Role of outer membrane barrier in efflux-mediated tetracycline resistance of Escherichia coli.

Accumulation of tetracycline in Escherichia coli was studied to determine its permeation pathway and to provide a basis for understanding efflux-mediated resistance. Passage of tetracycline across the outer membrane appeared to occur preferentially via the porin OmpF, with tetracycline in its magnesium-bound form. Rapid efflux of magnesium-chelated tetracycline from the periplasm was observed. In E. coli cells that do not contain exogenous tetracycline resistance genes, the steady-state level of tetracycline accumulation was decreased when porins were absent or when the fraction of Mg(2+)-chelated tetracycline was small. This is best explained by assuming the presence of a low-level endogenous active efflux system that bypasses the outer membrane barrier. When influx of tetracycline is slowed, this efflux is able to reduce the accumulation of tetracycline in the cytoplasm. In contrast, we found no evidence of a special outer membrane bypass mechanism for high-level efflux via the Tet protein, which is an inner membrane efflux pump coded for by exogenous tetA genes. Fractionation and equilibrium density gradient centrifugation experiments showed that the Tet protein is not localized to regions of inner and outer membrane adhesion. Furthermore, a high concentration of tetracycline was found in the compartment that rapidly equilibrated with the medium, most probably the periplasm, of Tet-containing E. coli cells, and the level of tetracycline accumulation in Tet-containing cells was not diminished by the mutational loss of the OmpF porin. These results suggest that the Tet protein, in contrast to the endogenous efflux system(s), pumps magnesium-chelated tetracycline into the periplasm. A quantitative model of tetracycline fluxes in E. coli cells of various types is presented.     

Light-Induced tetracycline accumulation by rhodopseudomonas sphaeroides

Light has been used as a primary energy source in studies of tetracycline transport by Rhodopseudomonas sphaeroides. Accumulation of the antibiotic occurs in light, while efflux occurs in dark. Both fluorescence enhancement and radioisotopic tracing have been used to monitor transport. K_m's obtained from both techniques are similar. Light-induced accumulation of tetracyclines is inhibited by a variety of inhibitors, including antimycin A, N-ethylmaleimide, carbonylcyanide m-chloro-phenylhydrazone, and 2,4-dinitrophenol. A rapid efflux is observed after loading when cells are placed in the dark or treated with inhibitors.     

Effect of osmotic shock on tetracycline resistance in Escherichia coli bearing an R-factor

1. Escherichia coli with an R-factor conferring resistance to tetracycline was induced to high-degree resistance by pre-exposure to the antibiotic. The degree of resistance was drastically lowered by subjecting the cells to osmotic shock. 2. Resistance to tetracycline was rapidly restored by incubating the shocked cells in a glucose-salts medium containing shock proteins prepared from tetracycline-sensitive and -resistant cells. Resistance was also restored by incubating the cells in a complex medium without shock protein. 3. The initial recovery of resistance was followed by a secondary fall in resistance when the cells were cultured in complex medium; this secondary fall was largely prevented by the addition of a low concentration (10mug/ml) of tetracycline to cells. The secondary fall was significantly less in shocked E. coli cells harbouring a mutant R-factor in which tetracycline resistance is largely constitutive. 4. Tetracycline resistance was also transiently depressed by treating R-factor-bearing cells with EDTA in tris buffer. 5. The significance of these results in relation to the mechanism of tetracycline resistance in R-factor-bearing cells is discussed.     

Stabilizing Effect of Colicin E2 on the “Spheroplast Membrane”

Cells of Escherichia coli W3110 and its thymineless mutant, both of which are colicin E2 sensitive, were treated with colicin E2, and then converted to spheroplasts. These spheroplasts seemed to be more stable than those from untreated cells; suspensions of spheroplasts of untreated cells were lysed spontaneously and the turbidity was reduced by approximately 45% on incubation with ethylenediaminetetraacetic acid-lysozyme, whereas suspensions of spheroplasts of colicin E2-treated cells showed 25% reduction in turbidity. This change was irreversible and 5 min of treatment with colicin E2 at 37 C was necessary for stabilization. This process was inhibited by 2,4-dinitrophenol or streptomycin. Cells harboring the colicin E2 factor were not affected by treatment in this way with colicin E2. Alteration of composition of phospholipids was not observed.     

Release of periplasmic enzymes from Escherichia coli by penicillin-ethylenediaminetetraacetate treatment.

