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1.
Nutrient Shock and Incubation Atmosphere Influence Recovery of Culturable Helicobacter pylori from Water
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Three different media—Columbia agar, Wilkins-Chalgren agar, and Helicobacter pylori special peptone agar—were prepared in a diluted version and compared to the standard medium formulation in order to study a possible nutrient shock effect observed when recovering H. pylori from water by counting the number of CFU. This same parameter was subsequently used to evaluate the influence of the incubation atmosphere by using a modular atmosphere-controlled system to provide different atmospheres and by employing an established gas generation kit as a control. Both a low nutrient content of the media and a rapidly achieved microaerophilic incubation atmosphere proved to increase the numbers of environment-stressed H. pylori organisms recovered. An atmosphere of 5% CO2, 5% O2, and 3% H2 is recommended, although other atmospheres with a low oxygen concentration are also acceptable. Besides highlighting and assessing the importance of several factors in the culturability of H. pylori, this paper demonstrates the potential ability to develop an optimized technique for recovery of this pathogen from water. 相似文献
2.
CO2 fixation by a hydrogen-oxidizing bacterium, Cupriavidus necator, was evaluated in a packed bed bioreactor under a constant flow rate of gas mixtures (H2, O2, CO2). The overall energy efficiency depends on the efficiencies of CO2 fixation into carbohydrate and the reduced carbon into biomass and bioproducts, respectively. The efficiencies varied with the limiting gas substrate. Under O2 limitation, the efficiency (20–30%) of CO2 fixation increased with time and was higher than the overall efficiency (12–18%). Under H2 limitation, the efficiency of CO2 fixation declined with time while the biomass yield was quite similar to that under O2 limitation. A cellular metabolic model was suggested for the lithoautotrophic growth of C. necator, including CO2 fixation into carbohydrate followed by the main metabolic pathway of reduced carbon. Under CO2 limitation, most H2 energy was wasted, resulting in a very low biomass yield. Under a dual limitation of O2 and nitrogen, biosynthesis of poly(3-hydroxybutyrate) was triggered, and the energy efficiency or yield of biopolyester was lower than those of microbial cell mass. Compared with a green microalga Neochloris oleoabundans that produces lipid under nutrient limitation, C. necator exhibited a much higher (3–6 times) energy efficiency in producing biomass and bioproducts from CO2. 相似文献
3.
Weiss E 《Journal of bacteriology》1970,101(1):133-137
Two strains of Saccharomycopsis guttulata, JB-1 and JB-3, isolated from stomach contents of domestic rabbits, were grown under different gas phases, and their growth rates were compared. Strain JB-1 grew exponentially at a maximal growth rate under a continuous gas phase of 15% CO2, 2% O2 in nitrogen. High cell yields with low cell granulation were obtained. The growth rates were almost the same between oxygen concentrations of 0.25 and 20% at 15% CO2. Poor growth and early cell granulation occurred in the absence of oxygen at 15% CO2. Growth increased at 2% O2 in direct proportion to the carbon dioxide concentration up to 10 to 15% CO2. A very high carbon dioxide content (e.g. 98%) was somewhat inhibitory. Cell granulation always occurred during the maximal stationary phase in media at pH 4, but was relatively slight at pH 5.6 or higher. Strain JB-3 responded to various gas phases in a similar manner except that it grew slowly in the absence of oxygen at 15% CO2 (pH 4). The effect of an optimal gas phase on the growth of strain JB-1 was examined in relation to other environmental conditions. In the presence of 15% CO2, 2% O2, this strain grew exponentially in yeast autolysate-Proteose Peptone-glucose medium at 37 C at pH 2, 4, and 5.6 at approximately the same rate; the growth rate was somewhat lower at pH 6.2. Under similar conditions, strain JB-1 grew at 30 C and pH 4 at one-sixth its maximal growth rate. Cell granulation was greatly reduced at this temperature. With adequate CO2 strain JB-1 also grew at a reduced rate in a yeast autolysate medium previously reported not to support growth. Results indicate that continuous gassing with an optimal gas phase increases the growth rate to the extent that the growth rate surpasses the death rate by a significant margin; as a result, granulated cells can be avoided almost entirely in the log phase. 相似文献
4.
