A Pseudomonas stutzeri outer membrane protein inserts copper into N2O reductase

Among a set of frameshift mutagen (ICR-191; Polysciences, Inc.)-induced mutations that confer inability to grow anaerobically with N2O as the sole electron acceptor, one class was found that produced an inactive N2O reductase which lacked copper. All of these mutant strains failed to produce a 61,000-Mr protein located in the outer membrane. This protein, termed NosA, seems not to be responsible for bringing copper into the cell because the mutant strains and their parent were similarly sensitive to the copper content of the growth medium and no intermediate copper concentration in the medium permitted the mutant strains (nosA) to grow anaerobically with N2O as the sole electron acceptor. We conclude that NosA is necessary to insert copper into N2O reductase or to maintain it there.     

Properties of a Pseudomonas stutzeri outer membrane channel-forming protein (NosA) required for production of copper-containing N2O reductase.

A protein (NosA) in the outer membrane of Pseudomonas stutzeri that is required for copper to be inserted into N2O reductase has been extracted and purified to homogeneity. The purified protein could form channels in black lipid bilayers. Like N2O reductase, NosA contained copper and was only made anaerobically. In contrast to N2O reductase, its synthesis was repressed by exogenous copper (but not by Mn, Co, Ni, Zn, or Fe). Also in contrast to N2O reductase, NosA homologs were not immunologically detectable in Pseudomonas aeruginosa, Pseudomonas mendocina, Pseudomonas alcaligenes, or other strains of P. stutzeri.     

Characterization of Tn5 mutants deficient in dissimilatory nitrite reduction in Pseudomonas sp. strain G-179, which contains a copper nitrite reductase.

Tn5 was used to generate mutants that were deficient in the dissimilatory reduction of nitrite for Pseudomonas sp. strain G-179, which contains a copper nitrite reductase. Three types of mutants were isolated. The first type showed a lack of growth on nitrate, nitrite, and nitrous oxide. The second type grew on nitrate and nitrous oxide but not on nitrite (Nir-). The two mutants of this type accumulated nitrite, showed no nitrite reductase activity, and had no detectable nitrite reductase protein bands in a Western blot (immunoblot). Tn5 insertions in these two mutants were clustered in the same region and were within the structural gene for nitrite reductase. The third type of mutant grew on nitrate but not on nitrite or nitrous oxide (N2O). The mutant of this type accumulated significant amounts of nitrite, NO, and N2O during anaerobic growth on nitrate and showed a slower growth rate than the wild type. Diethyldithiocarbamic acid, which inhibited nitrite reductase activity in the wild type, did not affect NO reductase activity, indicating that nitrite reductase did not participate in NO reduction. NO reductase activity in Nir- mutants was lower than that in the wild type when the strains were grown on nitrate but was the same as that in the wild type when the strains were grown on nitrous oxide. These results suggest that the reduction of NO and N2O was carried out by two distinct processes and that mutations affecting nitrite reduction resulted in reduced NO reductase activity following anaerobic growth with nitrate.     

Molecular Basis for Antioxidant Enzymes in Mediating Copper Detoxification in the Nematode Caenorhabditis elegans

Antioxidant enzymes play a major role in defending against oxidative damage by copper. However, few studies have been performed to determine which antioxidant enzymes respond to and are necessary for copper detoxification. In this study, we examined both the activities and mRNA levels of SOD, CAT, and GPX under excessive copper stress in Caenorhabditis elegans, which is a powerful model for toxicity studies. Then, taking advantage of the genetics of this model, we assessed the lethal concentration (LC₅₀) values of copper for related mutant strains. The results showed that the SOD, CAT, and GPX activities were significantly greater in treated groups than in controls. The mRNA levels of sod-3, sod-5, ctl-1, ctl-2, and almost all gpx genes were also significantly greater in treated groups than in controls. Among tested mutants, the sod-5, ctl-1, gpx-3, gpx-4, and gpx-6 variants exhibited hypersensitivity to copper. The strains with SOD or CAT over expression were reduced sensitive to copper. Mutations in daf-2 and age-1, which are involved in the insulin/insulin-like growth factor-1 signaling pathway, result in reduced sensitivity to stress. Here, we showed that LC₅₀ values for copper in daf-2 and age-1 mutants were significantly greater than in N2 worms. However, the LC₅₀ values in daf-16;daf-2 and daf-16;age-1 mutants were significantly reduced than in daf-2 and age-1 mutants, implying that reduced copper sensitivity is influenced by DAF-16-related functioning. SOD, CAT, and GPX activities and the mRNA levels of the associated copper responsive genes were significantly increased in daf-2 and age-1 mutants compared to N2. Additionally, the activities of SOD, CAT, and GPX were greater in these mutants than in N2 when treated with copper. Our results not only support the theory that antioxidant enzymes play an important role in copper detoxification but also identify the response and the genes involved in these processes.     

