Protection from Paraquat-Mediated Photo-Oxidative Stress by Glutathione in Poplar (Populus tremula×P. alba) Plants

Abstract: The sensitivity of hybrid poplar (Populus tremula × P. alba) to oxidative stress mediated by paraquat exposure was analysed with leaf discs from wild-type plants and plants expressing the bacterial cDNA of the enzymes of glutathione synthesis, namely gshI, encoding γ-glutamylcysteine synthetase (ECS), or gshII, encoding glutathione synthetase (GS), both in the cytosol. It was expected that leaf discs containing more than 2-fold elevated glutathione concentrations due to over-expression of ECS are less susceptible to paraquat exposure than wild-type plants and transformants over-expressing GS. However, neither over-expression of GS nor of ECS improved paraquat tolerance of the leaves. This result was surprising, because in wild-type plants reduced paraquat sensitivity of young compared with mature leaves coincided with ca. 30 % higher glutathione contents of the young leaves. Apparently, developmental changes in paraquat sensitivity of poplar leaves are controlled by factors different from glutathione contents. Feeding experiments with glutathione and its metabolic precursor γ-glutamylcysteine (EC) plus gly showed that glutathione can provide protection from paraquat-mediated photo-oxidative stress; but at least ca. 5-fold elevated glutathione levels are required for this effect in poplar leaves. Currently, such high glutathione levels have not been achieved by the application of plant molecular biology techniques. The significance of glutathione for the compensation of photo-oxidative stress is discussed.     

Anoxic function for the Escherichia coli flavohaemoglobin (Hmp): reversible binding of nitric oxide and reduction to nitrous oxide

The flavohaemoglobin Hmp of Escherichia coli is inducible by nitric oxide (NO) and provides protection both aerobically and anaerobically from inhibition of growth by NO and agents that cause nitrosative stress. Here we report rapid kinetic studies of NO binding to Fe(III) Hmp with a second order rate constant of 7.5 x 10(5) M(-1) s(-1) to generate a nitrosyl adduct that was stable anoxically but decayed in the presence of air to reform the Fe(III) protein. NO displaced CO bound to dithionite-reduced Hmp but, remarkably, CO recombined after only 2 s at room temperature indicative of NO reduction and dissociation from the haem. Addition of NO to anoxic NADH-reduced Hmp also generated a nitrosyl species which persisted while NADH was oxidised. These results are consistent with direct demonstration by membrane-inlet mass spectrometry of NO consumption and nitrous oxide production during anoxic incubation of NADH-reduced Hmp. The results demonstrate a new mechanism by which Hmp may eliminate NO under anoxic growth conditions.     

Flavohemoglobin Hmp affords inducible protection for Escherichia coli respiration, catalyzed by cytochromes bo' or bd, from nitric oxide

Respiration of Escherichia coli catalyzed either by cytochrome bo' or bd is sensitive to micromolar extracellular NO; extensive, transient inhibition of respiration increases as dissolved oxygen tension in the medium decreases. At low oxygen concentrations (25-33 microm), the duration of inhibition of respiration by 9 microm NO is increased by mutation of either oxidase. Respiration of an hmp mutant defective in flavohemoglobin (Hmp) synthesis is extremely NO-sensitive (I(50) about 0.8 microm); conversely, cells pre-grown with sodium nitroprusside or overexpressing plasmid-borne hmp(+) are insensitive to 60 microm NO and have elevated levels of immunologically detectable Hmp. Purified Hmp consumes O(2) at a rate that is instantaneously and extensively (>10-fold) stimulated by NO due to NO oxygenase activity but, in the absence of NO, Hmp does not contribute measurably to cell oxygen consumption. Cyanide binds to Hmp (K(d) 3 microm). Concentrations of KCN (100 microm) that do not significantly inhibit cell respiration markedly suppress the protection of respiration from NO afforded by Hmp and abolish NO oxygenase activity of purified Hmp. The results demonstrate the role of Hmp in protecting respiration from NO stress and are discussed in relation to the energy metabolism of E. coli in natural O(2)-depleted environments.     