Nitrogen Fixation and Hydrogen Metabolism in Relation to the Dissolved Oxygen Tension in Chemostat Cultures of the Wild Type and a Hydrogenase-Negative Mutant of Azorhizobium caulinodans 总被引:1,自引:0,他引:1
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Fred C. Boogerd Marijke M. A. Ferdinandy-van Vlerken Crispen Mawadza Annemieke F. Pronk Adriaan H. Stouthamer Henk W. van Verseveld 《Applied microbiology》1994,60(6):1859-1866
Both the wild type and an isogenic hydrogenase-negative mutant of Azorhizobium caulinodans growing ex planta on N2 as the N source were studied in succinate-limited steady-state chemostat cultures under 0.2 to 3.0% dissolved O2 tension. Production or consumption of O2, H2, and CO2 was measured with an on-line-connected mass spectrometer. In the range of 0.2 to 3.0%, growth of both the wild type and the mutant was equally dependent on the dissolved O2 tension: the growth yield decreased, and the specific O2 consumption and CO2 production increased. A similar dependency on the dissolved O2 tension was found for the mutant with 2.5% H2 in the influent gas. The H2/N2 ratio (moles of H2 evolved per mole of N2 consumed via nitrogenase) of the mutant, growing with or without 2.5% H2, increased with increasing dissolved O2 tensions. This increase in the H2/N2 ratio was small but significant. The dependencies of the ATP/N2 ratio (moles of ATP consumed per mole of N2 fixed) and the ATP/2e- ratio [moles of ATP consumed per mole of electron pairs transferred from NAD(P)H to nitrogenase] on the dissolved O2 tension were estimated. These dependencies were interpreted in terms of the physiological concepts of respiratory protection and autoprotection. 相似文献
5.
Extracellular DNA (eDNA) is an important component of the biofilm matrix produced by many bacteria. In general, the release of eDNA is associated with the activity of muralytic enzymes leading to obvious cell lysis. In the Gram-positive oral commensal Streptococcus gordonii, eDNA release is dependent on pyruvate oxidase generated hydrogen peroxide (H2O2). Addition of H2O2 to cells grown under conditions non-permissive for H2O2 production causes eDNA release. Furthermore, eDNA release is maximal under aerobic growth conditions known to induce pyruvate oxidase gene expression and H2O2 production. Obvious cell lysis, however, does not occur. Two enzymes have been recently associated with eDNA release in S. gordonii. The autolysin AtlS and the competence regulated murein hydrolase LytF. In the present report, we investigated the role of both proteins in the H2O2 dependent eDNA release process. Single and double mutants in the respective genes for LytF and AtlS released less eDNA under normal growth conditions, but the AtlS mutant was still inducible for eDNA release by external H2O2. Moreover, we showed that the AtlS mutation interfered with the ability of S. gordonii to produce eDNA release inducing amounts of H2O2. Our data support a role of LytF in the H2O2 eDNA dependent release of S. gordonii as part of the competence stress pathway responding to oxidative stress. 相似文献
6.
Carbon Dioxide-Induced Oscillations in Fluorescence and Photosynthesis: Role of Thylakoid Membrane Energization in Regulation of Photosystem II Activity 总被引:4,自引:4,他引:0
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The response of CO2 fixation to a sudden increase in ambient CO2 concentration has been investigated in intact leaf tissue from spinach (Spinacia oleracea) using a dual channel infrared gas analyzer. Simultaneous with these measurements, changes in fluorescence emission associated with a weak, modulated measuring beam were recorded. Application of brief (2-3 seconds) dark intervals enabled estimation of the dark fluorescence level (Fo) under both steady state and transient conditions. The degree of suppression of Fo level fluorescence in the light was strongly correlated with nonphotochemical quenching under all conditions. During CO2-induced oscillations in photosynthesis under 2% O2 the changes in nonphotochemical quenching anticipate changes in the rate of uptake of CO2. At such low levels of O2 and constant illumination, changes in the relative quantum efficiency of open photosystem II units were estimated as the ratio of the rate of CO2 uptake and the photochemical quenching coefficient. Under the same conditions the relative quantum efficiency of photosystem II was found to vary inversely with the degree of nonphotochemical quenching. The relationship between changes in the rate of CO2 uptake: photochemical quenching coefficient and nonphotochemical quenching was altered somewhat when the same experiment was conducted under 20% O2. The results suggest that electron transport coupled to reduction of O2 occurs to varying degrees with time during oscillations, especially when ambient O2 concentrations are high. 相似文献
7.
