|Cancer||Cell death||Cell cycle||Cytoskeleton||Exo/endocytosis||Differentiation||Division||Organelles||Signalling||Stem cells||Trafficking|
Cell Biology International (2003) 27, 325336 (Printed in Great Britain)
Relationship of demethylation processes to veratric acid concentration and cell density in cultures of Rhodococcus erythropolis
Marzanna Paździoch‑Czochraa*, Elżbieta Malarczyka and Jan Sielewiesiukb
aDepartment of Biochemistry, M. Curie-Skłodowska University, 20-031 Lublin, Poland
bDepartment of Biophysics, Institute of Physics, M. Curie-Skłodowska University, 20-031 Lublin, Poland
The aim of this study was to investigate the correlation between veratrate degradation, veratric acid concentration and cell density in Rhodococcus erythropolis cultures. The optimum culture conditions for veratrate demethylation proved to be a cell density of A
Keywords: Rhodococcus erythropolis, Veratric acid, Free radicals, Formaldehyde, Demethylation.
*Corresponding author. Fax: +48-815-375-761.
Phenolic compounds are very common substances in the environment, and are produced by many organisms de novo, as well as resulting from the degradation of humic acids and lignins. Microbiological degradation of phenolic compounds, particularly xenobiotics from the degradation of lignin, has practical importance (Eriksson,1993; Harwood and Parales, 1996; Kirk, 1984). Besides the fungi Basidiomycetes, Ascomycetes and some Fungi imperfecti (Higuchi, 1990), the ligninolytic strains of Nocardia and Rhodococcus can also degrade methoxyphenolic compounds during the demethylation reaction (Eggeling and Sahm, 1980; Finnerty, 1992; Malarczyk, 1984, 1989; Malarczyk and Paździoch-Czochra, 2000). Demethylation in fungi proceeds with the production of phenolic compounds and formaldehyde, and is similar to demethylation in Pseudomonas (Bernhardt et al., 1970, 1975; Ribbons, 1970, 1971). HCHO is an intermediate product in that reaction and may act as a participant in the formation of methoxyl groups during methylation and as a product in the demethylation of methoxyl groups (Malarczyk, 1991; Malarczyk et al., 1987).
Demethylation of veratric acid (3,4-dimethoxybenzoic acid), a substrate for the 3-O and 4-O-demethylases (demethylating monooxygenase), is accompanied by oscillatory changes in the endogenous uptake of oxygen, which is consequently a substrate for inducible monooxygenase (Malarczyk, 1989; Malarczyk and Kochmańska-Rdest, 1990). Due to the cytoplasmic location of oxygenases, the turnover of aromatic compounds depends on the availability of O
Oscillation is a significant biological phenomenon, underlying cell function, properties and behaviour(Gilbert and Ferreira, 2000; Gilbert and Llyod, 2000). Oscillations are known to occur in many enzymatic reactions, such as glycolysis (Civelek et al., 1997) and the peroxidase–oxidase reaction (Hauser and Olsen, 1998), hormonal and neurotransmitter signalling (Goldbeter et al., 1990; Huser et al., 2000), as well as protein concentration levels and the activity of many enzymes (Calvert-Evers and Hammond, 2000; Ferreira et al., 1996a,b,c; Hammond et al., 2000; Pogue et al., 2000). A kinetic model for the relationship of oscillations to methoxyphenol transformations has been proposed for R. erythropolis cultures. This model was a four-membered cycle of enzymatic reactions with repression of enzyme synthesis in the presence of cyclic symmetry (Sielewiesiuk et al., 1999).