An organic solvent-stable alkaline protease from Pseudomonas aeruginosa strain K: Enzyme purification and characterization

The organic solvent-tolerant strain K protease was purified to homogeneity by ammonium sulphate precipitation and anion exchange chromatography with 124-fold increase in specific activity. The molecular mass of the purified enzyme as revealed by SDS-PAGE electrophoresis is 51,000 Da. The strain K protease was an alkaline metalloprotease with an optimum pH and temperature of 10 and 70 °C, respectively. The enzyme showed stability and activation in the presence of organic solvents with log P_a/w values equal or more than 4.0. After 14 days of incubation, the purified protease was activated 1.11, 1.82, 1.50, 1.75 and 1.80 times in 1-decanol, isooctane, decane, dodecane and hexadecane, respectively.     

Pseudomonas aeruginosa deficient in 8-oxodeoxyguanine repair system shows a high frequency of resistance to ciprofloxacin

The 8-oxodeoxyguanine (8-oxodG) repair system participates in the prevention and correction of mutations generated by oxidative DNA damage in prokaryotes and eukaryotes. In this study, we report that Pseudomonas aeruginosa strains deficient in this repair mechanism by inactivation of the mutT, mutM and mutY genes generate a high frequency of cells resistant to the antibiotic ciprofloxacin. In the mutT strain, the increase in ciprofloxacin resistance achieved at threefold minimal inhibitory concentration was about 1600-fold over the wild-type (WT) level, similar to the frequency achieved by the mismatch repair-deficient mutS strain. Molecular analysis of WT, mutT and mutY clones resistant to ciprofloxacin indicated that the nfxB gene was mutated in the majority of the cases, while mutS- derived resistant clones were mainly mutated in gyrA and parC genes. Cell viability analysis after treatment with paraquat or hydrogen peroxide indicated that 8-oxodG repair-deficient strains were considerably more susceptible to oxidative stress than the parental strain. Finally, it is shown that the ciprofloxacin resistance frequency of WT and repair-deficient strains increased significantly after cell exposure to paraquat. Thus, oxidative stress is strongly implicated in the emergence of ciprofloxacin-resistant mutants in P. aeruginosa , and the 8-oxodG repair pathway plays an important role in the prevention of these mutations.     

Morganella morganii urease: purification, characterization, and isolation of gene sequences

Morganella morganii, a very common cause of catheter-associated bacteriuria, was previously classified with the genus Proteus on the basis of urease production. M. morganii constitutively synthesizes a urease distinct from that of other uropathogens. The enzyme, purified 175-fold by passage through DEAE-Sepharose, phenyl-Sepharose, Mono-Q, and Superose 6 chromatography resins, was found to have a native molecular size of 590 kilodaltons and was composed of three distinct subunits with apparent molecular sizes of 63, 15, and 6 kilodaltons, respectively. Amino-terminal analysis of the subunit polypeptides revealed a high degree of conservation of amino acid sequence between jack bean and Proteus mirabilis ureases. Km for urea equalled 0.8 mM. Antiserum prepared against purified enzyme inhibited activity by 43% at a 1:2 dilution after 1 h of incubation. All urease activity was immunoprecipitated from cytosol by a 1:16 dilution. Antiserum did not precipitate ureases of other species except for one Providencia rettgeri strain but did recognize the large subunits of ureases of Providencia and Proteus species on Western blots (immunoblots). Thirteen urease-positive cosmid clones of Morganella chromosomal DNA shared a 3.5-kilobase (kb) BamHI fragment. Urease gene sequences were localized to a 7.1-kb EcoRI-SalI fragment. Tn5 mutagenesis revealed that between 3.3 and 6.6 kb of DNA were necessary for enzyme activity. A Morganella urease DNA probe did not hybridize with gene sequences of other species tested. Morganella urease antiserum recognized identical subunit polypeptides on Western blots of cytosol from the wild-type strain and Escherichia coli bearing the recombinant clone which corresponded to those seen in denatured urease. Although the wild-type strain and recombinant clone produced equal amounts of urease protein, the clone produced less than 1% of the enzyme activity of the wild-type strain.     

