Method for the isolation of Escherichia coli relaxed mutants, utilizing near-ultraviolet irradiation.

Near-ultraviolet radiation (300 to 380 nm) induces a transient growth inhibition in "stringent" (rel+) strains of Escherichia coli, whereas "relaxed" (rel-) strains are only mildly affected. This difference permits rapid isolation of large numbers of relaxed mutants of E. coli.     

Inactivation of adenosine 5''-triphosphate synthesis and reduced-form nicotinamide adenine dinucleotide dehydrogenase activity in Escherichia coli by near-ultraviolet and violet radiations.

Near-ultraviolet (near-UV) light (300 to 380 nm) is a significant component of sunlight and has a variety of effects on biological systems. The present work is an attempt to identify chromophores (molecular absorbers of light) and targets (critical damaged molecules) for inhibition of adenosine triphosphate (ATP) synthesis in Escherichia coli by near UV. The fluence of 334 nm required for 37% survival of net ATP synthesis (F37) in E. coli AB2463 in succinate medium is 140 kJ/m2. The action spectrum for this inactivation is almost structureless, exhibiting a smooth transition from high efficiency at 313 nm to low efficiency at 405 nm. The action spectrum for inhibition of net ATP synthesis is consistent with the chromophore being either ubiquinone Q-8 or vitamin K2. The fluence required is consistent with ubiquinone Q-8 also being a target molecule. The activity of reduced nicotinamide adenine dinucleotide dehydrogenase in extracts of E. coli B is also inactivated by near UV and shows an F37 of about 40 kJ/m2. The action spectrum for this effect is quite structureless; it shows high efficiency at 313 nm and low efficiency at 435 nm. The data do not suggest a target molecule for this action, although it is possible that ubiquinone Q-8 absorbs the near-UV energy and then passes it on to some other target molecule. The data further indicate that inactivation of the oxidative phosphorylation system is not a primary factor in near-UV-induced growth delay in E. coli.     

Replication of lambda plasmid in amino acid-starved strains of Escherichia coli.

We found that lambda plasmid replication, as measured by the increase in plasmid content per bacterial mass, proceeds for hours in an amino acid-starved, relaxed mutant of Escherichia coli K-12, whereas is inhibited in its wild-type stringent partner. Replication of lambda plasmid in amino acid-starved, relaxed cells reveals absolute lambda O dependence and is not inhibited by chloramphenicol at 200 micrograms/ml. The replication also occurs in wild-type cells treated with chloramphenicol. We conclude that lambda plasmid replication is under stringent control, probably as a result of the action of ppGpp, the signal for the stringent response, on RNA polymerase.     

Specificity of the lethal and mutagenic actions of pico-second laser pulses of 532-nm wavelength

A study was made of the lethal effect of pulse laser (second harmonic Nd+3:YAG laser of 532 nm, pulse length 3.3.10(-11) s, peak intensity from 4.10(12) to 1.10(14) W/m2) on HeLa cells at the phases of active and stationary growth, and lethal and mutagenic effects of this radiation on E. coli cells. As was shown, HeLa cells at both growth phases and E. coli cells exhibited low sensitivity to laser radiation at lambda = 532 nm.     

Biological function and site II Ca2+-induced opening of the regulatory domain of skeletal troponin C are impaired by invariant site I or II Glu mutations

To investigate the roles of site I and II invariant Glu residues 41 and 77 in the functional properties and calcium-induced structural opening of skeletal muscle troponin C (TnC) regulatory domain, we have replaced them by Ala in intact F29W TnC and in wild-type and F29W N domains (TnC residues 1-90). Reconstitution of intact E41A/F29W and E77A/F29W mutants into TnC-depleted muscle skinned fibers showed that Ca(2+)-induced tension is greatly reduced compared with the F29W control. Circular dichroism measurements of wild-type N domain as a function of pCa (= -log[Ca(2+)]) demonstrated that approximately 90% of the total change in molar ellipticity at 222 nm ([theta](222 nm)) could be assigned to site II Ca(2+) binding. With E41A, E77A, and cardiac TnC N domains this [theta](222 nm) change attributable to site II was reduced to < or =40% of that seen with wild type, consistent with their structures remaining closed in +Ca(2+). Furthermore, the Ca(2+)-induced changes in fluorescence, near UV CD, and UV difference spectra observed with intact F29W are largely abolished with E41A/F29W and E77A/F29W TnCs. Taken together, the data indicate that the major structural change in N domain, including the closed to open transition, is triggered by site II Ca(2+) binding, an interpretation relevant to the energetics of the skeletal muscle TnC and cardiac TnC systems.     

