O Vasyliv, S Hnatush - Effect of transition metal compounds on catalase activity of sulfur-reducing bacterial desulfuromonas acetoxidans cells - страница 1
ISSN 0206-5657. Вісник Львівського університету. Серія біологічна. 2011. Випуск 57. С. 207-215 Visnyk of the Lviv University. Series BIology. 2011. Issue 57. P. 207-215
EFFECT OF TRANSITION METAL COMPOUNDS ON CATALASE ACTIVITY OF SULFUR-REDUCING BACTERIAL DESULFUROMONAS ACETOXIDANS CELLS
O. Vasyliv, S. Hnatush
Ivan Franko National University of Lviv 4, Hrushevskyi St., Lviv 79005, Ukraine e-mail: oresta. vasyliv@gmail. com
Desulfuromonas acetoxidans are gram-negative obligatory anaerobic sulfur-reducing bacteria. Aerobes and facultative anaerobes possess specific enzymatic antioxidant system which defenses them against toxic and mutagenic compounds or detrimental influence of the oxygen. One of the most crucial components of it is catalase. It's assumed that lack or low activity of this system' enzymes of the obligatory anaerobic microorganisms, such as sulfur- or sulfate-reducing bacteria, causes they high sensitivity to the influence of oxygen or toxic compounds, such as transition metals. The influence of FeSO4, FeCl3x6H2O, MnO2, MnCl2x4H2O, NiCl2x6H2O and CoCl2x6H2O on catalase activity of sulfur-reducing bacterial D. acetoxidans cells has been investigated. The highest activity of investigated enzyme has been observed under the influence of 0.5 mM, 1.5 mM and 2.0 mM of NiCl2x6H2O, FeSO4 and CoCl2x6H2O respectively on the second day, 1.5 mM of MnO2 on the third day and 0.5 mM of FeCl3x6H2O and 1.0 mM of MnCl2x4H2O on the fourth day of growth in comparison with control samples.
Key words: Desulfuromonas acetoxidans, sulfur bacteria, antioxidant defense, cata-lase, transition metals.
Nowadays applying of ecological biotechnologies is one of the most crucial directions in solution of the problem of different toxic metal ions environmental pollution, which is a result of negative anthropogenic influence. Investigations of effective biological mechanisms of harmful compounds detoxification by different microorganisms is one of the ways of it's development . Transition metals, such as Ferrum, Manganese, Nickel, Cobalt, Argentum, Cooper, Cadmium, Zinc, Molybdenum etc are necessary in the micromolar concentrations for normal functionality of microorganisms while their growth and functional activity are inhibited under the influence of millimolar concentrations of these metals . Despite that an exorbitant quantity of bacteria possesses effective defensive mechanisms that protect them against negative toxic xenobiotics influence. It determines their resistance towards high metal ion concentrations in the surrounding environment .
Nickel, Ferrum, Manganese and Cobalt are required as trace elements by many bacteria. Thus these metals are under specific investigations. Ferrum ions support effectivity of cell transport systems work, take part in the intracellular energy production and cell division . Nickel and Cobalt are similar to Ferrum by their electrochemical properties. It allows to assume that these elements could substitute iron under it's restricted concentrations in the environment. These elements are essential nutrients for selected microorganisms where they participate in a variety of cellular processes. Nickel ion is specifically incorporated into nickel-dependent enzymes, often via complex assembly processes requiring accessory proteins and additional non-protein components, in some cases accompanied by nucleotide triphosphate hydrolysis. To date, nine nickel-containing enzymes are known: urease, Ni-Fe-hydrogenase, carbon monoxide dehydro-genase, acetyl-CoA decarbonylase/synthase, methyl coenzyme M reductase, certain superoxide
© Василів О., Гнатуш С., 2011
О. Василів, С. Гнатуш
dismutases, some glyoxylases, aci-reductone dioxygenase, and methylenediurease . Cobalt is a constituent of vitamin B12 and other corrinoids that are widespread coenzymes . This metal also possesses a significant role in the regulation of enzymes activity, especially these which are involved in the regulation of aminoacids transamination . It was investigated that Ferrum and Manganese can be used as a final electron acceptors in the processes of anaerobic respiration, such as dissimilative Fe3+- and Mn4+ - reduction that is carried by sulfur-reducing Desulfuromo-nas acetoxidans bacteria.
D. acetoxidans are gram-negative obligatory anaerobic sulfur-reducing bacteria that inhabit sulfur containing aquatic environments. These bacteria belong to the д - Proteobacteria class .