Isolation and properties of extracellular lipase of native (B-10) and mutant (M-1) Serratia marcescens strains

The cultivation conditions of wild-type strain V-10 and mutant strain M-1 (overproducer of endonuclease and chitinase) of Serratia marcescens optimal for extracellular lipase biosynthesis were determined. The strain V-10 displayed the maximal lipase yield (840 AU/ml) after 10-12 h of cultivation; the strain M-1 (33 AU/ml), after 25-30 h. The data showed that extracellular lipases from V-10 and M-1 can be precipitated in a weakly acid medium (pH 5.0 and 4.5, respectively). This property was used to obtain partially purified lipase preparations. The effect of the ionic composition of the reaction mixture on the activities of these enzymatic preparations was studied. Both preparations displayed highest activities in weakly alkaline media (pH 8.0); however, the wild-type strain lipase displayed a higher thermal stability and stability at alkaline pH compared with M-1 lipase. Both lipases were activated by various anionic and nonionic surfactants and inactive in the presence of cetyltrimethylammonium bromide.     

Purification and identification of an FMN-dependent NAD(P)H azoreductase from Enterococcus faecalis

Azoreductases reduce the azo bond (N=N) in azo dyes to produce colorless amine products. Crude cell extracts from Enterococcus faecalis have been shown to utilize both NADH and NADPH as electron donors for azo dye reduction. An azoreductase was purified from E. faecalis by hydrophobic, anion exchange and affinity chromatography. The azoreductase activity of the purified preparation was tested on a polyacrylamide gel after electrophoresis under native conditions and the protein that decolorized the azo dye (Methyl Red) with both NADH and NADPH was identified by mass spectrometry to be AzoA. Previously, the heterologously expressed and purified AzoA was shown to utilize NADH only for the reduction of Methyl Red. However, AzoA purified from the wild-type organism was shown to utilize both coenzymes but with more than 180-fold preference for NADH over NADPH as an electron donor to reduce Methyl Red. Also, its specific activity was more than 150-fold higher than the previous study on AzoAwhen NADH was used as the electron donor. The catalytic efficiency for Methyl Red reduction by AzoA from E. faecalis was several orders of magnitude higher than other azoreductases that were purified from a heterologous source.     

Mutant Strain of Bradyrhizobium japonicum with Increased Symbiotic N(2) Fixation Rates and Altered Mo Metabolism Properties

Mutant strains of Bradyrhizobium japonicum that required higher levels of molybdate than the wild-type strain for growth on NO(3)-containing medium were obtained after transposon Tn5 mutagenesis of the wild-type strain. The mutant strains expressed more than fivefold-greater nitrate reductase activities in the range of 0.1 to 1.0 mM added molybdate compared with activities expressed upon incubation in non-Mo-supplemented medium, whereas the nitrate reductase activity of the wild-type strain (JH) was not markedly influenced by Mo supplementation. In free-living culture, mutant strains JH310 and JH359 expressed substantial nitrogenase activity, even in medium treated to remove molybdate, and nitrogenase activity was influenced little by Mo supplementation, whereas the wild-type strain required 100 nM added Mo for highest nitrogenase activity. Double-reciprocal plots of Mo uptake rates versus Mo concentration showed that both bacteroids and free-living cells of mutant strain JH359 had about the same affinity for Mo as did the parent strain. Bacteroids of both the mutants and the wild type also exhibited similar Mo accumulation rates over a 9-min period under very-low-Mo (4 nM) conditions. Nitrogenase activities for strain JH359 and for the wild-type strain in free-living culture were both strongly inhibited by tungsten; thus, the nitrogenase activities of both strains are probably the result of a "conventional" Mo-containing nitrogenase. Soybeans inoculated with strain JH359 and grown under either Mo-supplemented or Mo-deficient conditions had greater specific acetylene reduction rates and significantly greater plant fresh weight than those inoculated with the wild-type strain. Under Mo-deficient conditions, the acetylene reduction rates and plant fresh weights were up to 35 and 58% greater, respectively, for mutant-nodulated plants compared with wild-type-strain-nodulated plants.     