Real-time kinetics of the interaction between the two subunits,Escherichia coli thioredoxin and gene 5 protein of phage T7 DNA polymerase

T7 phage DNA polymerase is a tight 1:1 complex of the gene 5 protein (g5p) (80 kDa) of phage T7 and thioredoxin (12 kDa) from the Escherichia coli host. The holoenzyme is essential for the replication of the phage. We estimated the real-time kinetics and thermodynamics of the interaction of g5p with thioredoxin (wild type and mutants) using surface plasmon resonance. Thioredoxin was immobilized on a CM5 sensor chip through a six-carbon spacer (6-amino-n-hexanoic acid) using standard amine coupling. Reduced thioredoxin bound g5p but oxidized thioredoxin did not. The association and dissociation phases of the complex fit a two-exponential model with an apparent equilibrium dissociation constant (KD) of 2.2 nm for thioredoxin with 4.7 x 104.M-1.s-1 and 10.5 x 10-5.s-1 as the corresponding association (ka) and dissociation (kd) rate constants. The strong binding of g5p to thioredoxin is therefore due to fast association and very slow dissociation, a situation similar to antigen-antibody interactions. Thioredoxin mutants P34S, D26A, K57M, D26A/K57M, W31F, W31Y, K36A, K36E, and Y49F had KD values in the range of 1 to 8 nm, whereas mutant W28A had a KD of 12.5 nm. No detectable interaction was observed for mutants P40G, W31H, W31A, and C35A. The effect of temperature on KD and the changes in enthalpy (-DeltaH = 20.2 kcal.m-1) and entropy (TDeltaS =-8.4 kcal.m-1) upon formation of the complex suggested that the interaction is driven by an increase in enthalpy and opposed by a decrease in entropy.     

Effect of light fluence rate on mammalian cells photosensitization by chloroaluminium phthalocyanine tetrasulphonate

Phthalocyanines are photosensitizers evaluated for use in photodynamic therapy of cancer. As such, the dependence of the bioresponse on the light fluence rate may be of clinical importance. The effect of the fluence rate of white light from 0.165 to 3.3 kJ m-2 min-1 was studied in Chinese hamster cells and human lymphocytes, using as endpoints colony-forming ability and inhibition of [3H]thymidine incorporation following mitogenic stimulation and dye-photoactivation, respectively. Using Chinese hamster cells exposed to photoexcited chloroaluminium phthalocyanine tetrasulphonate in PBS solution, cytotoxicity was diminished as the fluence rate was reduced. In human lymphocytes changing the fluence rate between 0.33 and 3.3 kJ m-2 min-1 affected the response in a way similar to that of Chinese hamster cells. Human lymphocytes, when exposed to incremental increasing light fluences, 4 h after a conditioning dose, were able to recover from phthalocyanine-induced photodamage, as evidenced by the reappearance of a shoulder on the dose-effect curve. This recovery process during a protracted light exposure, could explain the reduced sensitivity to phthalocyanine photosensitization, compared to exposure at high fluence rates.     

Investigation of the aurovertin binding site of Escherichia coli F1-ATPase by fluorescence spectroscopy and site-directed mutagenesis.

(1) Previous mutational analyses have shown that residue beta R398 of the beta-subunit is a key residue for binding of the inhibitory antibiotic aurovertin to Escherichia coli F1Fo-ATP synthase. Here, we studied purified F1 from the beta R398C and beta R398W mutants. ATPase activity in both cases was resistant to aurovertin inhibition. The fluorescence spectrum (lambda exc = 278 or 295 nm) of beta R398W F1 showed a significant red-shift as compared to wild-type and beta R398C enzymes, indicating that residue beta R398 lies in a polar environment. On the basis of this and previous evidence, we propose that aurovertin binding to F1-ATPase involves a specific charged donor-acceptor H-bond between residue beta R398 and the 7-hydroxyl group of aurovertin. (2) The fluorescent substrate analog lin-benzo-ADP was shown to bind to beta R398W F1 catalytic sites with the same Kd values as to wild-type F1, and with the same quenching of the fluorescence of the analog. Fluorescence energy transfer was seen between the beta R398W residue and bound lin-benzo-ADP. Analysis of transfer efficiency at varying stoichiometry of bound lin-benzo-ADP showed that interaction occurred between one beta R398W residue and one catalytic-site-bound analog molecule at a distance of approximately 23 A. The relationships of the aurovertin and catalytic sites in the primary and tertiary structure are discussed.     

Suppression of mutations conferring penicillin tolerance by interference with the stringent control mechanism of Escherichia coli.