When Escherichia coli cells are converted into spheroplasts by penicillin treatment, none of the periplasmic enzymes is released. However, incubation of the penicillin-induced spheroplasts in a medium containing ethylenediaminetetraacetic acid caused the release of endonuclease I and cyclic phosphodiesterase but not of beta-galactosidase.     

Reconstitution of binding protein dependent ribose transport in spheroplasts derived from a binding protein negative escherichia coli K12 mutant and from salmonella typhimurium

Highly purified ribose-binding protein from Escherichia coli has been used to reconstitute a binding-protein-dependent ribose transport in spheroplasts derived from a binding-protein-deficient mutant of E coli K 12, and in spheroplasts derived from Salmonella typhimurium. The cross-species reconstitution was nearly as efficient as the reconstitution of the E coli strain from which the binding protein was derived. Antibody raised against the ribose binding protein completely prevented reconstitution, whereas it had no effect on whole cells. The reconstitution procedure has been improved by generating spheroplasts from cells grown in a rich medium and by reducing the background uptake in spheroplasts through a special washing procedure. Rapid purification of ribose binding protein by high pressure liquid chromatography is also described.     

New Type of R Factors Incapable of Inactivating Chloramphenicol

Four R factors conferring chloramphenicol (CM) resistance were isolated from Escherichia coli strains of clinical origin. Strains carrying the factors were found to be incapable of inactivating the drug in the presence of acetyl coenzyme A. E. coli W3630 carrying R(70), one of these factors, became sensitive to CM after treatment with glycine, indicating that the spheroplasts of W3630 R(70) (+) were sensitive to the drug and suggesting that the cell membrane is important for CM resistance. The observation that cell-free protein synthesis in W3630 R(70) (+) was inhibited by CM is also compatible with a decrease in permeability. CM resistance in W3630 R(70) (+) appeared to be inducible, because (i) preincubation with subinhibitory concentrations of CM prevented the prolonged lag noted for growth in the presence of 25 mug of CM per ml, and (ii) the preincubation effect was lost after overnight growth in CM-free medium. By contrast, E. coli W3630 cml(+), in which the resistance determinant is integrated into the chromosome, was capable of rapid inactivation of CM. E. coli W3630 cml(+) R(70) (+), which contains the proposed permeability determinant (episomal) as well as levels of the inactivating enzyme (chromosomal) that are comparable with W3630 cml(+), was capable of brief inactivation of CM when inoculated into drug-containing medium. The absence of continued inactivation on more prolonged incubation favors the hypothesis that the R(70) factor inhibited further penetration of CM and that this property possesses the characteristics of induction.     

Transfection of Escherichia coli and Salmonella typhimurium Spheroplasts: Host-Controlled Restriction of Infective Bacteriophage P22 Deoxyribonucleic Acid

Under proper conditions, one infective center was obtained for 3 x 10(8) molecules of P22 phage deoxyribonucleic acid (DNA) when lysozyme-ethylenediaminetetraacetic acid spheroplasts of Escherichia coli were transfected in the presence of 25 mug of protamine sulfate per ml. A 3- to 50-fold B-specific and K-specific E. coli restriction of the incoming P22 DNA was observed. When P22 DNA-infected E. coli spheroplasts were plated with infertile r(LT) (+)m(LT) (+)Salmonella typhimurium indicator, an additional 70-fold restriction was observed. In the presence of protamine sulfate, penicillin spheroplasts of S. typhimurium SB1330 could be transfected b P22 DNA with efficiencies sometimes approaching those obtained with the E. coli spheroplasts; thus, facilitation of transfection by protamine sulfate is not limited to E. coli or to lysozyme-ethylenediaminetetraacetic acid spheroplasts. The application of these results to studies of transfection among other genuses and to studies of in vitro host-controlled restriction and modification for the two loci in S. typhimurium and the one locus in E. coli is discussed.     

Inducible synthesis of beta-galactosidase in disrupted spheroplast of Escherichia coli

A membrane preparation obtained from osmotic lysate of spheroplasts of Escherichia coli cells showed an activity of synthesizing beta-galactosidase which was dependent upon oxidative phosphorylation. The synthesis was inhibited by the addition of actinomycin D or of chloramphenicol. The beta-galactosidase synthesized in the membrane preparation was completely released into the medium, while that synthesized in the spheroplasts and intact cells remained within the cells. The minimum concentration of the inducer, methyl-beta-d-thiogalactoside, required for the induction of beta-galactosidase was 5 x 10(-5)m for intact cells, 3 x 10(-4)m for spheroplasts and 1 x 10(-3)m for membrane preparation. Incorporation of labeled glucose into insoluble components in membrane preparation was extremely low compared with that in intact cells or in spheroplasts. Based on these and other observations, the nature of this membrane preparation is discussed in relation to the structure of E. coli cells.     