Effects of paraquat on Escherichia coli: differences between B and K-12 strains.

Escherichia coli B and K-12 are equally susceptible to the bacteriostatic effects of aerobic paraquat, but they differed strikingly when the lethality of paraquat was evaluated. E. coli B suffered an apparent loss of viability when briefly exposed to paraquat, whereas E. coli K-12 did not. This difference depended on the ability of the B strain, but not the K-12 strain, to retain internalized paraquat; the B strain was killed on aerobic tryptic soy-yeast extract plates during the incubation which preceded the counting of colonies. This difference in retention of paraquat between strains was demonstrated by delayed loss of viability, by growth inhibition, and by cyanide-resistant respiration after brief exposure to paraquat, washing, and testing in fresh medium. This difference was also shown by using [14C]paraquat. This previously unrecognized difference between E. coli B and K-12 has been the cause of apparently contradictory reports and should lead to some reevaluation of the pertinent literature.     

Flavohemoglobin Hmp,but not its individual domains,confers protection from respiratory inhibition by nitric oxide in Escherichia coli

Escherichia coli possesses a two-domain flavohemoglobin, Hmp, implicated in nitric oxide (NO) detoxification. To determine the contribution of each domain of Hmp toward NO detoxification, we genetically engineered the Hmp protein and separately expressed the heme (HD) and the flavin (FD) domains in a defined hmp mutant. Expression of each domain was confirmed by Western blot analysis. CO-difference spectra showed that the HD of Hmp can bind CO, but the CO adduct showed a slightly blue-shifted peak. Overexpression of the HD resulted in an improvement of growth to a similar extent to that observed with the Vitreoscilla hemeonly globin Vgb, whereas the FD alone did not improve growth. Viability of the hmp mutant in the presence of lethal concentrations of sodium nitroprusside was increased (to 30% survival after 2 h in 5 mM sodium nitroprusside) by overexpressing Vgb or the HD. However, maximal protection was provided only by holo-Hmp (75% survival under the same conditions). Cellular respiration of the hmp mutant was instantaneously inhibited in the presence of 13.5 microM NO but remained insensitive to NO inhibition when these cells overexpressed Hmp. When HD or FD was expressed separately, no significant protection was observed. By contrast, overexpression of Vgb provided partial protection from NO respiratory inhibition. Our results suggest that, despite the homology between the HD from Hmp and Vgb (45% identity), their roles seem to be quite distinct.     

Identification of the gene appA for the acid phosphatase (pH optimum 2.5) of Escherichia coli

Summary A strain of Escherichia coli exhibiting reduced activity of the periplasmic enzyme acid phosphoanhydride phosphohydrolase (pH 2.5 acid phosphatase) was isolated. The mutation designated appA1 was located at 22.5 min on the E. coli genetic map. Acid phosphatase purified from an appA ^–transductant showed less than ten percent of the specific activity of an isogenic appA ⁺strain. The mutant enzyme was highly thermolabile and its K_m for paranitrophenyl phosphate was increased about 20-fold. The mutant protein cross-reacted with antibody to the wild-type enzyme and had the same molecular weight and concentration in extracts as the wild-type enzyme. These findings strongly suggest that appA is the structural gene of the acid phosphatase.Abbreviations PNPP paranitrophenyl phosphate - cAMP 3-5-cyclic adenosine monophosphate - Nitrosoguanidine N-methyl-N'-nitro-N-nitrosoguanidine - TCY tetracycline - KAN kanamycin - STR streptomycin     

Role of sigma(B) in heat, ethanol, acid, and oxidative stress resistance and during carbon starvation in Listeria monocytogenes