Mutations in Escherichia coli previously reported (R. E. Harkness and E. E. Ishiguro, J. Bacteriol. 155:15-21, 1983; L. C. Shimmin, D. Vanderwel, R. E. Harkness, B. R. Currie, A. Galloway, and E. E. Ishiguro, J. Gen. Microbiol. 130:1315-1323, 1984) as conferring a temperature-dependent tolerance to lysis induced by inhibitors of peptidoglycan synthesis were suppressed by treatment with inhibitors of the stringent response or by introduction of a relA mutation. The relA+ derivatives of the mutants exhibited a stringent response at the nonpermissive temperature. The consequent inhibition of the autolytic enzyme system (W. Kusser and E. E. Ishiguro, J. Bacteriol. 164:861-865, 1985) was apparently responsible for the lysis-tolerant phenotypes of these mutants.     

System analysis of Phycomyces light-growth response. Photoreceptor and hypertropic mutants.

The light-growth responses of Phycomyces behavioral mutants, defective in genes madB, madC, and madH, were studied with the sum-of-sinusoids method of system identification. Modified phototropic action spectra of these mutants have indicated that they have altered photoreceptors (P. Galland and E.D. Lipson, 1985, Photochem. Photobiol. 41:331). In the two preceding papers, a kinetic model of the light-growth response system was developed and applied to wild-type frequency kernels at several wavelengths and temperatures. The present mutant studies were conducted at wavelength 477 nm. The log-mean intensity was 6 X 10(-2)W m-2 for the madB and madC night-blind mutants, and 10(-4)W m-2 for the madH hypertropic mutant. The prolonged light-growth responses of the madB and madC mutants are reflected in the reduced dynamic order of their frequency kernels. The linear response of the hypertropic mutant is essentially normal, but its nonlinear behavior shows modified dynamics. The behavior of these mutants can be accounted for by suitable modifications of the parametric model of the system. These modifications together support the hypothesis that an integrated complex mediates sensory transduction in the light responses and other responses of the sporangiophore.     

Influence of amino acid starvation on guanosine 5''-diphosphate 3''-diphosphate basal-level synthesis in Escherichia coli.

We observed that the synthesis of basal-level guanosine 5'-diphosphate 3'-diphosphate (ppGpp) in both relA mutants and relA+ relC strains of Escherichia coli decreased in response to amino acid limitation and that this was accompanied by an increase in ribonucleic acid (RNA) synthesis. Addition of the required amino acid to starved cultures of relaxed bacteria resulted in the resumption of ppGpp synthesis and a concomitant decrease in RNA production. Our results indicate that relA mutants retain a stringent factor-independent ribosomal mechanism for basal-level ppGpp synthesis. They also suggest that in relA+ bacteria, stringent factor-mediated ppGpp synthesis and the production of basal-level ppGpp are mutually exclusive. These findings substantiate the hypothesis that there are two functionally discrete mechanisms for ppGpp synthesis in E. coli. Through these studies we have also obtained new evidence which indicates that ppGpp serves as a modulator of RNA synthesis during balanced growth as well as under conditions of nutritional downshift and starvation.     

An Escherichia coli gene required for bacteriophage P2-lambda interference.

The gene old of bacteriophage P2 is known to (i) cause interference with phage lambda growth; (ii) kill recB- mutants of Escherichia coli after P2 infection; and (iii) determine increased sensitivity of P2 lysogenic cells to X-ray irradiation. In all of these phenomena, inhibition of protein synthesis occurs. We have isolated bacterial mutants, named pin (P2 interference), able to suppress all of the above-mentioned phenomena caused by the old+ gene product and the concurrent protein synthesis inhibition. Pin mutations are recessive, map at 12 min on the E. coli map, and identify a new gene. Satellite bacteriophage P4 does not plate on pin-3 mutant strains and causes cell lethality and protein synthesis inhibition in such mutants. P4 mutants able to grow on pin-3 strains have been isolated.     

Threshold and adaptation in Phycomyces. Their interrelation and regulation by light

The absolute light sensitivity of Phycomyces sporangiophores was determined by analyzing the intensity dependence of the phototropic bending rate and of the light growth and dark growth responses to step changes of the intensity. We found that the different methods give approximately the same results for the wild-type strain, as well as for several behavioral mutants with defects in the genes madA, madB, and madC. A crucial factor in the determination of thresholds is the light intensity at which the strains grow during the 4 d after inoculation and prior to the experiment. When the wild-type strain grows in the dark, its threshold for the bending rate is 10(-9) W X m-2, compared with 2 X 10(-7) W X m-2 when it is grown under continuous illumination. Further, the maximal bending rate is twice as high in dark-grown strains. This phenomenon is further complicated by the fact that the diameter and growth rate of the sporangiophores also depend on the illumination conditions prior to the experiment: light-grown sporangiophores have an increased diameter and an increased growth rate compared with dark-grown ones. Some of the behavioral mutants, however, are indifferent to this form of light control. Another factor that is controlled by the growth conditions is adaptation: the kinetics of dark adaptation are slower in light-grown sporangiophores than in dark-grown ones. We found empirically a positive correlation between the slower dark adaptation constant and the threshold of the bending rate, which shows that the two underlying phenomena are functionally related.     

