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Öğe Cell growth and oxygen uptake of Escherichia coli and Pseudomonas aeruginosa are differently effected by the genetically engineered Vitreoscilla hemoglobin gene(Elsevier Science Bv, 2001) Geckil, H; Stark, BC; Webster, DAVitreoscilla hemoglobin is a good oxygen trapping agent and its presence in genetically engineered Escherichia coli helps this bacterium to grow better. Here, the potential use of this hemoglobin, for improving the growth and the oxygen transfer properties of Pseudomonas aeruginosa as well as Escherichia coli, was investigated. To stably maintain it in both bacteria, a broad-host range cosmid vector (pHG1), containing the entire coding sequence for Vitreoscilla hemoglobin gene and its native promoter on a 2.3 kb fragment, was constructed. Though at different levels, both bacteria produced hemoglobin and while the oxygen uptake rates of vgb-bearing strains were 2-3-fold greater than that of non-vgb-bearing strains in both bacteria, the growth advantage afforded by the presence of Vitreoscilla hemoglobin was somewhat varied. As an alternative to the traditional method of the improvement of oxygen transfer properties of the environment in which cells are grown, the genetic manipulation applied here improved the oxygen utilization properties of cells themselves. (C) 2001 Published by Elsevier Science B.V.Öğe Cloning and expression of the Vitreoscilla hemoglobin gene in Enterobacter aerogenes(Pleiades Publishing Inc, 2004) Erenler, SO; Gencer, S; Geckil, H; Stark, BC; Webster, DAThe hemoglobins found in unicellular organisms show a great deal of chemical reactivity, protecting cells against oxidative stress, and hence have been implicated in a wider variety of potential functions than those traditionally associated with animal and plant hemoglobins. There are well-documented studies showing that bacteria expressing Vitreoscilla hemoglobin (VHb), the first prokaryotic hemoglobin characterized, have better growth and oxygen uptake rates than their VHb counterparts. Here, the expression of VHb, its effect on the growth and antioxidant enzyme status of cells under different culture conditions was studied by cloning the complete regulatory and coding sequences (vgb) for VHb in Enterobacter aerogenes. Contrary to what has been reported for Escherichia coli, the expression of vgb in E.aerogenes decreased several fold under 10% of atmospheric oxygen (approximate to2% oxygen) and its growth was not greatly improved by the presence of VHb. Measured either as viable cells or total cell mass, untransformed E. aerogenes grew better than the recombinant strains. At the late exponential phase, however, the vgb-bearing strain was determined to have a higher cell number and total cell mass than the strain bearing only the plasmid vector with no vgb insert. The VHb expressing strain also had an oxygen uptake rate several fold higher than its counterparts. Given that oxidative stress may occur upon elevated oxygen exposure and be balanced by the action of antioxidative compounds, the level of antioxidative response of E. aerogenes expressing VHb was also studied. The VHb expressing strain had substantially (1.5-2.6-fold) higher catalase activity than strains not expressing VHb. Both VHb+ and VHb- strains, however, showed similar levels of superoxide dismutase activity. The activity of both enzymes was also growth phase dependent. Stationary phase cells of all strains showed 2-5-fold higher activity for these enzymes than cells at the exponential phase.Öğe Degradation of benzene, toluene and xylene by Pseudomonas aeruginosa engineered with the Vitreoscilla hemoglobin gene(Wiley, 2005) Kahraman, H; Geckil, HThis study concerns the potential use of Pseudomonas aeruginosa expressing the Vitreoscilla hemoglobin gene for the degradation of important harmful aromatic compounds such as benzene, toluene, and xylene (BTX). The use of these compounds by both strains was determined as the production of cell mass (viable cell number) in a minimal medium containing any one of the BTX compounds as the sole carbon and energy source. Furthermore, the BTX degradation capability of both strains was monitored by measuring the production of 3-methylcatechol, a common intermediate. For the cells of the logarithmic phase, which were grown at high aeration/high agitation or low aeration/low agitation, the engineered strain showed a better growth rate than the host strain. With the benzene in the medium, the recombinant strain exhibited a higher (up to 4-fold) cell density than the parental wild-type strain at this phase. In contrast, regarding the cells of the late stationary phase under high aeration/high agitation conditions, the host strain had generally higher viable cell numbers than the recombinant strain. At this phase this difference was, however, less significant under the conditions of low aeration/low agitation. Similarly, in toluene containing medium (at high aeration/high agitation) the recombinant strain showed a higher cell density which was from a 15-fold to almost one order of magnitude greater than its