To determine the contribution of sigma B (sigma(B)) to survival of stationary-phase Listeria monocytogenes cells following exposure to environmental stresses, we compared the viability of strain 10403S with that of an isogenic nonpolar sigB null mutant strain after exposure to heat (50 degrees C), ethanol (16.5%), or acid (pH 2.5). Strain viabilities were also determined under the same conditions in cultures that had been previously exposed to sublethal levels of the same stresses (45 degrees C, 5% ethanol, or pH 4.5). The DeltasigB and wild-type strains had similar viabilities following exposure to ethanol and heat, but the DeltasigB strain was almost 10,000-fold more susceptible to lethal acid stress than its parent strain. However, a 1-h preexposure to pH 4.5 yielded a 1,000-fold improvement in viability for the DeltasigB strain. These results suggest the existence in L. monocytogenes of both a sigma(B)-dependent mechanism and a pH-dependent mechanism for acid resistance in the stationary phase. sigma(B) contributed to resistance to both oxidative stress and carbon starvation in L. monocytogenes. The DeltasigB strain was 100-fold more sensitive to 13.8 mM cumene hydroperoxide than the wild-type strain. Following glucose depletion, the DeltasigB strain lost viability more rapidly than the parent strain. sigma(B) contributions to viability during carbon starvation and to acid resistance and oxidative stress resistance support the hypothesis that sigma(B) plays a role in protecting L. monocytogenes against environmental adversities.     

Tripolyphosphatase from Methanobacterium thermoautotrophicum (strain ΔH)

Abstract A low-melecular-mass polyphosphatase (tripolyphosphatase, PPPi) from the archaeon Methanobacterium thermoautotrophicum (strain ΔH) was purified 340-fold and characterized. The tripolyphosphatase showed an optimal activity at pH 9.7 (at 60°C). Though the highest activities were measured with tripolyphosphate, tetrapolyphosphate (57%), phosphate glass type 5 (41%) and phosphate glass type 15 (20%) could also be used as substrates. However, tripolyphosphatase was unable to use pyrophosphate. The enzyme was dependent on the presence of Mg²⁺. In the presence of 2 mM PPPi, an optimal activity was found at 6 mM Mg²⁺. The K _m for PPPi was estimated at 0.37 mM. In addition, the enzyme was inhibited by KF (50% at 6 mM) and appeared to be very heat stable: after an incubation of 2 h at 85°C about 85% of the activity was still present.     

Cooperative conformational changes in a G-protein-coupled receptor dimer, the leukotriene B(4) receptor BLT1

We have used an isolated receptor, the leukotriene B(4) receptor BLT1, to analyze the mechanism of receptor activation in a G-protein-coupled receptor dimer. The isolated receptor is essentially a dimer whether the agonist is present or not, provided the detergent used stabilizes the inactive dimeric assembly. We have produced a receptor mutant where Cys(97) in the third transmembrane domain has been replaced by a serine. This mutation leads to an approximately 100-fold decrease in the affinity for the agonist. 5-Hydroxytryptophan has then been introduced at position 234 in the C97A mutant sixth transmembrane domain. Agonist binding to the labeled receptor is associated with variations in the fluorescence properties of 5-hydroxytryptophan due to specific agonist-induced conformational changes. The C97A mutant labeled with 5-hydroxytryptophan has then been associated with a wild-type receptor in a dimeric complex that has been subsequently purified. The purified complex activates its G-protein partner in a similar manner as the wild-type homodimer. Due to the difference in the affinity for the agonist between the wild-type and mutant protomers in this dimer, we have been able to reach a state where one of the protomers, the mutant, is in its unliganded state, whereas the other, the wild type, is loaded with the agonist. We show that agonist binding to the wild-type receptor induces specific changes in the conformation of the unliganded protomer, as evidenced by the variations in the emission of the 5-hydroxytryptophan residue in the mutant receptor. These data provide a direct demonstration for agonist-induced cooperative conformational changes in a GPCR dimer.