Gas Exchange in the Filamentous Cyanobacterium Nostoc punctiforme Strain ATCC 29133 and Its Hydrogenase-Deficient Mutant Strain NHM5 总被引:1,自引:0,他引:1
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Nostoc punctiforme ATCC 29133 is a nitrogen-fixing, heterocystous cyanobacterium of symbiotic origin. During nitrogen fixation, it produces molecular hydrogen (H2), which is recaptured by an uptake hydrogenase. Gas exchange in cultures of N. punctiforme ATCC 29133 and its hydrogenase-free mutant strain NHM5 was studied. Exchange of O2, CO2, N2, and H2 was followed simultaneously with a mass spectrometer in cultures grown under nitrogen-fixing conditions. Isotopic tracing was used to separate evolution and uptake of CO2 and O2. The amount of H2 produced per molecule of N2 fixed was found to vary with light conditions, high light giving a greater increase in H2 production than N2 fixation. The ratio under low light and high light was approximately 1.4 and 6.1 molecules of H2 produced per molecule of N2 fixed, respectively. Incubation under high light for a longer time, until the culture was depleted of CO2, caused a decrease in the nitrogen fixation rate. At the same time, hydrogen production in the hydrogenase-deficient strain was increased from an initial rate of approximately 6 μmol (mg of chlorophyll a)−1 h−1 to 9 μmol (mg of chlorophyll a)−1 h−1 after about 50 min. A light-stimulated hydrogen-deuterium exchange activity stemming from the nitrogenase was observed in the two strains. The present findings are important for understanding this nitrogenase-based system, aiming at photobiological hydrogen production, as we have identified the conditions under which the energy flow through the nitrogenase can be directed towards hydrogen production rather than nitrogen fixation. 相似文献
8.
E. G. NISBET N. V. GRASSINEAU C. J. HOWE P. I. ABELL M. REGELOUS R. E. R. NISBET 《Geobiology》2007,5(4):311-335
The evolutionary history of oxygenesis is controversial. Form I of ribulose 1,5‐bisphosphate carboxylase/oxygenase (Rubisco) in oxygen‐tolerant organisms both enables them to carry out oxygenic extraction of carbon from air and enables the competitive process of photorespiration. Carbon isotopic evidence is presented from ~2.9 Ga stromatolites from Steep Rock, Ontario, Canada, ~2.9 Ga stromatolites from Mushandike, Zimbabwe, and ~2.7 Ga stromatolites in the Belingwe belt, Zimbabwe. The data imply that in all three localities the reef‐building autotrophs included organisms using Form I Rubisco. This inference, though not conclusive, is supported by other geochemical evidence that these stromatolites formed in oxic conditions. Collectively, the implication is that oxygenic photosynthesizers first appeared ~2.9 Ga ago, and were abundant 2.7–2.65 Ga ago. Rubisco specificity (its preference for CO2 over O2) and compensation constraints (the limits on carbon fixation) may explain the paradox that despite the inferred evolution of oxygenesis 2.9 Ga ago, the Late Archaean air was anoxic. The atmospheric CO2:O2 ratio, and hence greenhouse warming, may reflect Form I Rubisco's specificity for CO2 over O2. The system may be bistable under the warming Sun, with liquid oceans occurring in either anoxic (H2O with abundant CH4 plus CO2) or oxic (H2O with more abundant CO2, but little CH4) greenhouse states. Transition between the two states would involve catastrophic remaking of the biosphere. Build‐up of a very high atmospheric inventory of CO2 in the 2.3 Ga glaciation may have allowed the atmosphere to move up the CO2 compensation line to reach stability in an oxygen‐rich system. Since then, Form I Rubisco specificity and consequent compensation limits may have maintained the long‐term atmospheric disproportion between O2 and CO2, which is now close to both CO2 and O2 compensation barriers. 相似文献
9.
