The Escherichia coli SOS gene dinF protects against oxidative stress and bile salts

DNA is constantly damaged by physical and chemical factors, including reactive oxygen species (ROS), such as superoxide radical (O₂ ⁻), hydrogen peroxide (H₂O₂) and hydroxyl radical (•OH). Specific mechanisms to protect and repair DNA lesions produced by ROS have been developed in living beings. In Escherichia coli the SOS system, an inducible response activated to rescue cells from severe DNA damage, is a network that regulates the expression of more than 40 genes in response to this damage, many of them playing important roles in DNA damage tolerance mechanisms. Although the function of most of these genes has been elucidated, the activity of some others, such as dinF, remains unknown. The DinF deduced polypeptide sequence shows a high homology with membrane proteins of the multidrug and toxic compound extrusion (MATE) family. We describe here that expression of dinF protects against bile salts, probably by decreasing the effects of ROS, which is consistent with the observed decrease in H₂O₂-killing and protein carbonylation. These results, together with its ability to decrease the level of intracellular ROS, suggests that DinF can detoxify, either direct or indirectly, oxidizing molecules that can damage DNA and proteins from both the bacterial metabolism and the environment. Although the exact mechanism of DinF activity remains to be identified, we describe for the first time a role for dinF.     

Active accumulation of tetracycline by Escherichia coli

1. At low concentrations of tetracycline (10mug/ml) net accumulation of the drug by Escherichia coli cells ceased after 7-10min. 2. At higher concentrations of tetracycline (>30mug/ml) the period of net accumulation of the drug was significantly extended. 3. The efflux of tetracycline from E. coli cells transferred from medium containing 10mug of tetracycline/ml to drug-free medium was a rapid temperature-dependent process and was accelerated by 2,4-dinitrophenol. 4. As the concentration of tetracycline in the preloading phase was increased, the rate of subsequent efflux of the drug progressively declined. The efflux of drug from cells preloaded in medium containing 200mug of tetracycline/ml was negligible, although efflux was readily provoked by 2,4-dinitrophenol, by N-ethylmaleimide or by omission of glucose from the medium. 5. The initial rate of uptake of tetracycline by E. coli cells was linearly proportional to the concentration of tetracycline in the medium up to the maximum concentration of drug obtainable under the experimental conditions used (400mug/ml, 0.83mm). 6. Although N-ethylmaleimide strongly inhibited the accumulation of tetracycline by E. coli, no evidence was obtained for the direct involvement of thiol groups in the transport process. It was concluded that N-ethylmaleimide inhibited accumulation by interruption of the energy supply of the cells. 7. Osmotic shock of E. coli cells did not significantly affect the influx of tetracycline, but promoted both efflux of tetracycline and cell lysis in cells treated with a high concentration of tetracycline. 8. A study of the distribution of tetracycline among the subcellular fractions of penicillin-induced spheroplasts preincubated with various concentrations of tetracycline indicated that 60-70% of the accumulated tetracycline was in the high-speed supernatant fraction. Sephadex chromatography showed that the tetracycline of this fraction was present as the free drug. Sephadex chromatography of a detergent extract of the membrane fraction, however, indicated that a significant proportion of the tetracycline radioactivity of this fraction was apparently bound to some macromolecular component. 9. Cellulose phosphate paper chromatography of cold-acid extracts of spheroplasts preloaded with tetracycline indicated that the accumulated drug was chemically unchanged. 10. Membrane preparations isolated from osmotically lysed penicillin-induced spheroplasts showed a temperature-dependent binding of tetracycline that was not energy-dependent and was not inhibited by N-ethylmaleimide. The binding process was stimulated by omitting Mg(2+) from the medium, but conversely was profoundly inhibited by EDTA. 11. The relevance of these findings to the probable mechanism of active tetracycline accumulation by E. coli is discussed.     

Activation potassium efflux from Escherichia coli by glutathione metabolites

The mechanism by which N-ethylmaleimide (NEM) elicits potassium efflux from Escherichia coli has been investigated. The critical factor is the formation of specific glutathione metabolites that activate transport systems encoded by the kefB and kefC gene products. Formation of N-ethyl-succinimido-S-glutathione (ESG) leads to the activation of potassium efflux via these transport systems. The addition of dithiothreitol and other reducing agents to cells reverses this process by causing the breakdown of ESG and thus removing the activator of the systems. Chlorodinitrobenzene, p-chloromercuribenzoate and phenylmaleimide provoke similar effects to NEM. lodoacetate, which leads to the formation of S-carboxymethyl-glutathione, does not activate the systems but does prevent the action of NEM. It is concluded that the KefB and KefC systems are gated by glutathione metabolites and that the degree to which they are activated is dependent upon the nature of the substituent on the sulphydryl group.     

Energetics of calcium efflux from cells of Escherichia coli.

Intact cells of a H+-translocating ATPase-deficient strain of Escherichia coli were starved of endogenous energy reserves and passively loaded with 45CaCl2. Energy-dependent efflux of calcium was observed upon addition of glucose or respiratory substrates. Addition of cyanide or uncouplers prevented efflux. It is concluded that calcium efflux in intact cells is coupled to the proton motive force via secondary calcium-proton exchange.     

