that transport systems both dependent on and independent of periplasmic binding proteins could be studied under various conditions of energy supply for transport. Our results suggest that the systems transporting ornithine, phenylalanine and proline are those most likely to undergo inactivation by direct reaction of MalNEt with the transport apparatus, rather than merely via side effects such as interruption of their energy supply. The inhibition of proline transport is specifically enhanced by the presence of proline, competitive inhibitors of proline transport, or carbonylcyanide p-trifluoromethyoxyphenylhydrazone during MalNEt treatment. The other eight systems tested showed no analogous effects.     

Glutathione-dependent conversion of N-ethylmaleimide to the maleamic acid by Escherichia coli: an intracellular detoxification process

The electrophile N-ethylmaleimide (NEM) elicits rapid K(+) efflux from Escherichia coli cells consequent upon reaction with cytoplasmic glutathione to form an adduct, N-ethylsuccinimido-S-glutathione (ESG) that is a strong activator of the KefB and KefC glutathione-gated K(+) efflux systems. The fate of the ESG has not previously been investigated. In this report we demonstrate that NEM and N-phenylmaleimide (NPM) are rapidly detoxified by E. coli. The detoxification occurs through the formation of the glutathione adduct of NEM or NPM, followed by the hydrolysis of the imide bond after which N-substituted maleamic acids are released. N-ethylmaleamic acid is not toxic to E. coli cells even at high concentrations. The glutathione adducts are not released from cells, and this allows glutathione to be recycled in the cytoplasm. The detoxification is independent of new protein synthesis and NAD(+)-dependent dehydrogenase activity and entirely dependent upon glutathione. The time course of the detoxification of low concentrations of NEM parallels the transient activation of the KefB and KefC glutathione-gated K(+) efflux systems.     

Formation of ion channels in the Escherichia coli cytoplasmic membrane after exposure to bacteriophages T4 and lambda

The effects of phage T4 and lambda on the ion permeability of the E. coli cytoplasmic membrane were studied. It was shown that the phage-induced depolarization of the membrane is coupled with a simultaneous increase in a transmembrane pH gradient. Hence, the total value of the proton-motive force remains unchanged at moderate multiplicity of infection. The rise in the pH gradient occurs due to an increase in the activity of the redox H+-pump of the E. coli membrane. Analysis of the temperature dependence showed that the stimulating effect of the phage is observed at 6-8 degrees C. Apart from the phages, gramicidin is also capable of stimulating the H+-pump under these conditions, while the stimulating effect of valinomycin is diminished. These data suggest that the ion-permeable channels are formed in the membrane during the interaction of E. coli cells with the phages. The experimental results demonstrate that the channels are permeable to ions of monovalent metals. The phage can also increase the permeability of cell membranes to protons; however, the permeability to monovalent ions is higher when these ions are in excess.     

Cooperative mechanosensitive ion channels in Escherichia coli.

A patch-clamp investigation was carried out on giant Escherichia coli spheroplasts. The membrane exhibited stretch-induced as well as "spontaneous" activity, with similar characteristics, i.e., a large number of conductance values arising from the cooperative behavior of channels in functional clusters. It appears likely that the same molecular species are responsible for both stretch-induced and "spontaneous" current conduction; the channel multiplexes can either respond to membrane stretch or function in an activate state, presumably brought about by the previous application of the mechanical stimulus.     

Thiamine transport in Escherichia coli: the mechanism of inhibition by the sulfhydryl-specific modifier N-ethylmaleimide

Active transport of thiamin (vitamin B(1)) into Escherichia coli occurs through a member of the superfamily of transporters known as ATP-binding cassette (ABC) transporters. Although it was demonstrated that the sulfhydryl-specific modifier N-ethylmaleimide (NEM) inhibited thiamin transport, the exact mechanism of this inhibition is unknown. Therefore, we have carried out a kinetic analysis of thiamin transport to determine the mechanism of inhibition by NEM. Thiamin transport in vivo exhibits Michaelis-Menten kinetics with K(M)=15 nM and V(max)=46 U mg(-1). Treatment of intact E. coli KG33 with saturating NEM exhibited apparent noncompetitive inhibition, decreasing V(max) by approximately 50% without effecting K(M) or the apparent first-order rate constant (k(obsd)). Apparent noncompetitive inhibition is consistent with an irreversible covalent modification of a cysteine(s) that is critical for the transport process. A primary amino acid analysis of the subunits of the thiamin permease combined with our kinetic analysis suggests that inhibition of thiamin transport by NEM is different from other ABC transporters and occurs at the level of protein-protein interactions between the membrane-bound carrier protein and the ATPase subunit.     

Inactivation of Escherichia coli acetate kinase by N-ethylmaleimide. Protection by substrates and products

Acetate kinase (ATP:acetate phosphotransferase, EC 2.7.2.1) from Escherichia coli exhibited a time-dependent loss of activity when incubated with N-ethylmaleimide at micromolar concentrations. However, prolonged incubation did not eliminate all catalytic activity and generally about 15% of its initial activity remained. When incubated with 7.2 microM N-ethylmaleimide, acetate kinase was inactivated with a rate constant of 0.063 min-1. Adenine nucleotides, ATP, ADP and AMP, protected the enzyme against such inactivation, but acetate up to 3.0 M and in the presence of 0.2 M MgCl2 and acetyl phosphate at 24 mM did not interfere with the rate of inactivation. While both acetate and acetyl phosphate did not affect the protection rendered by AMP, the presence of acetyl phosphate altered ADP protection. However, both substrates prevented ATP from protecting the enzyme. These data suggest that the binding sites for acetate and acetyl phosphate are different from that of the adenosine binding domain, but are in close vicinity to the phosphoryl binding regions of the nucleotides.     