Photosynthetic CO2 and O2 exchange was studied in two moss species, Hypnum cupressiforme Hedw. and Dicranum scoparium Hedw. Most experiments were made during steady state of photosynthesis, using 18O2 to trace O2 uptake. In standard experimental conditions (photoperiod 12 h, 135 micromoles photons per square meter per second, 18°C, 330 microliters per liter CO2, 21% O2) the net photosynthetic rate was around 40 micromoles CO2 per gram dry weight per hour in H. cupressiforme and 50 micromoles CO2 per gram dry weight per hour in D. scoparium. The CO2 compensation point lay between 45 and 55 microliters per liter CO2 and the enhancement of net photosynthesis by 3% O2versus 21% O2 was 40 to 45%. The ratio of O2 uptake to net photosynthesis was 0.8 to 0.9 irrespective of the light intensity. The response of net photosynthesis to CO2 showed a high apparent Km (CO2) even in nonsaturating light. On the other hand, O2 uptake in standard conditions was not far from saturation. It could be enhanced by only 25% by increasing the O2 concentration (saturating level as low as 30% O2), and by 65% by decreasing the CO2 concentration to the compensation point. Although O2 is a competitive inhibitor of CO2 uptake it could not replace CO2 completely as an electron acceptor, and electron flow, expressed as gross O2 production, was inhibited by both high O2 and low CO2 levels. At high CO2, O2 uptake was 70% lower than the maximum at the CO2 compensation point. The remaining activity (30%) can be attributed to dark respiration and the Mehler reaction. 相似文献
10.
A versatile plasmid shuttle vector system was constructed, which is useful for genetic complementation of Helicobacter pylori strains or mutants with cloned genes of homologous or heterologous origin. The individual plasmid vectors consist of the minimal essential genetic elements, including an origin of replication for Escherichia coli, a H. pylori-specific replicon originally identified on a small cryptic H. pylori plasmid, an oriT sequence and a multiple cloning site. Shuttle plasmid pHel2 carries a chloramphenicol resistance cassette (cat GC) and pHel3 contains a kanamycin resistance gene (aphA-3) as the selectable marker; both are functional in E. coli and H. pylori. The shuttle plasmids were introduced into the H. pylori strain P1 by natural transformation. A efficiency of 7.0?×?10?7 and 4.7?×?10?7 transformants per viable recipient was achieved with pHel2 and pHel3, respectively, and both vectors showed stable, autonomous replication in H. pylori. An approximately 100-fold higher H. pylori transformation rate was obtained when the shuttle vectors for transformation were isolated from the homologous H. pylori strain, rather than E. coli, indicating that DNA restriction and modification mechanisms play a crucial role in plasmid transformation. Interestingly, both shuttle vectors could also be mobilized efficiently from E. coli into different H.?pylori recipients, with pHel2 showing an efficiency of 2.0?×?10?5 transconjugants per viable H. pylori P1 recipient. Thus, DNA restriction seems to be strongly reduced or absent during conjugal transfer. The functional complementation of a recA-deficient H. pylori mutant by the cloned H. pylorirecA + gene, and the expression of the heterologous green fluorescent protein (GFP) in H.?pylori demonstrate the general usefulness of?this system, which will significantly facilitate the molecular analysis of H. pylori virulence factors in the future. 相似文献
11.
Methanol and the O-methyl group of vanillate did not support the growth of Clostridium formicoaceticum in defined medium under CO2-limited conditions; however, they were growth supportive when fumarate was provided concomitantly. Fumarate alone was not growth supportive under these conditions. Fumarate reduction (dissimilation) to succinate was the predominant electron-accepting, energy-conserving process for methanol-derived reductant under CO2-limited conditions. However, when both reductant sinks, i.e., fumarate and CO2, were available, reductant was redirected towards CO2 in defined medium. In contrast, in undefined medium with both reductant sinks available, C. formicoaceticum simultaneously engaged fumarate dismutation and the concomitant usage of CO2 and fumarate as reductant sinks. With Clostridium aceticum, fumarate also substituted for CO2, and H2 became growth supportive under CO2-limited conditions. Fumarate dissimilation was the predominant electron-accepting process under CO2-limited conditions; however, when both reductant sinks were available, H2-derived reductant was routed towards CO2, indicating that acetogenesis was the preferred electron-accepting process when reductant flow originated from H2. Collectively, these findings indicate that fumarate dissimilation, not dismutation, is selectively used under certain conditions and that such usage of fumarate is subject to complex regulation. 相似文献
12.