Sulfur- and sulfate-reducing bacteria (SRB) have a significant meaning in the maintenance of ecological stability of surrounding environment. They are able to control transfer of metals in aquatic sediments by the hardly soluble metal sulfids creation. The process, which is held by the sulfur cycle reducive stage bacteria, represents an effective mechanism of biological remediation of overconcentrated heavy metals environmental pollution . SRB possess a unique potential of spontaneous removal of metals and sulfates from the surrounding environment during the process of sulfides bioprecipitation.
Reactive oxygen species (ROS) such as superoxide radical, hydrogen peroxide, hydroxyl radical etc are toxic to all living cells. They are produced as a result of prolonged influence of oxygen or toxic xenobiotics, such as heavy metal ions, on the cell. ROS cause amino-acid residues modification, proteins' sulfhydryl groups oxidation, peptides' connections cleavage, release of metal from metaloproteids, nucleic acids depolymerization, point mutations, polysaccharides and unsaturated fatty acids oxidation etc [2, 6]. Aerobs and facultative anaerobs possess an enzymatic antioxidant defensive system that prevents the consequences of this toxic and mutagenic compounds and toxic oxygen influence. One of the most important components of it is catalase (hydrogen peroxide: hydrogen peroxide oxidoreductase). This enzyme causes the two-electron H2O2 cleavage with O2 and H2O production. In this case H2O2 is used as electrons donor. Catalase belongs to the enzymes that support their activity during long time and almost don't need the activation energy. It was investigated that catalase could join four NADFH+ molecules. As a result it is defended against inactivation and enzyme activity enhances. Catalase hardly influxes inside the cell and loses it's activity in the extracellular surrounding, especially because of high proteolytic enzymes activity .
Aerobic microorganisms possess an effective cascade functioning complex of defensive enzymatic and non-enzymatic systems that cleavage reactive oxygen species and synthesize DNA reparation enzymes and antioxidant defense regulations .
Obligatory anaerobic microorganisms, such as sulfur- and sulfate-reducing bacteria possess an increased sensitivity to the oxygen. This fact is explained by the lack or low activity of antioxidant defensive system enzymes. It was investigated that this bacteria are able to live in the environments that contain oxygen because of possession the alternative defensive mechanism against oxidative stress. It includes:
• specific rubredoxin oxidoreductase that possesses a superoxide dismutase activity, causes an intercellular O2 - reduction with H2O production and also serves as terminal oxidase;
• Rubrerythrin that possesses NADH-peroxidase activity;
• Similar to rubredoxin proteins that are used as intermediate electron donors . Catalase activity has been observed among the several species of sulfate-reducing bacteria
of Desulfovibrio genus. There are D. desulfuricans, D. vulgaris, D. oxyclinae and D. gigas .
Despite that, it isn't investigated enough in sulfur-reducing bacteria.
That's the aim of our work was to investigate the influence of different valences transition metals compounds, such as FeSO4, FeCl3x6H2O, MnO2, MnCl2x4H2O, NiCl2x6H2O and CoCl2x6H2O on catalase activity of bacterial Desulfuromonas acetoxidans cells.
MATERIALS AND METHODS
The object of investigations was sulfur-reducing Desulfuromonas acetoxidans bacterium that was extracted from Yavoriv sulfur aquatic field (Lviv region), obtained in the pure culture and identificated at the Department of Microbiology of Ivan Franko National University of Lviv, Ukraine. Bacteria were cultivated in the modified Postgait C medium [3, 5] during four days under the anaerobic conditions and temperature +30°С with addition from 0.5 to 2.5 mM of FeSO4, FeCl3x6H2O, MnO2, MnCl2x4H2O, NiCl2x6H2O and CoCl2x6H2O into the growth medium. Control samples didn't contain any investigated metal compounds. After the second, third and fourth day of bacterial growth cell-free extracts were obtained by the following technique. Cells were washed by 0.9% NaCl solution and disintegrated on the ultrasonic homogenizer at 22 kHz at 0°C during five minutes. Cell debris were sedimented by centrifugation at 5635-8800g at 4°C during 30 minutes. Catalase activity was measured spectrophotometrically with wave length 410 nm by the quantity of cleavaged hydrogen peroxide . Reaction mixture contains 2,8 ml of 0,5% Н2О2 solution and 0.1-0.2 ml of cell-free extract, diluted by n times with concentration 1 mg of protein per 1 ml. Incubation was carried out during 5 minutes. Reaction was stopped by 1 ml of 6% (NH4)2MoO4 solution. Probe that contains Н2О instead of cell-free extract solution served as a control.