Veratric acid has two methoxyl groups that are removed by 3-O- and 4-O-demethylases. Two isomeric vanillic acids (vanillic and isovanillic) resulting from partial demethylation of veratrate and protocatechuic acids (products of total demethylation) appear in the incubation medium. Each reaction follows the scheme proposed by Ribbons (1970, 1971) and is the sum of many intermediate reactions. The mechanism of free radical-dependent demethylation of veratrate by R. erythropolis cells involves the activation of NADH oxidase and 3-O/4-O-demethylases, the production of free radicals, and the production of two pools of formaldehyde—one as the result of stress conditions and the other as the result of the demethylation process (Malarczyk and Paździoch-Czochra, 2000). The cooperation between two multiprotein membrane complexes, NAD(P)H oxidase and 3-O/4-O-demethylases, in R. erythropolis cells and their competition for two substrates, NAD(P)H and O
2 Materials and methods
2.1 Biological material
R. erythropolis (Nocardia sp. DSM 1069) was cultivated as described previously (Malarczyk and Paździoch-Czochra, 2000).
2.2 Induction experiments
After culture, a suspension of cells of density A 1.
2.3 Preparation of cell homogenates
The cell suspension (10
2.4 Determination of oxygen uptake
Oxygen uptake by the Rhodococcus cells was monitored with a biological oxygen monitor (YSI model 5300). During measurements, the standard vessel contained 3
2.5 Determination of phenolic compound concentration
The concentration of phenolic compounds was determined spectrophotometrically based on a colorimetric reaction with diazosulphanilamide (DASA), according to Malarczyk (1989). A volume of 0.2
2.6 Determination of formaldehyde concentration
Formaldehyde concentration was determined spectrophotometrically with a Merck (Darmstadt, Germany) test, correcting the volume of the sample to 1
2.7 Determination of superoxide radicals
Relative concentrations of superoxide radicals were assessed spectrophotometrically in alkaline medium by detection of the superoxide radical anion-dependent formation of formazan from nitrotetrazolium blue (NBT). The reaction was carried out in 3
2.8 Determination of hydrogen peroxide concentration
The concentration of H
2.9 Determination of superoxide dismutase-like activity
Superoxide dismutase (SOD)-like activity was calculated on a percentage basis by the auto-oxidation inhibition of pyrogallol. Briefly, 0.2
Changes in oxygen levels during incubation of R. erythropolis cells with veratrate (0.01, 0.02, 0.04%). The cells were suspended in phosphate buffer at densities A
Concentration of vanillic acids during incubation of R. erythropolis cells with veratrate (0.01, 0.02, 0.04%). The cells were suspended in phosphate buffer at densities A
Concentration of protocatechuic acid during incubation of R. erythropolis cells with veratrate (0.01, 0.02, 0.04%). The cells were suspended in phosphate buffer at densities A
3.1 Endogenous oxygen uptake
Cells of R. erythropolis transferred to phosphate buffer, pH 7.5, in the logarithmic growth phase participated very actively in the transformation of methoxyphenolic compounds. Although the cells did not show typical features of cell growth, endogenous oxygen uptake showed oscillatory changes (maximum and minimum oxygen uptake) with transient oxygen burst events dependent upon the time of incubation, the concentration of veratric acid and the density of cells in the incubation medium (Fig. 1). The highest oxygen uptake was observed between 2.5 and 3
3.2 The demethylation process
The concentrations of vanillic acids and protocatechuic acid were monitored as the activity of 3-O- and 4-O-demethylases. We were only able to show the presence of all the products of veratrate demethylation in the cells and incubation medium in the presence of 0.02% veratrate (A
Concentration of formaldehyde during incubation of R. erythropolis cells with veratrate (0.01, 0.02, 0.04%). The cells were suspended in phosphate buffer at densities A
Full-size table (<1K)
The cells were suspended in phosphate buffer at densities A660=0.5; A660=1; A660=2.
−, lack; +, presence; ±, small amounts; ++, accumulation.
3.3 Production of reactive oxygen species and superoxide dismutase activity
In all cases, high levels of hydrogen peroxide were observed in the incubation medium (Fig. 5). Increasing the cell density caused a decrease in hydrogen peroxide. In contrast, increasing the concentration of veratric acid maintained a high level of hydrogen peroxide throughout the incubation period.