Abstract. Elongation responses of intact coleoptiles of rice (Oryza sativa L. ev. Sasanishiki) explants to various gases were examined under submerged conditions in continuously flowing gas-saturated incubation media. Reduced O2 tension (hypoxia). CO2 and especially C2H4 significantly stimulated coleoptile elongation; the optimal concentrations of O2, CO2 and C2H4 when applied singly were 0.07 m3 m-3, 0.10 m3 m-3, and 3 cm3, respectively. However, in addition to these gases other as yet unknown factors were involved in the enhanced elongation of rice coleoptiles under water. The actions of CO2 and C2H4, unlike that of hypoxia, were accompanied by increases in dry weight of the coleoptiles. The effect of C2H4 occurred independently of O2 concentrations, whereas that of CO2 occurred above 0.08 m3 m-3O2. Maximum elongation of rice coleoptiles under submerged conditions was obtained when the flowing medium was saturated with a gas mixture containing 0.10 m3 m-3 O2, 0.10 m3 m-3 CO2 and 10 cm3 m-3 C2H4, greatly surpassing elongation in static media. However, elongation in static media was greater than that in a closed atmosphere. The intercellular C2H4 concentration in explants growing in static media was higher than that in a closed atmosphere. These results showed that the coleoptile elongation of rice seedlings under water may be regulated by the accumulation of CO2 and C2H4 in and around the seedlings under hypoxic conditions. 相似文献
13.
Regulation of growth in rice seedlings 总被引:1,自引:0,他引:1
Etiolated rice seedlings (Oryza sativa L.) exhibited marked morphological differences when grown in sealed containers or in containers through which air was passed continuously. Enhancement of coleoptile and mesocotyl growth and inhibition of leaf and root growth in the sealed containers (“enclosure syndrome”) were accompanied by accumulation of CO2 and C2H4 in and depletion of O2 from the atmosphere. Ethylene (1 μl 1?1), high levels of CO2, and reduced levels of O2 contributed equally to the increase in coleoptile and mesocotyl growth. The effect of enclosure could be mimicked by passing a gas mixture of 3% O2, 82% N2, 15% CO2 (all v/v), and 1 μl l?1) C2H4 through the vials containing the etiolated seedlings. The effects of high CO2 and low O2 concentrations were not mediated through increased C2H4 production. The enclosure syndrome was also observed in rice seedlings grown under water either in darkness or in light. The length of the rice coleoptile was positively correlated with the depth of planting in water-saturated vermiculite. The length of coleoptiles of wheat, barley, and oats was not affected by the depth of planting. In rice, the length of coleoptile was determined by the levels of O2, CO2, and ethylene, rather than by light. This regulatory mechanism allows rice seedlings to grow out of shallow water in which the concentration of O2 is limiting. 相似文献
14.
Kinetics of the Oxyhydrogen Reaction in the Presence and Absence of Carbon Dioxide in Scenedesmus obliquus 总被引:2,自引:2,他引:0
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The oxyhydrogen reaction in the presence and absence of CO2 was studied in H2-adapted Scenedesmus obliquus by monitoring the initial rates of H2, O2, and 14CO2 uptake and the effect of inhibitors on these rates with gas-sensing electrodes and isotopic techniques. In the presence of 0.02 atmosphere O2, the pH2 was varied from 0 to 1 atmosphere. Whereas the rate of O2 uptake increased by only 30%, the rate of H2 uptake increased severalfold over the range of pH2 values. At 0.1 atmosphere H2 and 0.02 atmosphere O2, rates for H2 and O2 uptake were between 15 and 25 micromoles per milligram chlorophyll per hour. As the pH2 was changed from 0 to 1 atmosphere, the quotient H2:O2 changed from 0 to roughly 2. This change may reflect the competition between H2 and the endogenous respiratory electron donors. Respiration in the presence of glucose and acetate was also competitive with H2 uptake. KCN inhibited equally respiration (O2 uptake in the absence of H2) and the oxyhydrogen reaction in the presence and absence of CO2. The uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone accelerated the rate of respiration and the oxyhydrogen reaction to a similar extent. It was concluded that the oxyhydrogen reaction both in the presence and absence of CO2 has properties in common with components of respiration and photosynthesis. Participation of these two processes in the oxyhydrogen reaction would require a closely linked shuttle between mitochondrion and chloroplast. 相似文献
15.