parental strain during the logarithmic phase where the cell density of P aeruginosa remained nearly constant. Contrary to the results with benzene and toluene, both strains exhibited similar growth characteristics when they were grown in the presence of xylene. The positive effect of the oxygen uptake by the recombinant system on the BTX metabolizing activity was also apparent in a high accumulation of 3-methylcatechol in the cultures of the recombinant strain. At certain points of incubation, the hemoglobin expressing strain showed a significantly (p < 0.05) higher 3-methylcatechol accumulation than the host strain. These results demonstrated the possible potential of the Vitreoscilla hemoglobin as an efficient oxygen uptake system for the bioremediation of some compounds of environmental concern.Öğe Effects of metals on seed germination, root elongation, and Coleoptile and hypocotyl growth in Triticum aestivum and Cucumis sativus(Springer-Verlag, 2002) Munzuroglu, O; Geckil, HA simple, fast, and easy-to-perform method was carried out for the quantification of the inhibitory effects of metals on wheat and cucumber. The method uses seed germination, root elongation, and hypocotyl and coleoptile growth in these plants as parameters in the presence of varying concentrations of metals. Metals selected for this study were Hg, Cd, Co, Cu, Pb, and Zn. Although effective concentrations of these metals for a certain degree of inhibition were different, both plants had a reduced seed germination rate, root, and hypocotyl or coleoptile length with increasing concentrations. Mercury was determined to be the most inhibitory metal on these parameters. This metal caused a complete inhibition of germination in wheat and cucumber seeds at certain concentrations-greater than or equal to1.5 mM in cucumber and at 1.7 mM in wheat. No other metal caused this kind of inhibition even at the highest concentration (8.0 mM) applied. Though this metal possessed a higher inhibition of germination in cucumber than in wheat seeds, the inhibitory effects of other metals used were the reverse, being higher in wheat. With some exceptions, all metals in selected concentrations caused a significant (p < 0.01 or p < 0.05) decrease in germination rate of both plants compared to control group seeds.Öğe Effects of simulated acid rain on the pollen germination and pollen tube growth of apple (Malus sylvestris Miller cv. Golden)(Akademiai Kiado, 2003) Munzuroglu, O; Obek, E; Geckil, HThe pollens of apple flowers have been treated with simulated acid rain solutions in range of pHs 2.9 to 5.0 in order to determine the threshold proportion values that lead the observed symptoms of detriments of acid rain. Compared to controls (pH 6.5), pollen germination decreased by 41.75% at pH 3.3 and pollen tube elongation decreased by 24.3% at pH 3.4. Acid rain threshold proportion value was around pH 3.3 and 3.4 for apple pollen germination and pollen tube elongation, respectively. Furthermore, pollen tube elongation was determined to be more sensitive to acid rain than pollen germination. The pH values below 3.1 resulted in complete destruction of pollen tubes. Pollen germination entirely stopped at around pH 3.0. Finally, it has been shown that the acid rain has a blocking effect on pollen germination and pollen tube elongation in apple. The conclusion is that not only pH value but also the quantity of acid rain is important factor in germination. The results were found statistically significant through the LSD test at levels of p < 0.05 and p < 0.01.Öğe Enhanced production of acetoin and butanediol in recombinant Enterobacter aerogenes carrying Vitreoscilla hemoglobin gene(Springer, 2004) Geckil, H; Barak, Z; Chipman, DM; Erenler, SO; Webster, DA; Stark, BCMicrobial production of butanediol and acetoin has received increasing interest because of their diverse potential practical uses. Although both products are fermentative in nature, their optimal production requires a low level of oxygen. In this study, the use of a recombinant oxygen uptake system on production of these metabolites was investigated. Enterobacter aerogenes was transformed with a pUC8-based plasmid carrying the gene (vgb) encoding Vitreoscilla (bacterial) hemoglobin (VHb). The presence of vgb and production of VHb by this strain resulted in an increase in viability from 72 to 96 h in culture, but no overall increase in cell mass. Accumulation of the fermentation products acetoin and butanediol were enhanced (up to 83%) by the presence of vgb/VHb. This vgb/VHb related effect appears to be due to an increase of flux through the acetoin/butanediol pathway, but not at the expense of acid production.