Phosphatidylserine synthesis required for the maximal tryptophan transport activity in Saccharomyces cerevisiae

Saccharomyces cerevisiae cho1/pss mutants, which are severely impaired in phosphatidylserine (PS) synthesis, do not have detectable amounts of PS in their lipid fractions. Their derivatives with mutations that cause defects in tryptophan synthesis grew poorly in a medium containing 5 micrograms/ml of L-tryptophan, a concentration that met the requirements of tryptophanauxotrophic CHO1/PSS strains. The rates of tryptophan uptake of trp1 cho1/pss mutants were low at low tryptophan concentrations. This defect in the use of tryptophan was restored either by expression of CHO1/PSS or by introduction of a gene encoding tryptophan transporter, TAT1 or TAT2. These results indicate that PS synthesis is required for the maximal tryptophan-transporting activity of S. cerevisiae at low tryptophan concentrations.     

Dissociation between macrophage tumoricidal capacity and suppressive activity: analysis with macrophage-defective mouse strains

Macrophages (M phi diameter) from three mouse strains with genetically distinct M phi diameter deficits (C3H/HeJ, A/J, and P/J) were unable to develop high cytolytic and cytotoxic activity against tumor cells in vitro when exposed to agents (MAF and IFN-beta) that strongly increased the tumoricidal capacity of M phi diameter from nondefective C3H/HeN mice. Nevertheless, the tumoricidal deficits of M phi diameter from the defective strains did not affect their suppressive capacity on Con A-induced lymphoproliferation, nor their ability to react to IFN-beta by decreasing suppressive activity. In fact, natural suppressive activity and IFN-beta-induced changes in the suppression of M phi diameter from C3H/HeJ, A/J, and P/J mice were highly comparable to those of C3H/HeN M phi diameter, thus stressing the dissociation between the mechanisms governing M phi diameter suppression and M phi diameter tumoricidal activity. Analysis of the modulation by MAF and IFN-beta of M phi diameter ability to release the oxygen metabolites O2- and H2O2, molecules possibly involved in the effector mechanism of both M phi diameter cytotoxicity and suppression, revealed a close correlation with the patterns of suppressive activity in both nondefective and defective strains. In contrast, no correlation between the production of oxygen-reactive species and M phi diameter tumoricidal activity was observed. The ability of MAF- and IFN-beta-treated M phi diameter to produce PGE, a molecule of major importance in M phi diameter-mediated suppression and possibly involved also in the regulation of M phi diameter tumoricidal activity, again paralleled M phi diameter suppressive capacity. Thus, the mechanisms controlling M phi diameter antitumor activity appeared to be clearly distinct from those involved in M phi diameter suppression.     

Identification of the lipopolysaccharide core region as the receptor site for a cytotoxin-converting phage, phi CTX, of Pseudomonas aeruginosa.