Enzyme activity was calculated by the formula:
A 1™°_= А£,4ю'4'п
' min-mg of protein 110,6-t-V-C where АЕ410 - difference between Е410 of control sample and Е410 of probe; 4 - the overall volume of reaction mixture, ml; n - solution of cell-free extract, times; 110.6 - constant, determined by the callibration curve; t - time of incubation, minutes, V - volume of cell-free extract, added to the reaction mixture, ml; C - protein concentration in the probe, mg/ml.
Protein concentration in the cell-free extract was determined by Lauri method .
The crucial statistical indexes were calculated on the base of direct data, such as arithmetical mean (M) and standard deviation of arithmetical mean (m). Student coefficient was calculated for the estimation of validity of difference between statistical characteristics of two alternative blocks of data. The difference was claimed to be valid under the index of validity Р>0,95 . Statistical calculation of results were carried out by the Origin and Excel programs.
RESULTS AND DISCUSSION
Catalase activity of sulfur-reducing D. acetoxidans bacteria cell-free extracts has been investigated under the influence of different concentrations of ferrous (II) sulfate, ferrous (III) chloride hexahydrate, manganese (IV) oxide, manganese (II) chloride tetrahydrate, nickel (II) chloride hexahydrate and cobalt (II) chloride hexahydrate during four days of cultivation.
Addition of different FeCl3x6H2O concentrations caused the highest activity of investigated enzyme (61,36±0,77 umol/minxmg of protein) on the fourth day of growth under the influence of 0.5 mM of investigated metal salt hydrate (fig. 1, A). It increased by 2.2 times on the second, by 3.8 times on the third and by 5 times on the fourth days of growth under such influence of ferrous (III) chloride hexahydrate on investigated bacterial cells in comparison with control samples. Increase of FeCl3x6H2O concentration by 2.5 mM in the growth medium caused the catalase activity decrease by 2.5 times on the second day of cultivation and increase by 2.7 and 3.9 times respectively on the third and fourth day of growth in comparison with control samples.
21° ISSN 0206-5657. Вісник Львівського університету. Серія біологічна. 2011. Випуск 57
Obviously, increase of duration of ferrous (III) chloride hexahydrate influence on D. acetoxidans cells caused the enhance of catalase activity. It is possibly could be explained by reactive oxygen species production, especially of hydrogen sulfide creation under the influence of investigated metal salt. It was investigated that existence of unconjugated Ferrous ions, sulfides and sulfhy-dryl groups in bacterial growth medium enhances peroxide radicals production that significantly increases bacterial antioxidant defensive system activity .
Under the influence of ferrous (II) sulfate the highest catalase activity was observed on the second day of bacterial growth with addition of 1.5 mM of metal salt in the growth medium. It equaled 28,52±0,34 umol/minxmg of protein (fig. 1, B). Increase of metal salt concentration in the growth medium from 0.5 to 1.5 mM caused the enhance of catalase activity by 2.1-2.5 times on the second and by 42-80% on the third day of growth respectively in comparison with control samples. Activity of investigated enzyme decreased by 65% under the influence of 0.5 mM of FeSO4 and by 3.3 times under the influence of 1.5 mM of investigated Ferrous salt respectively on the fourth day of growth. Catalase activity increased by 2.4 and 1.5 times on the second and third days and decreased by 3 times on the fourth day of growth respectively under the influence of 2.5 mM of FeSO4 in comparison with control samples. Possibly, increase of duration of investigated metal salt influence doesn't cause toxic effect on investigated bacterial cells. As a result, catalase activity as one of the mechanisms of antioxidant defensive system decreased.
Concentration of FeCl3 x 6]H,O in the medium, ml Concentration of FeSO4 in the medium, ml
Fig. 1. The influence of (A) ferrous (III) chloride hexahydrate and (B) ferrous (II) sulfate on catalase actvity of cell-free D. acetoxidans extracts during four days of cultivation.
Obviously, Fe2+ as a final products of dissimilative Fe3+- reduction are necessary for supporting of normal cell functionality of D. acetoxidans bacteria .
Results of nickel (II) chloride hexahydrate influence on catalase activity of sulfur-reducing D. acetoxidans bacteria are presented on the fig. 2, A. The maximal activity of investigated enzyme was observed on the second day of growth under the influence of 0.5 mM of NiCl2x6H20. It equaled 30,49±0,63 umol/minxmg of protein. Catalase activity was higher by 2.7; 2 and 1.7 times respectively on the second, third and fourth days of growth in comparison with control samples under the influence of this metal salt hydrate concentration. Increase of NiCl2x6H2O concentration to 2.5 mM caused the increase of catalase activity by 67 and 32% respectively on the second and third days of growth compared with control samples. On the fourth day of growth it decreased to the control samples level.