Öğe Genetic engineering of Enterobacter aerogenes with the Vitreoscilla hemoglobin gene(Elsevier, 2003) Geckil, H; Gencer, S; Kahraman, H; Erenler, SOHemoglobins in unicellular organisms, like the one here in the bacterium Vitreoscilla, have greater chemical reactivity than their homologues in multicellular organisms. They can catalyze redox reactions and may protect cells against oxidative stress. The ability of Vitreoscilla hemoglobin to complement deficiencies of terminal cytochrome oxidases in Escherichia coli also suggests that this hemoglobin can receive electrons during respiration. In this study, a recombinant strain of Enterobacter aerogenes engineered to produce the Vitreoscilla Hb was investigated with regard to its susceptibility to oxidative stress. The culture response to oxidative stress produced by exogenously applied hydrogen peroxide was characterized in terms of cell growth, survival and the activities of two key antioxidant enzymes (catalase and superoxide dismutase). The influence of the physiological state of the cells and different media upon these culture dynamics was determined. Results showed that the hemoglobin-expressing strain is quite distinct in terms of growth/survival properties and activity of antioxidant enzymes from that of non-hemoglobin counterparts. (C) 2003 Editions scientitiques et medicales Elsevier SAS. All rights reserved.Öğe Membrane permeabilization of gram-negative bacteria with a potassium phosphate/hexane aqueous phase system for the release of L-asparaginase: an enzyme used in cancer therapy(Elsevier Sci Ltd, 2005) Geckil, H; Ates, B; Gencer, S; Uckun, M; Yilmaz, IA fast, efficient and reproducible recovery procedure for periplasmic L-asparaginase from two distinctly related gram-negative bacteria, Enterobacter aerogenes and Pseudomonas aeruginosa, is presented. As the method uses inexpensive organic solvent hexane and an aqueous salt solution, it is also highly cost-effective in comparison with the currently available techniques used for the release of this enzyme. As hexane is a highly water immiscible organic solvent, it can be removed easily from the top of the aqueous phase by a simple evaporation. Also, various organic solvents and other membrane partitioning compounds were compared for their efficiency on L-asparaginase/protein release. The degree to which the enzyme was released was different for two bacteria, suggesting that they possess different permeability characteristics. The most efficient enzyme release from both bacteria was determined to be in 50 mM potassium phosphate with 1% hexane. Enzyme recoveries up to three-fold with respect to sonication have been achieved with this system. (C) 2004 Elsevier Ltd. All rights reserved.Öğe Production of L-asparaginase in Enterobacter aerogenes expressing Vitreoscilla hemoglobin for efficient oxygen uptake(Springer-Verlag, 2004) Geckil, H; Gencer, SThis study is the first utilizing Vitreoscilla hemoglobin in a heterologous bacterium, Enterobacter aerogenes, to determine the effect of such a highly efficient oxygen-uptake system on the production of L-asparaginase, an enzyme that has attracted considerable attention due to its anti-tumor activity. Here, we show that the Vitreoscilla hemoglobin expressing strain has from 10-fold to more than two orders of magnitude lower L-asparaginase activity than the wild type or the control without the Vitreoscilla hemoglobin gene under different aeration conditions. Aeration and agitation were also determining factors for enzyme production. The enzyme activity was reduced considerably under both full aerobic and anaerobic conditions, while the highest enzyme activity was determined in cultures under low aeration and low agitation. Also, the effect of different concentrations of glucose on enzyme production showed catabolic repression. Glucose at 1% caused almost total inhibition of enzyme activity, while at 0.1% it showed a slightly stimulatory effect on enzyme production, compared with glucose-free medium.Öğe Protective role of melatonin given either before ischemia or prior to reperfusion on intestinal ischemia-reperfusion damage(Wiley, 2004) Ates, B; Yilmaz, I; Geckil, H; Iraz, M; Birincioglu, M; Fiskin, KTissue injury resulting from ischemia-reperfusion is of fundamental importance. Experimental evidence suggests that the generation of reactive oxygen species is significantly responsible for this type of injury. In the present study, besides investigating the protective role of melatonin on tissue damage caused by intestinal ischemia-reperfusion, the protective activity of this compound was also analyzed in both pre- and post ischemia melatonin-treated rats. The activities of the main antioxidative enzymes, catalase, superoxide dismutase and glutathione peroxidase in the intestine showed significant (P < 0.05) increases in melatonin-treated animals that were subjected to ischemia/reperfusion compared with those subjected only to ischemia/reperfusion. Also, results clearly indicate that the level of malondialdeyhde, an index of lipid peroxidation, decreased significantly (P < 0.05) when rats subjected to intestinal/reperfusion were given melatonin either before ischemia or before reperfusion.