Concentration of hydrogen peroxide during incubation of R. erythropolis cells with veratrate (0.01, 0.02, 0.04%). The cells were suspended in phosphate buffer at densities A
The production of superoxide radical anions was very violent in the culture with cell density A
Concentration of free radicals during incubation of R. erythropolis cells with veratrate (0.01, 0.02, 0.04%). The cells were suspended in phosphate buffer at densities A
SOD-like activity during incubation of R. erythropolis cells with veratrate (0.01, 0.02, 0.04%). The cells were suspended in phosphate buffer at densities A
Rhodococcus and Nocardia are microorganisms that are able to decompose phenolic compounds (Bell et al., 1998; Finnerty, 1992; Hopper, 1991). Our earlier study showed that, although R. erythropolis cells can degrade veratric acid, contact of cells with veratrate acts as a chemical stress-inducer and causes the production of superoxide radicals and a pool of stress HCHO—as a stress response, as well as NADH oxidase and demethylase activation, and episodes of oxidative burst. These are all events that are oscillatory, or periodic, in character (Malarczyk and Paździoch-Czochra, 2000). It is therefore interesting to note the way in which changes in cell density influence the dynamics of veratrate degradation and the appearance of oscillations.
The results of these experiments showed the difference in quantity between the products of partial demethylation of veratric acid (vanillic and isovanillic acids) and those of total demethylation (protocatechuic acid). Among the three cell densities studied, only A
The three different concentrations of veratrate examined in this study influenced the demethylation process to varying degrees. For the lowest concentration of this compound (0.01%), only one oxidative burst was observed, but for the other two concentrations (0.02 and 0.04%) there were two episodes of violent oxygen uptake. In the case of 0.01% veratrate, protocatechuic acid was not detected in the medium. For 0.04% veratrate, only small amounts of protocatechuic acid were detected in the incubation medium, as opposed to high levels of vanillic acid. The latter was maintained at a constant level, because the transformation of vanillic acid into protocatechiuc acid (product of total demethylation) was not observed. In the presence of 0.01 and 0.04% veratrate, only small amounts of superoxide radicals were seen, which may have disturbed the mechanism of free radical-dependent demethylation.
Changes in the concentration of veratric acid and density of the cell suspension influenced the production of HCHO and O
The adaptation mechanism to new environmental conditions is activated in the cells of R. erythropolis. According to the phases of stress syndrome (Selye, 1956; Tyihak et al., 1998), the cells of R. erythropolis adopted an alarm phase metabolism and reached maximum resistance of stress factors after contact with 0.02% veratrate. In the alarm phase, intensive demethylation of precursors rich in methoxyl groups occurs, which appear as an extra pool of HCHO and superoxide radical anions. The violent production of these particles at moments of stress between plants and microbes is common. It has been proven that the amount of HCHO dramatically increases in biotic stress, e.g. in infected Nicotiana tobacum tobacco leaves (Burgyan et al., 1982), in cells of Citrullus vulgaris after infection with Fusarium oxysporum (Sardi and Balla, 1997), and in abiotic stress, e.g. in bean leaves after heat shock (Tyihak et al., 1989), in Quercus cerris after cold shock (Albert et al., 1997), and in some Basidiomycetes after heat shock and treatment with cadmium ions (Jarosz-Wilkołazka et al., 1998, 2001).
The synchrony of enzymatic-dependent demethylation and non-enzymatic methylation is due to the presence of reactive oxygen species (Malarczyk and Paździoch-Czochra, 2000). The appearance of a rhythmic, cyclic metabolic process is due to the adaptation of cells to new environmental conditions and the activation of adaptation processes. The quantitative correlation between cells and their environment, the cell density and, in consequence the availability of substrate, influences the cells' reactions, so that they are able to react to stressful conditions, either by the rhythmic, oscillatory transformation of substrates and products or with a chaotic, short-term response.
Our study shows that an over-abundance of accumulated cells of R. erythropolis results in a lack of protocatechuic acid. The latter is important for dearomatization, giving the cells access to carbon energy from its aromatic ring. Conversely, the scattering of cells when density is low hinders effective cooperation between them. The production of reactive oxygen species is common to all the cultures examined in this study. Their number seems to directly influence the course of events; too few reactive oxygen species cannot initiate the demethylation process, and too many can inhibit it.