Possibly, high concentrations of hydrogen peroxide are produced on the first stages of different concentrations of nickel (II) chloride hexahydrate influence on investigated bacteria.
As a result, high catalase activity was observed during the second day of D. acetoxidans bacteria growth under the influence of NiCl2x6H20.
Addition of different CoCl2>6H2O concentrations into the growth medium caused the highest catalase activity of sulfur-reducing D. acetoxidans bacteria under the influence of 2.0 mM of investigated metal salt hydrate on the second day of growth. It equaled 38,68±0,59 umol/ minxmg of protein (fig. 2, B).
] 2 day ] 3 day ] 4 day
] 2 day ] 3 day ] 4 day
0,5 1,0 1,5 2,0 2,5 Concentration of NiCl2 x 6H2O in the medium, ml
Concentration of CoCl2 x 6H2O in the medium, ml
Fig. 2. The influence of (А) nickel (II) chloride hexahydrate and (B) cobalt (II) chloride hexahydrate on catalase activity of cell-free D. acetoxidans extracts during four days of cultivation.
Catalase activity increased by 3.4; 2.6 times and by 86% respectively on the second, third and fourth days of growth compared with control samples under the influence of investigated cobalt (II) chloride hexahydrate concentration. Unlike NiCl2>6H2O influence on investigated enzyme activity of D. acetoxidans, increase from 0.5 to 2.5 mM of cobalt salt hydrate concentration caused the increase of investigated enzyme activity in comparison with control samples during the whole investigated period of cultivation. It increased by 3, 2.6 and 1.8 times respectively on the second, third and fourth days of growth in comparison with control samples under the influence of 2.5 mM of cobalt (II) chloride hexahydrate.
Obviously, increase of Cobalt ions concentration in the bacterial growth medium causes the intracellular ROS content enhance on the first stages of metal-bacteria interaction. It is caused cell defensive mechanisms activation, especially activation of antioxidant system as a reaction to stress, caused by transition metal long-term exposure. Possibly, high Co2+ concentrations are more toxic to investigated bacterial cells in comparison with Ni2+.
Catalase activity changes of sulfur-reducing D. acetoxidans bacteria under the influence of manganese (IV) oxide have been investigated. MnO2 and FeCl3>6H2O are strong prooxidants. That's why catalase activity was the most high under the influence of these compounds in comparison with all other investigated transition metals influence. Addition of manganese (IV) oxide into the growth medium caused the maximal activity of investigated enzyme under the influence of 1.5 mM of Mn4+ on the third day of growth. It equalled 56,32±0,67 umol/minxmg of protein (fig. 3, A). Under such conditions investigated enzyme activity increased by 4.6 times in comparison with control samples. Increase of MnO2 concentration from 0.5 to 2.5 mM caused the inhibition of catalase activity by 1.4-1.8 times on the second, 4.3-4.1 times on the third and 2.3-2.2 times on the fourth days of cultivation in comparison with control samples. Obviously, existence of MnO2 in the cultural medium causes the intensive reactive oxygen species production. It causes an activation of catalase synthesis as the mechanism of cell defense against an oxidative stress.
Decrease of investigated enzyme activity possibly is caused by partial manganese (IV) oxide cleavage and Mn4+ involving in the process of Mn4+ - reduction, which is held by sulfur-reducing D. acetoxidans bacteria. In this process they support themselves with the energy and Mn2+ that are needed for bacterial cells normal physiological and biochemical functionality [8, 9].
Under the influence of manganese (II) chloride tetrahydrate the highest catalase activity of sulfur-reducing D. acetoxidans bacteria was observed on the fourth day of growth with addition of 1.0 mM of metal salt hydrate in the growth medium. It was 36,62±0,70 umol/minxmg of protein under such growth conditions (fig. 3, B). Investigated enzyme activity increased by 2.9 times under the influence of this concentration of MnCl2>4H2O in comparison with control samples. Increase from 0.5 to 2.5 mM of metal chloride hexahydrate concentration in the growth medium caused the increase of catalase activity by 2.6-2.3, 2.6-2.4 and 2.8-2.7 times respectively on the second, third and fourth days of cultivation in comparison with control samples. Under the influence of MnCl2>4H2O catalase activity was lower in comparison with the influence of MnO2 that is strong prooxidant.
о ад В x с
"о в в
] 2 day ] 3 day ] 4 day
Concentration of MnO2 in the medium, ml
k 0,5 1,0 1,5 2,0 2,5
Concentration of MnCl2 x 4H2O in the medium, ml
Fig. 3. The influence of (А) manganese (IV) oxide and (B) manganese (II) chloride hexahydrate on catalase actvity of cell-free D. acetoxidans extracts during four days of cultivation.