Öğe The synthesis of indole-3-acetic acid by the industrially important white-rot fungus Lentinus sajor-caju under different culture conditions(Cambridge Univ Press, 2003) Yurekli, F; Geckil, H; Topcuoglu, FThe synthesis of plant growth hormone auxin, indole-3-acetic acid (IAA), is not only confined to flowering plants. Yeasts and other fungi are also known to produce this hormone and in many cases at higher levels than plants. The main concern of this study was to determine the physical and chemical conditions necessary for optimal biosynthesis of this hormone by Lentinus sajor-caju. Glucose was determined to be superior to sucrose as carbon and energy source. The synthesis of IAA in a nitrogen-free medium or in a medium with low external phosphate was substantially reduced. Light exposed and non-agitated cultures grown in dark had also reduced levels of IAA compared to agitated cultures grown in dark. The highest (0.18 mg ml(-1)) IAA level was determined in cultures grown in glucose containing medium (pH 7.5) on a rotary shaker (150 rpm) at 30 degreesC in dark. The biological activity of IAA obtained from the extra-cellular culture of Lentinus sajor-caju was determined using oat coleptile growth test.Öğe The vitamin and selenium contents of apricot fruit of different varieties cultivated in different geographical regions(Elsevier Sci Ltd, 2003) Munzuroglu, O; Karatas, F; Geckil, HHigh-pressure liquid chromatographic and fluorometric methods were utilized to determine the vitamin (A, C, E and beta-carotene) and selenium levels in apricot fruit. The vitamins (A, C, and E), beta-carotene and selenium levels of both fresh and sulphurized (sulphur-dried) apricot fruit from several common cultivars and wild type, grown in different regions, were analysed. The effects of post-harvest processing, regional difference and cultivar type on the levels of these nutrients were investigated. Cultivated varieties possessed significantly higher vitamin C (P < 0.01), vitamin A and E (P < 0.05) than the wild type. The vitamin C levels of fresh and sulphur-dried apricot from cultivated forms, for example, were 1.8-2.7- and 2.9-4.6-fold, respectively, higher than those of wild type ones. Among cultivated varieties, H cultivar was determined to have significantly highest vitamin A, E and beta-carotene contents (P<0.05). In general, the vitamin, beta-carotene and selenium levels were determined to be significantly different, both between varieties and between different regions within the same variety. Cultivars in region ES, a high altitude region, for example, had significantly (P < 0.05) higher vitamin C content than the same cultivars grown in other regions. The selenium content, however, was significantly (P<0.01) higher only in fresh fruit from variety HB in region ES than the other two regions. (C) 2003 Elsevier Ltd. All rights reserved.Öğe Vitreoscilla hemoglobin expressing Enterobacter aerogenes and Pseudomonas aeruginosa respond differently to carbon catabolite and oxygen repression for production of L-asparaginase, an enzyme used in cancer therapy(Elsevier Science Inc, 2004) Geckil, H; Gencer, S; Uckun, MThe production of antileukemic enzyme L-asparaginase in two distinctly related bacteria, Enterobacter aerogenes, Pseudomonas aeruginosa, and in their recombinants expressing the Vitresocilla hemoglobin (VHb) has been studied. Both bacteria showed a substantially different degree of carbon catabolite repression of the enzyme production. E. aerogenes grown under catabolite repression had more than 20-fold lower L-asparaginase activity than the controls. This figure was only 1.6-fold for P. aeruginosa. In the medium with restricted nutrient content, however, the inhibitory effect of glucose on the enzyme production was less pronounced. The presence of VHb, an efficient oxygen uptake system, had also different effects in both bacteria. Under conditions of no catabolite repression, this protein caused about 7-fold lower L-asparaginase activity in E. aerogenes, but similar or even slightly stimulatory effect in P aeruginosa. The use of a relatively poor carbon source, mannitol, caused a lower L-asparaginase level and no glucose type catabolite repression. (C) 2004 Elsevier Inc. All rights reserved.Öğe Vitreoscilla hemoglobin renders Enterobacter aerogenes highly susceptible to heavy metals(Springer, 2004) Geckil, H; Arman, A; Gencer, S; Ates, B; Yilmaz, HRWhen expressed in heterologous microorganisms Vitreoscilla hemoglobin (VHb) acts as oxygen storage and causes a higher oxygen uptake. In this study, the effect of this protein on growth, sensitivity and antioxidant properties of Enterobacter aerogenes exposed to metal stress was investigated. The strain expressing VHb was more sensitive to mercury and cadmium as the minimal inhibitory concentration (MIC) for these metals was up to 2-fold lower in this strain than the host and the recombinant strain carrying a comparable plasmid. At lower concentrations than MIC, the metals partially limited growth and caused an inhibition proportional to metal concentration applied. The growth pattern of VHb expressing strain was also distinctly different from other two non-hemoglobin strains. The hemoglobin containing strain showed substantially higher superoxide dismuates (SOD) activity than the non-hemoglobin strains, while catalase levels were similar in all strains. All strains exposed to copper, however, showed similar MIC values, growth patterns, and SOD and catalase levels.