A temperate phage, phi CTX, is a cytotoxin-converting phage of Pseudomonas aeruginosa. In this study, we characterized the lipopolysaccharide (LPS) structures of phi CTX-resistant mutants derived from phi CTX-sensitive strains. phi CTX infectivity was neutralized by LPS preparations derived from sensitive strains but not by those from resistant strains. phi CTX-resistant mutants had lower-molecular-weight rough (R)-type LPS than the parental strains and lacked the reactivity of some anti-LPS core monoclonal antibodies. Some LPS core components were lacking or significantly decreased in the resistant mutants. These results suggested that a receptor site of the cytotoxin-converting phage phi CTX was the LPS core region and that especially L-rhamnose and D-glucose residues in the outer core were involved in phage binding. The host range of phi CTX was nearly O-serotype dependent, probably because of the diversity of the LPS core structure among P. aeruginosa strains. phi CTX bound to most strains of Homma serotypes A, G, and I but not to strains of serotypes B and E. Furthermore, we found that a genetic locus specifying phi CTX sensitivity (and consequently participating in the biosynthesis of part of the LPS core) existed in or near the locus participating in the determination of O-serotype specificity (somA), which has been mapped between leu-10 and eda-9001. phi CTX, as well as anti-LPS core monoclonal antibodies, will be a good tool for structural characterization of the P. aeruginosa LPS core region.     

The Cu(II)-reductase NADH dehydrogenase-2 of Escherichia coli improves the bacterial growth in extreme copper concentrations and increases the resistance to the damage caused by copper and hydroperoxide

NADH dehydrogenase-2 (NDH-2) from Escherichia coli respiratory chain is a membrane-bound cupric-reductase encoded by ndh gene. Here, we report that the respiratory system of a ndh deficient strain suffered a faster inactivation than that of the parental strain in the presence of tert-butyl hydroperoxide due to endogenous copper. The inactivation was similar for both strains when copper concentration increased in the culture media. Furthermore, several ndh deficient mutants grew less well than the corresponding parental strains in media containing either high or low copper concentrations. A mutant strain complemented with ndh gene almost recovered the parental phenotype for growing in copper limitation or excess. Then, NDH-2 gives the bacteria advantages to diminish the susceptibility of the respiratory chain to damaging effects produced by copper and hydroperoxides and to survive in extreme copper conditions. These results suggest that NDH-2 contributes in the bacterial oxidative protection and in the copper homeostasis.     

High irradiance-induced changes in carotenoid composition and increase in non-photochemical quenching of Chl a fluorescence in primary wheat leaves

The effect of acclimation to high irradiance stress (HIS, 250 Wm-2) in wheat leaves grown under three different irradiances was investigated by HPLC analyses of pigments, chlorophyll a fluorescence parameters and photochemical activities of chloroplasts. Significant loss of beta-carotene was observed compared to the xanthophylls in all three types of seedlings exposed to HIS. However, the effect of HIS on neoxanthin and lutein contents was not significant. The loss of partial electron transport (Asc-DCPIP to MV, PSI activity) was less than the whole chain (H2O to MV) and PS II activity (H2O to DCPIP) suggesting that PS I is less susceptible to HIS compared to PS II. The percent of reductions in Fv/Fm and phi PS II were less in plants grown under high irradiance (HI-1, 30 Wm-2 and HI-2, 45 Wm-2) compared to those grown under moderate irradiance (MI, 15 Wm-2). On the other hand, the percent of NPQ increased more in the leaves of HI plants compared to the leaves of MI when exposed to HIS which suggests a more efficient non-radiative dissipation of excess excitation energy in HI plants compared to MI. These observations suggest that plants grown under relatively high irradiance are better adapted to HIS condition.     

Blocking of bacteriophages phi W and phi 5 with lipopolysaccharides from Escherichia coli K-12 mutants.

In the preceding paper we presented a formula for the composition of lipopolysaccharides (LPS) from Escherichia coli K-12. This formula contains four regions defined from analyses of LPS from four key strains, the parent and mutants which had lost one, two, or three regions of their carbohydrates. Support for the formula was derived from the susceptibility of the key mutants to several bacteriophages. One of these, phage phi W, was found specific for strains which had lost region 4. In this paper we described inactivation in vitro of phage phi W and its host-range mutant phi 5, using LPS devoid of regions 2 to 4. The blocking of phi W was found to require about 0.15 M concentrations of monovalent cations and to be inhibited by low concentrations of calcium and magnesium ions. One particle of phage phi W required 2 times 10-16 g of LPS devoid of region 4 for stoichiometric inactivation. Phage phi 5 was blocked by both heptose-less LPS (devoid of regions 2 to 4) and glucose-less LPS (devoid of regions 3 to 4) but was unaffected by LPS devoid of region 4. LPS from a heptose-less mutant of Salmonella minnesota showed the same inactivation ability as did LPS from heptose-less strains of E. coli K-12. Lipid A was prepared from LPS containing all four regions. Such lipid A was found to inactivate phi 5, whereas both the polysaccharide moiety as well as the intact LPS were without effect. It is suggested that lipid A is part of the receptor site for phage phi 5.     