Catalase activity of D. acetoxidans bacteria almost didn't change during four days of bacterial growth under the influence of different manganese (II) chloride hexahydrate concentrations. Possibly it could be explained by the fact that increase of MnCl2x4H2O concentration and duration of it's influence cause the toxic effect on cell's enzyme functionality, activity of reparation systems, microbial transport systems etc. Possibly, catalase activity increases as one of cell defensive mechanisms against negative influence of reactive oxygen species which are produced because of MnCl2>4H2O long-term exposure on the cells of investigated bacteria.
Thus, the highest catalase activity of sulfur-reducing D. acetoxidans bacteria was observed under the influence of 1.5 mM of MnO2 and 0.5 mM of FeCl3>6H2O respectively on the third and fourth days of cultivation in comparison with control samples. Under such conditions it was higher by 2 times in comparison with the influence of other investigated transition metal compounds. Under the influence of NiCl2>6H2O, FeSO4 and CoCl2>6H2O the highest catalase activity was observed on the second day of growth with addition of 0.5 mM, 1.5 mM and 2.0 mM of investigated metal salts respectively. Under the influence of MnCl2>4H2O the maximal activity of investigated enzyme was observed with addition of 1.0 mM of metal salt hydrate into the growth medium. Obviously, ferrous (III) chloride hexahydrate and manganese (IV) oxide that are strong oxidants, served as prooxidants towards sulfur-reducing bacterial D. acetoxidans cells.
As a result, the activity of their antioxidant defensive system, in particular catalase activity, was maximal under such influence. Effect of other investigated transition metal compounds caused lower investigated enzyme activity. Possibly, it shows that NiCl2>6H2O, FeSO4 and CoCl2>6H2O possess less toxic effect toward the cells of investigated bacteria in comparison with FeCl3 >6H2O та MnO2.
It was shown that sulfur-reducing Desulfuromonas acetoxidans bacteria possess antioxidant defensive system activity, in particular catalase functioning, under the derimental influence of different concentrations of transition metals, despite that there are obligatory anaerobic microorganisms. The influence of such transition metal compounds as FeSO4, FeCl3>6H2O, MnO2, MnCl2x4H2O, NiCl2x6H2O and CoCl2x6H2O on catalase activity of investigated bacteria has been investigated. The maximal activity of investigated enzyme has been observed under the influence of 0.5 mM, 1.5 mM and 2.0 mM of NiCl2>6H2O, FeSO4 and CoCl2>6H2O respectively on the second day of growth, 1. 5 mM of MnO2 on the third day of growth and 0. 5 mM of FeCl3>6H2O and 1.0 mM of MnCl2>4H2O on the fourth day of bacterial cultivation in comparison with control samples. Under the influence of different concentrations of ferrous (III) chloride hexahydrate and manganese (IV) oxide catalase activity was higher by 2 times in comparison with the influence of other investigated transition metal compounds.
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ВПЛИВ СПОЛУК ПЕРЕХІДНИХ МЕТАЛІВ НА АКТИВНІСТЬ КАТАЛАЗИ У КЛІТИНАХ СІРКОВІДНОВЛЮВАЛЬНИХ БАКТЕРІЙ
О. Василів, С. Гнатуш
Львівський національний університет імені Івана Франка вул. Грушевського, 4, Львів 79005, Україна e-mail: email@example.com
Desulfuromonas acetoxidans - грамнегативні облігатно анаеробні сірковіднов-лювальні бактерії. Для захисту від токсичних і мутагенних сполук й усунення токсичного впливу кисню в аеробів та факультативних анаеробів існує ензиматична система антиоксидантного захисту, однією з найважливіших складових якої є каталаза. Припускають, що відсутність чи низька активність ферментів даної системи в облігатно анаеробних мікроорганізмів, зокрема сірко- та сульфатредукуючих бактерій, зумовлює їхню підвищену чутливість до кисню та дії токсичних сполук, зокрема перехідних металів. Досліджено вплив FeSO4, FeCl3>6H2O, MnO2, MnCl2>4H2O, NiCl2>6H2O та CoCl2>6H2O на активність каталази сірковідновлювальних бактерій D. acetoxidans. Виявлено максимальну активність досліджуваного ферменту за впливу 0,5 мM, 1,5 мM та 2,0 мM відповідно NiCl2>6H2O, FeSO4 та CoCl2>6H2O на другу добу, 1,5 мM MnO2 на третю добу та 0,5 мM FeCl3>6H2O і 1,0 мM MnCl2>4H2O на четверту добу культивування, порівняно з контролем.