An active type IV secretion system encoded by the F plasmid sensitizes Escherichia coli to bile salts

F(+) strains of Escherichia coli infected with donor-specific bacteriophage such as M13 are sensitive to bile salts. We show here that this sensitivity has two components. The first derives from secretion of bacteriophage particles through the cell envelope, but the second can be attributed to expression of the F genes required for the formation of conjugative (F) pili. The latter component was manifested as reduced or no growth of an F(+) strain in liquid medium containing bile salts at concentrations that had little or no effect on the isogenic F(-) strain or as a reduced plating efficiency of the F(+) strain on solid media; at 2% bile salts, plating efficiency was reduced 10(4)-fold. Strains with F or F-like R factors were consistently more sensitive to bile salts than isogenic, plasmid-free strains, but the quantitative effect of bile salts depended on both the plasmid and the strain. Sensitivity also depended on the bile salt, with conjugated bile salts (glycocholate and taurocholate) being less active than unconjugated bile salts (deoxycholate and cholate). F(+) cells were also more sensitive to sodium dodecyl sulfate than otherwise isogenic F(-) cells, suggesting a selectivity for amphipathic anions. A mutation in any but one F tra gene required for the assembly of F pili, including the traA gene encoding F pilin, substantially restored bile salt resistance, suggesting that bile salt sensitivity requires an active system for F pilin secretion. The exception was traW. A traW mutant was 100-fold more sensitive to cholate than the tra(+) strain but only marginally more sensitive to taurocholate or glycocholate. Bile salt sensitivity could not be attributed to a generalized change in the surface permeability of F(+) cells, as judged by the effects of hydrophilic and hydrophobic antibiotics and by leakage of periplasmic beta-lactamase into the medium.     

Energetics of sodium efflux from Escherichia coli

When energy-starved cells of Escherichia coli were passively loaded with 22Na+, efflux of sodium could be initiated by addition of a source of metabolic energy. Conditions were established where the source of energy was phosphate bond energy, an electrochemical proton gradient, or both. Only an electrochemical proton gradient was required for efflux from intact cells. These results are consistent with secondary exchange of Na+ for H+ catalyzed by a sodium/proton antiporter.     

Functional and biochemical characterization of Escherichia coli sugar efflux transporters.

A family of bacterial transporters, the SET (sugar efflux transporter) family, has been recently reported (Liu, J. Y., Miller, P. F., Gosink, M., and Olson, E. R. (1999) Mol. Microbiol. 31, 1845-1851). In this study, the biochemical and cell biological properties of the three Escherichia coli members (SetA, SetB, and SetC) of the family are characterized. We show that both SetA and SetB can transport lactose and glucose. In addition, SetA has broad substrate specificity, with preferences for glucosides or galactosides with alkyl or aryl substituents. Consistent with the observed in vitro substrate specificities, strains that hyperexpress SetA or SetB are desensitized to lactose analogues as measured by induction of the lac operon. In addition, strains that hyperexpress SetA are resistant to the growth inhibitory sugar analogue o-nitrophenyl-beta-D-thiogalactoside. Strains disrupted for any one or all of the set genes are viable and show no defects in lactose utilization nor increased sensitivity to inducers of the lac operon and nonmetabolizable sugar analogues. The data suggest that the set genes are either poorly expressed under normal laboratory growth conditions or are redundant with other cellular gene products.     

Cloning of the ethidium efflux gene from Escherichia coli

The gene specifying the ethidium efflux system of Escherichia coli has been cloned on a 3.2 kbp HindIII fragment and located on a 1.2 kbp fragment within this. Cross-resistance studies indicate that the system has a broad specificity for monovalent cations and the gene shows no hybridisation with similar genes found in Staphylococci.     

AcrD of Escherichia coli is an aminoglycoside efflux pump

AcrD, a transporter belonging to the resistance-nodulation-division family, was shown to participate in the efflux of aminoglycosides. Deletion of the acrD gene decreased the MICs of amikacin, gentamicin, neomycin, kanamycin, and tobramycin by a factor of two to eight, and DeltaacrD cells accumulated higher levels of [(3)H]dihydrostreptomycin and [(3)H]gentamicin than did the parent strain.     

Manganese Active Transport in Escherichia coli

Manganese was accumulated by cells of Escherichia coli by means of an active transport system quite independent of the magnesium transport system. When the radioisotope (54)Mn was used, manganese transport showed saturation kinetics with a K(m) of 2 x 10(-7)m and a V(max) of 1 to 4 nmoles/min per 10(12) cells at 25 C. The manganese transport system is highly specific; magnesium and calcium did not stimulate, inhibit, or compete with manganese for cellular uptake. Cobalt and iron specifically interfered with (54)Mn uptake, but only when added at concentrations 100 times higher than the K(m) for manganese. Active transport of manganese is temperature- and energy-dependent: uptake of (54)Mn was inhibited by cyanide, dinitrophenol, and m-chlorophenyl carbonylcyanide hydrazone (CCCP). Furthermore, the turnover or exit of manganese from intact cells was inhibited by energy poisons such as dinitrophenol and CCCP.     