Characterization of cytosine permeation in Saccharomyces cerevisiae.

Cytosine permeation in Saccharomyces cerevisiae has been studied. Cytosine uptake is mediated by a permease which is also responsible for purines transport. The Km for the transport of various substrates of this permease have been determined. By means of appropriate selective techniques, mutants with altered Km and mutants lacking the permease have been selected. Cytosine transport is active and is inhibited by 2,4-dinitrophenol, an uncoupler of oxidative phosphorylation, and by N-ethylmaleimide, a reagent of--SH group. Internal labeled cytosine is chased by addition of unlabeled cytosine in the medium. These results support the hypothesis of a carrier-mediated transport, with reduced internal affinity, allowing the release and accumulation of cytosine in the inner compartment. The efflux of cytosine from cytosine permease-less cells has also been studied and shows first order kinetics. A diffusion coefficient of 5.7 per 10- minus 8 cm per S- minus 1 has been evaluated for this efflux.     

Uptake of tetracycline by membrane preparations from Escherichia coli

1. A membrane fraction from Escherichia coli has been prepared essentially free from ribosomes by treatment of the membranes with Triton X-100 at 0 degrees C followed by differential centrifugation. 2. The ribosome-free membrane vesicles absorbed tetracycline by a reversible temperature-dependent process with an apparent K(m) of 0.029mm at pH7.5 and 37 degrees C. 3. The absorption process was negligible below 25 degrees C and had an optimum at 40 degrees C; a pH optimum at 7.5 was observed. 4. The absorption of tetracycline was strongly inhibited by EDTA and ATP; ADP inhibited less strongly and AMP had no effect. 5. There was no significant difference in the rates or extent of uptake of tetracycline by membranes prepared from tetracycline-sensitive and tetracycline-resistant, R-factor-bearing E. coli.     

Filamentous Cells of Escherichia coli Formed in the Presence of Mitomycin

The effects of mitomycin C on cell elongation of Escherichia coli B were studied. Filament formation was most marked in cultures treated with a moderate level (1 mug/ml) of the antibiotic, becoming less obvious at higher levels (10 mug/ml). Cells treated with a bacteriostatic concentration (0.1 mug/ml or less) of mitomycin C were also significantly elongated. The filamentous or elongated cells appeared to lack septa, since their spheroplasts were considerably larger than those formed from normal cells. The appearance of empty spheres also indicated some defects in the surfaces of the filamentous cells. Electron micrographs of the filaments revealed a characteristic difference in the arrangement of the nuclei in the filaments formed in the presence of low (0.1 mug/ml) and high (5 mug/ml) concentrations of mitomycin C. The filaments formed by the low level of mitomycin C had normal well-defined nuclear bodies distributed along the long axis, whereas those formed by the elevated level of the antibiotic contained smaller nuclei. The latter were characteristically confined to the center of the cells and did not extend out to the tips of the filaments.     

Unidirectional K+ fluxes in rat thymocytes stimulated by concanavalin A.

Unidirectional K+ fluxes were estimated in isolated rat thymocytes by 42K exchange kinetics. The cells were either preloaded with isotope and the release of it measured during incubation for one hour at 38 degrees C, or the cellular uptake of isotope during a similar incubation was measured. The influx rate of untreated thymocytes was: 2.3-10(-12) moles cm-2-s-1 and efflux rate: 1.8-10(-12) moles cm-2-s-1. When con A was added to the cells, influx was raised 74% and efflux 65%. Maximal effect was obtained when the concentration of con A was 15 mug/ml, but concentrations as low as 0.75 mug/ml were effective. Hydrocortisone resistant thymocytes responded at least was well as untreated cells to con A, which also raised RNA synthesis rate in the former cells 2.5 times. Using an extracellular marker, 51CrEDTA, intracellular concentrations of some ions was estimated in the thymocytes after one hour incubation: Na+: 30 mmoles/kg water, K+: 177 mmoles/kg water and Cl-:43 mmoles/kg water. Cellular water content: 69%. These values were not found significantly altered when con A was present. Since con A raised influx and efflux to the same extent and no net flux of K+ could be detected, it is proposed that both active and passive transport of K+ was increased by con A. The increased fluxes induced by con A, can apparently not be reversed by removal of con A from the incubation medium or by addition of the inhibiting hapten, alpha-methyl-D-mannoside.     