Regulation of Electron Transport in Photosystems I and II in C3, C3-C4, and C4 Species of Panicum in Response to Changing Irradiance and O2 Levels

Regulation of the quantum yields of linear electron transport and photosystem II photochemistry ([phi]II) with changing irradiance and gas-phase O2 concentration was studied in leaf tissue from Panicum bisulcatum (C3), Panicum milioides (C3-C4), and Panicum antidotale (C4) at 200 [mu]bars of CO2 and 25[deg]C using infrared gas analysis and chlorophyll fluorescence yield measurements. When the O2 level was increased from 14 to 213 mbars at high irradiance, [phi]II increased by as much as 115% in P. bisulcatum but by no more than 17% in P. antidotale. Under the same conditions [phi]II increased to an intermediate degree in P. milioides. Measurements of accumulation of the photooxidized form of the photosystem I reaction center (P700+) based on the light-dependent in vivo absorbance change at 830 nm indicate that the steady-state concentration of P700+ varied in an antiparallel manner with [phi]II when either the irradiance or O2 concentration was changed. Hence, O2-dependent changes in [phi]II were indicative of variations in linear photosynthetic electron transport. These experiments revealed, however, that a significant capacity was retained for in vivo regulation of the apparent quantum yield of photosystem I ([phi]I) independently of [phi]II+ Coordinate regulation of quantum yields of photosystems I and II (expressed as [phi]I:[phi]II in response to changing irradiance and O2 level differed markedly for the C3 and C4 species, and the response for the C3-C4 species most closely resembled that observed for the C4 species. The fraction of total linear electron transport supporting photorespiration at 213 mbars of O2 was negligible in the C4 species and was 13% lower in the C3-C4 species relative to the C3 species as calculated from fluorescence and gas-exchange determinations. At high photon-flux rates and high O2 concentration, the potential benefit to light use for net CO2 uptake arising from lower photorespiration in P. milioides was offset by a reduced capacity for total CO2- and O2-dependent noncyclic electron transport in this species compared with P. bisulcatum.     

The O:34-antigen lipopolysaccharide as an adhesin in Aeromonas hydrophila

Abstract We compared the ability of different Aeromonas hydrophila strains from serogroup O:34 grown at different temperatures to adhere to Hep-2 cells. We found a high level of adhesion when the strains were grown at 20 °C but not when they were grown at 37 °C. We previously described that these strains were able to form the O-antigen lipopolysaccharide when they grow at low temperature but not at high temperature. We also obtained by transposon mutagenesis mutants only devoid of the O-antigen lipopolysaccharide ( rfb mutants), and they showed significantly lower levels of adhesion to Hep-2 cells than the smooth strains. All these results prompted us to conclude that the O-antigen LPS, in these strains, is an important adhesin.     

Rhizobium japonicum mutants that are hypersensitive to repression of H2 uptake by oxygen.

The synthesis of an H2 oxidation system in free-living Rhizobium japonicum wild-type strain SR is repressed by oxygen. Maximal H2 uptake rates were obtained in strain SR after derepression in 11 microM or less dissolved oxygen. Oxygen levels above 45 microM completely repressed H2 uptake in strain SR. Five R. japonicum mutant strains that are hypersensitive to repression or H2 oxidation by oxygen were derived from strain SR. The mutants were obtained by screening H2 uptake-negative mutants that retained the ability to oxidize H2 as bacteroids from soybean nodules. As bacteroids, the five mutant strains were capable of H2 oxidation rates comparable to that of the wild type. The mutants did not take up H2 when derepressed in 22 microM dissolved oxygen, whereas strain SR had substantial activity at this oxygen concentration. The O2 repression of H2 uptake in both the wild-type and two mutant strains, SR174 and SR200, was rapid and was similar to the effect of inhibiting synthesis of H2 uptake system components with rifampin. None of the mutant strains was able to oxidize H2 when the artificial electron acceptors methylene blue or phenazine methosulfate were provided. The mutant strains were not sensitive to killing by oxygen, they took up O2 at rates similar to strain SR, and they did not produce an H2 uptake system that was oxygen labile. Cyclic AMP levels were comparable in strain SR and the five mutant strains after subjection of the cultures to the derepression conditions.     