13C NMR and fluorescence analysis of tryptophan dynamics in wild-type and two single-Trp variants of Escherichia coli thioredoxin.

The rotational motion of tryptophan side chains in oxidized and reduced wild-type (WT) Escherichia coli thioredoxin and in two single-tryptophan variants of E. coli thioredoxin was studied in solution in the temperature range 20-50 degrees C from 13C-NMR relaxation rate measurements at 75.4 and 125.7 MHz and at 20 degrees C from steady-state and time-resolved trp fluorescence anisotropy measurements. Tryptophan enriched with 13C at the delta 1 and epsilon 3 sites of the indole ring was incorporated into WT thioredoxin and into two single-trp mutants, W31F and W28F, in which trp-28 or trp-31 of WT thioredoxin was replaced, respectively, with phenylalanine. The NMR relaxation data were interpreted using the Lipari and Szabo "model-free" approach (G. Lipari and A. Szabo. 1982. J. Amer. Chem. Soc. 104:4546-4559) with trp steady-state anisotropy data included for the variants at 20 degrees C. Values for the correlation time for the overall rotational motion (tau m) from NMR of oxidized and reduced WT thioredoxin at 35 degrees C agree well with those given by Stone et al. (Stone, M. J., K. Chandrasekhar, A. Holmgren, P. E. Wright, and H. J. Dyson. 1993. Biochemistry. 32:426-435) from 15N NMR relaxation rates, and the dependence of tau m on viscosity and temperature was in accord with the Stokes-Einstein relationship. Order parameters (S2) near 1 were obtained for the trp side chains in the WT proteins even at 50 degrees C. A slight increase in the amplitude of motion (decrease in S2) of trp-31, which is near the protein surface, but not of trp-28, which is partially buried in the protein matrix, was observed in reduced relative to oxidized WT thioredoxin. For trp-28 in W31F, order parameters near 1 (S2 > or = 0.8) at 20 degrees C were found, whereas trp-31 in W28F yielded the smallest order parameters (S2 approximately 0.6) of any of the cases. Analysis of time-resolved anisotropy decays in W28F and W31F yielded S2 values in good agreement with NMR, but gave tau m values about 60% smaller. Generally, values of tau e, the effective correlation time for the internal motion, were < or = 60 ps from NMR, whereas somewhat longer times were obtained from fluorescence. The ability of NMR and fluorescence techniques to detect subnanosecond motions in proteins reliably is examined.     

Mixed culture kinetics of stringent and relaxed Escherichia coli cells in glucose-limited chemostat

The mixed culture kinetics of stringent and relaxed Escherichia coli cells were investigated in a glucose-limited chemostat at different dilution rates. Independent of the dilution rate the stringent cells competed out the relaxed cells. But the number of generations necessary for displaying the relaxed cells by the stringent ones increased with increasing dilution rate. The results are discussed as a consequence of the regulatory role of guanosine-5'-diphosphate-3'-diphosphate (ppGpp) which is known to be present at different concentrations in stringent and relaxed cells under conditions of nutrient limitation. In addition, it is postulated that the coefficient of the maintenance metabolism according to PIRT (1965) is slower in stringent cells than in relaxed cells of E. coli.     

The iron-oxygen reconstitution reaction in protein R2-Tyr-177 mutants of mouse ribonucleotide reductase. Epr and electron nuclear double resonance studies on a new transient tryptophan radical.

The ferrous iron/oxygen reconstitution reaction in protein R2 of mouse and Escherichia coli ribonucleotide reductase (RNR) leads to the formation of a stable protein-linked tyrosyl radical and a mu-oxo-bridged diferric iron center, both necessary for enzyme activity. We have studied the reconstitution reaction in three protein R2 mutants Y177W, Y177F, and Y177C of mouse RNR to investigate if other residues at the site of the radical forming Tyr-177 can harbor free radicals. In Y177W we observed for the first time the formation of a tryptophan radical in protein R2 of mouse RNR with a lifetime of several minutes at room temperature. We assign it to an oxidized neutral tryptophan radical on Trp-177, based on selective deuteration and EPR and electron nuclear double resonance spectroscopy in H2O and D2O solution. The reconstitution reaction at 22 degrees C in both Y177F and Y177C leads to the formation of a so-called intermediate X which has previously been assigned to an oxo (hydroxo)-bridged Fe(III)/Fe(IV) cluster. Surprisingly, in both mutants that do not have successor radicals as Trp. in Y177W, this cluster exists on a much longer time scale (several seconds) at room temperature than has been reported for X in E. coli Y122F or native mouse protein R2. All three mouse R2 mutants were enzymatically inactive, indicating that only a tyrosyl radical at position 177 has the capability to take part in the reduction of substrates.