Survival of Escherichia coli in lake bottom sediment.

The survival of Escherichia coli in bottom sediment (Lake Onalaska, navigation pool no. 7, Mississippi River) was studied by using in situ dialysis culture of sterile (autoclaved) and unsterile sediment samples. Bags made from dialysis tubing were filled with either course sand sediment (28.8% fine) or organic, silty clay sediment (77.2% fine) and placed at the sediment-water interface. Bags representing sterile controls, unsterile uninoculated controls, autoclaved inoculated sediment, and unsterile inoculated sediment were studied during a 5-day period for each sediment type. Daily most-probable-number determinations indicated that E. coli populations in unsterile inoculated sediment fluctuated between 5.3 X 10(2) and 2.2 X 10(3) bacteria per g of silty clay and between 3.0 X 10(3) and 1.4 X 10(4) bacteria per g of sand. Autoclaved silty clay sediment inoculated with 1.0 X 10(6) bacteria per g increased to 2.2 X 10(8) bacteria per g in 3 days. During the same period, autoclaved sand sediment inoculated with 1.2 X 10(5) cells per g increased to 5.4 X 10(7) bacteria per g. By day 5, populations in both cultures had decreased by 1 log. The ability of E. coli to survive for several days in aquatic sediment in situ suggests that fecal coliforms in water may not always indicate recent fecal contamination of that water but rather resuspension of viable sediment-bound bacteria.     

Survival of recombination-deficient mutants of Escherichia coli during incubation with nalidixic acid.

The ability of several Escherichia coli strains deficient in recombination (rec) to survive in the presence of nalidixic acid was determined. Genetic blocks of the RecBC or the RecF pathways resulted in increased sensitivity to nalidixic acid when compared with the wild-type strain. Mutants lacking functional recA, recL, or recB recC recF genes showed the most rapid decrease in colony-forming ability when incubated with nalidixic acid. However, the uvrB gene also plays a role in maintaining cell viability.     

Survival of Escherichia coli during drying and storage in the presence of compatible solutes

Five different compatible solutes, sucrose, trehalose, hydroxyectoine, ectoine, and glycine betaine, were investigated for their protective effect on Escherichia coli K12 and E. coli NISSLE 1917 during drying and subsequent storage. Two different drying techniques, freeze-drying and air-drying, were compared. The highest survival rate was observed when the non-reducing disaccharides sucrose (for E. coli K12) and trehalose (for E. coli NISSLE 1917) were added. The two tetrahydropyrimidines, hydroxyectoine and ectoine, gave protection to freeze-dried E. coli NISSLE 1917 whereas E. coli K12 was protected only by hydroxyectoine. Glycine betaine seemed to be harmful for both strains of E. coli with both drying techniques. Air0drying gave much better survival rates than freeze-drying. The two strains of E. coli differed in their ability to take up compatible solutes.     

Managing hypoosmotic stress: aquaporins and mechanosensitive channels in Escherichia coli.

When Escherichia coli cells are subject to hypoosmotic shock they are subject to substantial flows of water that can be equivalent to a 4-5-fold increase in the pressure exerted from the cytoplasm on the membrane and peptidoglycan wall. The recently described aquaporin that facilitates rapid water movement across the cytoplasmic membrane is repressed during growth at high osmolarity. This may enable the cell to reduce the rate of pressure build up during transitions from high to low osmolarity. The presence of multiple mechanosensitive channels in the E. coli cell membrane is well documented. The recent identification of genes that inactivate the MscL and MscS channels has established their role in releasing the pressure built up by hypoosmotic shock. The isolation of specific mutations and the structural studies on MscL now pave the way to a molecular understanding of the mechanism of activation of mechanosensitive channels.     

Survival of Escherichia coli and Salmonella spp. in estuarine environments.

Survival of Escherichia coli and Salmonella spp. in estuarine waters was compared over a variety of seasonal temperatures during in situ exposure in diffusion chambers. Sublethal stress was measured by both selective-versus-resuscitative enumeration procedures and an electrochemical detection method. E. coli and Salmonella spp. test suspensions, prepared to minimize sublethal injury, were exposed in a shallow tidal creek and at a site 7.1 km further downriver. Bacterial die-off and sublethal stress in filtered estuarine water were inversely related to water temperature. Salmonella spp. populations exhibited significantly less die-off and stress than did E. coli at water temperatures of less than 10 degrees C. Although the most pronounced reductions (ca. 3 log units) in test bacteria occurred during seasonally warm temperatures in the presence of the autochthonous microbiota, 10(2) to 10(4) test cells per ml remained after 2 weeks of exposure to temperatures of greater than 15 degrees C. Reductions in test bacteria were associated with increases in the densities of microflagellates and plaque-forming microorganisms. These studies demonstrated the survival potential of enteric bacteria in estuarine waters and showed that survival was a function of interacting biological and physical factors.