Glycosylation of a Streptomyces coelicolor A3(2) cell envelope protein is required for infection by bacteriophage phi C31

Mutants of Streptomyces coelicolor A3(2) J1929 (Delta pglY) were isolated that were resistant to the Streptomyces temperate phage phi C31. These strains could be transfected with phi C31 DNA, but could not act as infective centres after exposure to phage. Thus, it was concluded that infection was blocked at the adsorption/DNA injection step. The mutants fell into three classes. Class I mutants were complemented by a gene, SCE87.05, isolated from the cosmid library of S. coelicolor A3(2). The product of SCE87.05 had good overall homology to a Mycobacterium tuberculosis hypothetical protein and regions with similarity to dolichol phosphate-D-mannose:protein O-D-mannosyltransferases. Concanavalin A (ConA) inhibited phi C31 infection of S. coelicolor J1929, and this could be partially reversed by the addition of the sugar, alpha-D-methyl-pyranoside. Moreover, glycosylated proteins from J1929, but not from the class I mutant DT1017, were detected using ConA as a probe in Western blots. Class I and II mutants were sensitive to phi C31hc, a previously isolated phage exhibiting an extended host range phenotype, conferred by h. A phage with the same phenotype, phi DT4002, was isolated independently, and a missense mutation was found in a putative tail gene. It is proposed that the phi C31 receptor is a cell wall glycoprotein, and that the phi C31h mutation compensates for the lack of glycosylation of the receptor.     

Revertible hydrogen uptake-deficient mutants of Rhizobium japonicum.

We have developed mutants of Rhizobium japonicum which are deficient in H2 uptake capacity (Hup-) and which spontaneously revert to the parent type at a frequency consistent with that of a single-point mutation (ca. 1.0 x 10(-09)). The mutagenesis by nitrous acid and the selection of the Hup- phenotype by using penicillin and chemolithotrophy as enrichment for chemolithotrophy-deficient strains are described. Two mutants retain low but reproducible levels of ribulose bisphosphate-dependent CO2 fixation when grown on a low-carbon medium under an atmosphere of 1% O2, 4% H2, 5% CO2, and 90% N2. Neither O2 nor the artificial electron acceptors phenazine methosulfate or methylene blue supported detectable H2 uptake by the free-living Hup- mutants or by their bacteroids. Plant growth experiments under bacteriologically controlled conditions were conducted to assess the mutants' performance as inocula for soybean plants. Plants inoculated with Hup- strains had lower dry weights and contained less total N than did plants inoculated with the parent Hup+ strain. Use of either the Hup- mutants or the Hup+ parent strain as inocula, however, did not significantly affect the acetylene-reducing activity or the fresh weight of nodules. These results, obtained with apparently isogenic lines of H2 uptake-deficient R. japonicum, provide strong support for a beneficial role of the H2 uptake phenotype in legume symbiosis.     

On the nature of suppression by Escherichia coli HF4714

The growth of 2 well characterized bacteriophage phi X174 mutants, phi XGam3 and phi XGms3 (TCG)Ser, was examined on 3 suppressor strains of Escherichia coli: CQ3 Su1, which inserts serine at amber codons; HF4714, a well known amber suppressor that has been used as the permissive strain for a number of phi X mutants; and CQ2 Su3, which inserts tyrosine. The data demonstrate HF4714 inserts glutamine and is Su2, not Su1 as has been reported in several recent papers.     

环境污染物对巨噬细胞的影响及生物监测意义

随着工农业的发展,环境污染问题日趋严重。作为常见的环境污染物,二氧化硫经呼吸道转化为亚硫酸盐能引起多种呼吸系统疾病,并可能有促癌作用;重金属汞能引起中枢神经系统和多种器官损害,同时具有一定的免疫毒性,其危害已引起人们广泛关注。生物监测(bi0H10nitoring)是使用活