An inducible β-lactamase in a strain ofEscherichia coli

The production of beta-lactamase (penicillin/cephalosporin beta-lactam amidohydrolase, E.C.3.5.2.6) was found to be inducible in a clinically isolated strain of Escherichia coli. This is the first report of an inducible beta-lactamase in E. coli. The optimal concentration of inducer was 400 mug/ml of ml of benzylpenicillin, or 800 mug/ml of 6-aminopenicillanic acid. About fiftyfold induction was achieved. Maximum induction took ninety minutes from the time of adding the inducer. Induction was abolished by the presence of chloramphenicol(10 mug/ml). The enzyme has a molecular wieght of 23,000, and is inhibited by rho-chloromercuribenzoate and by iodine. It is active against a wide range of substrates, including cephaloridine and cloxacillin.     

Relationships between the exchange of calcium and phosphate in isolated fat-cells.

The uptake and the washout of 45Ca2+ and 32Pi is described in free fat-cells and whole epididymal fat-pads from fed rats. 2. In isolated fat-cells, the uptake of 45Ca2+ proceeds with an initial rapid phase of about 1 min duration, followed by a slower subsequent accumulation. In contrast with the rapid phase, the slow phase is inhibited by 2,4-dinitrophenol, warfarin, oligomycin and verapamil, shows saturation, and presumably represents transport across the plasma membrane. 3. The washout of 45Ca2+ from preloaded cells consists of a rapid (1 min) initial phase and a slow phase which is non-monoexponential, suggesting that the radioactive isotope is released from several cellular pools. 4. When Pi is omitted from the incubation medium, the slow phase of 45Ca uptake is almost abolished, and the washout of 45Ca from preloaded fat-cells is markedly accelerated. At elevated extracellular concentrations of Pi (2,4-6.2mM), the uptake of 45Ca is stimulated by 2-10-fold, and the release of the radioactive isotope from preloaded cells is inhibited. In whole epididymal fat-pads, variations in the extracellular concentration of Pi have no detectable effect on the uptake or the washout of 45Ca. 5. In isolated fat-cells, the accumulation of 32Pi is inhibited by 2,4-dinitrophenol or the omission of glucose from the incubation medium. In a Ca2+-depleted buffer, the uptake of 32Pi is diminished, and hyperosmolarity, which stimulates 45Ca uptake, also accelerates the accumulation of 32Pi. 6. It is concluded that in free fat-cells, the uptake and release of Ca2+ and Pi take place by closely interrelated processes, which are dependent on mitochondrial energy production.     

Mechanism for the pyridoxal neutralization of isoniazid action of Mycobacterium tuberculosis

In Sauton's synthetic liquid medium, 10 mug of pyridoxal per ml completely protected Mycobacterium tuberculosis (H37R(a)) from the effects of a minimal inhibitory concentration of isoniazid (0.01 mug/ml). (14)C-labeled isoniazid was employed to study the nature of this protective effect. Uptake of the drug by cells in a Sauton environment containing 0.01 mug of (14)C-isoniazid per ml was inhibited 20 to 40% by 10 mug of pyridoxal per ml during the early hours of drug exposure. A stronger inhibition of uptake resulted when labeled isoniazid and pyridoxal were increased to 0.1 mug/ml and 50 to 100 mug/ml, respectively. Further studies revealed that certain Sauton nutrients are required to achieve this effect. When l-asparagine or salts (MgSO(4) and ferric ammonium citrate) or both were deleted from the menstruum, pyridoxal did not inhibit isoniazid incorporation by the tubercle bacilli. Pyridoxal also failed to inhibit uptake when (NH(4))(2)SO(4) was substituted for l-asparagine. Growth experiments in Sauton's medium modified to contain (NH(4))(2)SO(4) instead of l-asparagine were consistent with the latter finding. Pyridoxal did not prevent isoniazid growth inhibition in this medium. It is postulated that a large excess of pyridoxal in Sauton's medium protects tubercle bacilli from the effects of isoniazid through formation of an extracellular complex involving drug, vitamin, and certain medium constituents, thereby reducing the level of isoniazid available to the cells.     