Simultaneous measurement of oxygen and hydrogen exchange from the blue-green alga anabaena 总被引:16,自引:4,他引:12
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Two Clark-type polarographic electrodes were used to measure simultaneous H2 and O2 exchange from three species of the blue-green alga Anabaena. Maximum H2 photoevolution from N2-fixing cultures of Anabaena required only the removal of dissolved O2 and N2; no adaptation period was necessary. No correlation of H2 photoproduction with photosynthetic O2 evolution, beyond their mutual light requirement, was found. Hydrogen photoevolution has the following characteristics in common with N2 fixation in these organisms: DCMU insensitivity; similar white light dependency with very low dark production rates; maximum efficiency in photosystem I light; inhibition by N2, O2 and acetylene; and an apparent requirement for the presence of heterocysts. Growth on nitrate medium reduces, and on ammonium medium obliterates, both reactions. Cultures grown under limiting CO2 conditions have H2 photoproduction rates proportional to their growth rates. Hydrogenase activity is inferred from H2 uptake in the dark, but this activity apparently is independent of the photoevolution of H2 which is ascribed strictly to the nitrogenase system. 相似文献
16.
Photorespiration and Oxygen Inhibition of Photosynthesis in Chlorella pyrenoidosa 总被引:7,自引:6,他引:1
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The inhibition of photosynthesis by O2 in air-grown Chlorella pyrenoidosa was investigated using three experimental techniques (artificial leaf, aqueous method, and O2 electrode) to measure carbon assimilation. CO2 response curves were determined under different O2, pH, and temperature conditions. Regardless of the experimental technique and condition, O2 inhibition was not evident until a concentration of 50% was reached; Vmax values were reduced whereas Km (CO2) values were unaffected by the increasing O2 concentration. The response of photosynthesis to O2 was independent of CO2 and HCO3− concentrations as well as temperature. Relative rates of photosynthesis showed a 4 to 5% stimulation in 2% O2, a 12% inhibition in 50% O2, and a 24% inhibition in 100% O2. The inhibition by 50% O2 was still reversible after 20 minutes exposure whereas 100% O2 caused irreversible inhibition after only 4 minutes. 相似文献
17.
Zeaxanthin Synthesis, Energy Dissipation, and Photoprotection of Photosystem II at Chilling Temperatures 总被引:7,自引:5,他引:2
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When leaves of a mangrove, Rhizophora mangle, were exposed to an excess of light at chilling temperatures, synthesis of zeaxanthin through violaxanthin de-epoxidation as well as nonphotochemical fluorescence quenching were markedly reduced. The results suggest a protective role of energy dissipation against the adverse effects of high light and chilling temperatures: leaves of R. mangle that had been preilluminated in 2% O2, 0% CO2 at low photon flux density and showed a high level of zeaxanthin, and leaves that had been kept in the dark and contained no zeaxanthin, were both exposed to high light and chilling temperatures (5°C leaf temperature) in air and then held under control conditions in low light in air at 25°C. Measurements of chlorophyll a fluorescence at room temperature showed that the photochemical efficiency of PSII and the yield of maximum fluorescence of the preilluminated leaf recovered completely within 1 to 3 hours under the control conditions. In contrast, the fluorescence responses of the predarkened leaf in high light at 5°C did not recover at all. During a dark/light transient in 2% O2, 0% CO2 in low light at 5°C, nonphotochemical fluorescence quenching increased linearly with an increase in the zeaxanthin content in leaves of R. mangle. In soybean (Glycine max) leaves, which contained a background level of zeaxanthin in the dark, a similar treatment with excess light induced a level of nonphotochemical fluorescence quenching that was not paralleled by an increase in the zeaxanthin content. 相似文献
18.
Aflagellated mutants of Helicobacter pylori generated by genetic transformation of naturally competent strains using transposon shuttle mutagenesis 总被引:20,自引:9,他引:11
Three out of 10 Helicobacter pylori clinical isolates were found to be naturally competent for genetic transformation to streptomycin resistance by chromosomal DNA extracted from a spontaneous streptomycin-resistant H. pylori mutant. The frequency of transformation varied between 5 × 10?4 and 4 × 10?6, depending on the H. pylori isolate used. Transposon shuttle mutagenesis based on this natural competence was established using the flagellin gene flaA as the target. The cloned flaA gene was interrupted by insertion of TnMax1, a mini-Tn1721 transposon carrying a modified chloramphenicol-acetyltransferase gene, the catGC cassette. Natural transformation of competent H. pylori strains with plasmid constructs harbouring a catGC-inactivated flaA gene resulted in chloramphenicol-resistant transformants at an average frequency of 4 × 10?5. Southern hybridization experiments confirmed the replacement of the chromosomal H. pylori flaA gene by the cat-inactivated cloned gene copy via homologous recombination resulting in allelic exchange. Phenotypic characterization of the mutants demonstrated the absence of flagella under the electron microscope and the loss of bacterial motility. Immunoblots of cell lysates of the H. pylori mutants with an antiserum raised against the C-terminal portion of recombinant H. pylori major flagellin (FlaA) confirmed the absence of the 54kDa FlaA protein. This efficient transposon shuttle mutagenesis procedure for H. pylori based on natural competence opens up new possibilities for the genetic assessment of putative H. pylori virulence determinants. 相似文献
19.
《Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology》2001,1544(1-2):55-63
The α-carbonic anhydrase gene from Helicobacter pylori strain 26695 has been cloned and sequenced. The full-length protein appears to be toxic to Escherichia coli, so we prepared a modified form of the gene lacking a part that presumably encodes a cleavable signal peptide. This truncated gene could be expressed in E. coli yielding an active enzyme comprising 229 amino acid residues. The amino acid sequence shows 36% identity with that of the enzyme from Neisseria gonorrhoeae and 28% with that of human carbonic anhydrase II. The H. pylori enzyme was purified by sulfonamide affinity chromatography and its circular dichroism spectrum and denaturation profile in guanidine hydrochloride have been measured. Kinetic parameters for CO2 hydration catalyzed by the H. pylori enzyme at pH 8.9 and 25°C are kcat=2.4×105 s−1, KM=17 mM and kcat/KM=1.4×107 M−1 s−1. The pH dependence of kcat/KM fits with a simple titration curve with pKa=7.5. Thiocyanate yields an uncompetitive inhibition pattern at pH 9 indicating that the maximal rate of CO2 hydration is limited by proton transfer between a zinc-bound water molecule and the reaction medium in analogy to other forms of the enzyme. The 4-nitrophenyl acetate hydrolase activity of the H. pylori enzyme is quite low with an apparent catalytic second-order rate constant, kenz, of 24 M−1 s−1 at pH 8.8 and 25°C. However, with 2-nitrophenyl acetate as substrate a kenz value of 665 M−1 s−1 was obtained under similar conditions. 相似文献
20.
Investigation of the H(2) Oxidation System in Rhizobium japonicum 122 DES Nodule Bacteroids 总被引:5,自引:0,他引:5
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The H2-oxidizing complex in Rhizobium japonicum 122 DES bacteroids failed to catalyze, at a measurable rate, 2H1H exchange from a mixture of 2H2 and 1H2 in presence of 2H2O and 1H2O, providing no evidence for reversibility of the hydrogenase reaction in vivo. In the H2 oxidation reaction, there was no significant discrimination between 2H2 and 1H2, indicating that the initial H2-activation step in the over-all H2 oxidation reaction is not rate-limiting. By use of improved methods, an apparent Km for H2 of 0.05 micromolar was determined. The H2 oxidation reaction in bacteroids was strongly inhibited by cyanide (88% at 0.05 millimolar), theonyltrifluoroacetone, and other metal-complexing agents. Carbonyl cyanide m-chlorophenylhydrazone at 0.005 millimolar and 2,4-dinitrophenol at 0.5 millimolar inhibited H2 oxidation and stimulated O2 uptake. This and other evidence suggest the involvement of cytochromes and nonheme iron proteins in the pathway of electron transport from H2 to O2. Partial pressures of H2 at 0.03 atmosphere and below had a pronounced inhibitory effect on endogenous respiration by bacteroid suspensions. The inhibition of CO2 evolution by low partial pressures of H2 suggests that H2 utilization may result in conservation of oxidizable substrates and benefits the symbiosis under physiological conditions. Succinate, acetate, and formate at concentrations of 50 millimolar inhibited rates of H2 uptake by 8, 29, and 25%, respectively. The inhibition by succinate was noncompetitive and that by acetate and formate was uncompetitive. A concentration of 11.6 millimolar CO2 (initial concentration) in solution inhibited H2 uptake by bacteroid suspensions by 18%. Further research is necessary to establish the significance of the inhibition of H2 uptake by succinate, acetate, formate, and CO2 in the metabolism of the H2-uptake-positive strains of Rhizobium. 相似文献