AcrAB and related multidrug efflux pumps of Escherichia coli

The AcrAB system of Escherichia coli is a multidrug efflux system composed of an RND-type transporter AcrB and a periplasmic accessory protein AcrA, and pumps out a wide variety of lipophilic and amphiphilic inhibitors directly into the medium, presumably through the TolC outer membrane channel. AcrA, a highly elongated protein, is thought to bring the outer and inner membranes closer. It forms a trimer that interacts with a monomeric AcrB, which was shown by in vitro reconstitution to be a proton antiporter. Details of interaction between the     

Stimulation of respiration-linked proton efflux in Escherichia coli by carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP).

The proton efflux from intact, anaerobic $\text{Escherichia}\text{coli}$ cells following a small oxygen pulse is both slow ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>M</mtext><mtext>2</mtext>}}\text{～-10s}$) and inefficient ($\text{H}{}^{\mathrm{+}}\text{O}\text{～-0.5}$. Very low levels (<80 nM) of the proton ionophore carbonylcyanide-$\text{p}$-trifluoromethoxyphenylhydrazone (FCCP), which have no detectable effect upon active transport, cause a 3–5 fold stimulation in the extent of proton efflux without affecting the efflux rate. At slightly higher concentrations of FCCP (80 nM to 0.5 μM), a sharp inhibition of this increased proton efflux occurs, with the $\text{H}{}^{\mathrm{+}}\text{O}$ ratio obtained in the presence of 0.5 μM FCCP approximately equal to that obtained in the absence of FCCP. Still higher concentrations of FCCP (> 1 μM), which inhibit active transport, cause a further gradual decrease in the $\text{H}{}^{\mathrm{+}}\text{O}$ ratio. The unusual increase in the apparent efficiency of H⁺ efflux by <80 nM FCCP is not accompanied by an increase in the rate of membrane deenergization following an O₂ pulse, although such an increase is seen with the higher (uncoupling) FCCP concentrations.     

Active Subunits of Escherichia coli Glutamate Synthase

The large and small subunits of Escherichia coli glutamate synthase were isolated. The small subunit catalyzes the NH₃-dependent synthesis of glutamate. The large subunit exhibits glutaminase activity.     

Endotoxin-free Biologically Active Component of Escherichia coli

The proteinaceous component of gram-negative bacteria, which has been termed “protodyne,” enhances nonspecific host resistance while eliciting a slight pyrogenic response equivalent to 0.2% that of a typical endotoxin. Since this material still contains small amounts of carbohydrate and lipid, it was imperative to establish that its biological activities are not the result of endotoxin contamination. Evidence that the protective activity of protodyne does not result from endotoxin contamination has now been obtained by an evaluation of the Pronase digestion products of this substance. These digestion products were found to be nonpyrogenic and to contain no measurable amount of 2-keto-3-deoxyoctonate, an essential component of bacterial lipopolysaccharides.     

Fluorometric determination of ethidium bromide efflux kinetics in Escherichia coli

Background  

Efflux pump activity has been associated with multidrug resistance phenotypes in bacteria, compromising the effectiveness of antimicrobial therapy. The development of methods for the early detection and quantification of drug transport across the bacterial cell wall is a tool essential to understand and overcome this type of drug resistance mechanism. This approach was developed to study the transport of the efflux pump substrate ethidium bromide (EtBr) across the cell envelope of Escherichia coli K-12 and derivatives, differing in the expression of their efflux systems.     

Fluorescent derivatives of bile salts. I. Synthesis and properties of NBD-amino derivatives of bile salts.

In order to visualize bile salt transport, fluorescent bile salt derivatives were synthesized by introduction of the relatively small fluorescent 4-nitrobenzo-2-oxa-1,3-diazol (NBD)-amino group in either the 3-, 7-, or 12-position of the steroid structure, thus providing a complete set of diastereomeric derivatives, 3 alpha-NBD-amino-7 alpha,12 alpha-dihydroxy-5 beta-cholan-24-oic acid, 3 beta-NBD-amino-7 alpha,12 alpha-dihydroxy-5 beta-cholan-24-oic acid, 7 alpha-NBD-amino-3 alpha,12 alpha-dihydroxy-5 beta-cholan-24-oic acid, 7 beta-NBD-amino-3 alpha,12 alpha-dihydroxy-5 beta-cholan-24-oic acid, 12 alpha-NBD-amino-3 alpha,7 alpha-dihydroxy-5 beta-cholan-24-oic acid, 12 beta-NBD-amino-3 alpha,7 alpha-dihydroxy-5 beta-cholan-24-oic acid, as well as their taurine conjugates. Their optical properties with absorption maxima at about 490 nm and emission maxima at 550 nm make them suitable for fluorescent microscopic studies. Fluorescence of the NBD-derivatives is strongly dependent on polarity of the solvent, on the concentration of the bile salt derivatives, and only slightly on temperature.