Isoniazid Uptake in Relation to Growth Inhibition of Mycobacterium tuberculosis

(14)C-isoniazid (INH) was used to study the relationship between drug uptake or binding by Mycobacterium tuberculosis and growth inhibition of the organism, which is dependent upon the concentration of drug and the duration of exposure. When strain H37R(a), grown in modified Sauton's liquid medium, was treated with 0.1 mug of INH per ml for 2 to 6 hr, followed by 10 mug of nicotinic hydrazide (NH) per ml to block further INH uptake, growth was retarded but not completely inhibited upon continued incubation. NH itself did not retard growth. However, cells treated in a similar manner with INH alone grew normally when diluted 1:100 in fresh, drug-free media. Uptake data showed that bacilli exposed to 0.1 mug of INH per ml accumulated 5.5, 9.7, and 12 mmug/mg of dry cells at 2, 4, and 6 hr, respectively. Other experiments suggested that once isoniazid is bound, it is not rapidly lost when NH is added or when the cells are diluted in fresh media. In the presence of 1.0 mug of INH per ml, tubercle bacilli took up 10 to 37 mmug/mg of dry cells in 20 to 90 min. These cells were not markedly inhibited when diluted 1:40 in fresh NH-containing media and incubated for 6 days. Growth inhibition of tubercle bacilli by INH depends on the uptake of sufficient drug, but the evidence obtained in this study suggests that the absolute concentration of bound INH is not as important in the action of the drug as is the maintenance of a critical cellular concentration for a requisite period of time.     

Kinetic characteristics of the two glucose transport systems in Neurospora crassa

Glucose is transported across the cell membrane of Neurospora crassa by two physiologically and kinetically distinct transport systems. System II is repressed by growth of the cells in 0.1 m glucose. System I is synthesized constitutively. The apparent K(m) for glucose uptake by system I and system II are 25 and 0.04 mm, respectively. Both uptake systems are temperature dependent, and are inhibited by NaN(3) and 2,4-dinitrophenol. Glucose uptake by system II was not inhibited by fructose, galactose, or lactose. However, glucose was shown to be a noncompetitive inhibitor of fructose and galactose uptake. The transport rate of [(14)C]3-0-methyl-d-glucose (3-0-MG) was higher in cells preloaded with unlabeled 3-0-MG than in control cells. The rate of entry of labeled 3-0-MG was only slightly inhibited by the presence of NaN(3) in the medium. Further, NaN(3) caused a rapid efflux of accumulated [(14)C]3-0-MG. These data imply that the energetic step in the transport process prevents efflux.     

Inhibitory Effect of Fatty Acids on the Entry of the Lipid-Containing Bacteriophage PR4 into Escherichia coli

Various unsaturated fatty acids (notably palmitoleic acid and oleic acid) interfered with plaque production by the lipid-containing bacteriophage PR4 on lawns of Escherichia coli. Addition of fatty acid to give 50 mug/ml ( approximately 0.2 mM) at the time of infection prevented phage replication. If, however, the fatty acid was added after infection, normal amounts of phage were produced. If the fatty acid was added (to 50 mug/ml) to the host cell culture a long enough time before infection such that the fatty acid concentration in the growth medium at the time of infection was reduced to less, similar5 mug/ml (due to fatty acid incorporation by the host cells), normal phage replication occurred also. Neither palmitoleic acid nor oleic acid prevented PR4 attachment to E. coli. Several types of experiments indicated that it is the entry process of the virus that is inhibited by these fatty acids. Specifically, if the fatty acid was added at the time of infection, the host cells were not killed by the virus and no detectable amounts of viral protein were synthesized. In addition, experiments using purified radioisotope-labeled virions showed directly that entry is inhibited. Mutants of PR4 that did replicate in the presence of oleic acid arose spontaneously at a frequency of 10(-6). Three of these mutants that have been further characterized have protein and phospholipid compositions indistinguishable from those of wild-type PR4.     

Cephapirin: In Vitro Antibacterial Spectrum

Cephapirin, a new semisynthetic cephalosporin derivative, was found to have an antibacterial spectrum similar to that of cephalothin. Staphylococcus aureus was inhibited by cephapirin concentrations of 0.09 to 12.5 mug/ml. S. epidermidis, S. viridans, S. pyogenes, and Diplococcus pneumonia isolates were inhibited by less than 1 mug/ml. The Enterococcus required a concentration of 25 mug of antibiotic per ml for inhibition. Approximately 65% of Escherichia coli, and all Klebsiella, indole-negative Proteus, and Salmonella strains tested were inhibited by the drug. Serratia, Pseudomonas, indole-positive Proteus, and Erwinia strains were highly resistant. Inoculum size was not an important factor in determining the level of sensitivity of S. aureus to cephapirin. The antibiotic does not appear to be significantly bound to serum protein. In vitro development of resistance to the drug was demonstrated with two isolates of S. aureus.