. . Albert L, Nemeth Zsi, Varga SZ, Barna T. The cold shock in the early stage of European turkey oak (Quercus cerris L.). Collected abstracts of stress of life: stress and adaptation from molecules to man. International Congress, Budapest, Hungary, July 1–5, 1997. p. 180.
Arras T, Schirawski, J, Unden, G. Availability of O
Bernhardt F-H, Staudinger, H, Ulrich, V. The properties of p-anisate O-demethylase in cell-free extracts of Pseudomonas sp. Hoppe-Seyler's Z Physiol Chem 1970:351:467-78
Civelek VN, Deeney, JT, Fusonie, GE, Corkey, BE, Tornheim, K. Oscillations in oxygen consumption by permabilized clonal pancreatic β-cells (HIT) incubated in an oscillatory glycolyzing muscle extract. Diabetes 1997:46:51-6
Eggeling L, Sahm, H. Degradation of coniferyl alcohol and other lignin-related aromatic compounds by Nocardia sp. DSM 1069. Arch Microbiol 1980:126:141-8
Ferreira GMN, Hammond, KD, Gilbert, DA. Independent high-frequency oscillations in the amounts of individual isozymes of lactate dehydrogenase in Hl60 cells. Cell Biol Int 1996:20:607-11
Ferreira GMN, Hammond, KD, Gilbert, DA. Distinct, very high frequency oscillations in the activity and amount of active isozyme of lactate dehydrogenase in murine erythroleucaemic cells and a cell-free system. Cell Biol Int 1996:20:625-33
Ferreira GMN, Wolfle, H, Hammond, KD, Gilbert, DA. High frequency oscillations in the activity of phosphotyrosine phosphatase in murine erythroleucaemic cells: action of insulin and hexamethylene bisacetamide. Cell Biol Int 1996:20:599-605
Goldbeter A, Dupont, G, Berridge, MJ. Minimal model for signal-induced Ca+2oscillations and for their frequency encoding through protein phosphorylation. Proc Natl Acad Sci U S A 1990:87:1461-5
Hammond KD, Sacage, N, Littlewood, M. Rhythmic patterns in the expression of the ras oncogene in proliferating and differentiating erythroleukemia cells. Cell Biol Int 2000:24:8:529-37
Jarosz-Wilkołazaka A, Fink-Boots, M, Malarczyk, E, Leonowicz, A. Formaldehyde as the proof and an answer on various kind of stress in some Basidiomycetes. Acta Biol Hung 1998:49:2–4:393-403
Jarosz-Wilkołazaka A, Fink-Boots, M, Malarczyk, E, Leonowicz, A. . Jarosz-Wilkołazka A, Malarczyk E, Pirszel J, Skowroński T, Leonowicz A. Uptake of cadmium ions in white-rot fungus Trametes versicolor: effect of Cd (II) ions on the activity of laccase. Cell Biol Int 2002;26(7):605–613.
Malarczyk E. Substrate-induction of veratric acid O-demethylase in Nocardia sp. Acta Biochim Pol 1984:31:4:383-95
Malarczyk E, Rogalaski, J, Kochmańska-Rdest, J, Leonowicz, A. Formaldehyde as the possible substrate in formation and methylation of aromatic ring. The role of formaldehyde in biological system. Proc. II Intern. Formaldehyde Conference 1987:184-8
Malarczyk E, Rogalaski, J, Kochmańska-Rdest, J, Leonowicz, A. . Malarczyk E, Zińko E, Nowak G, Ziaja J, Kochmańska-Rdest J, Leonowicz A. The relation between the concentration of phenolic activity of manganese peroxidase in cultures of two species of Pleurotus during fruit body formation. Second TAPPI Biological Sciences Symposium, San Francisco, October 19–23, 1997.p. 391–3.
Marlkund S, Marklund, C. . Paździoch M, Malarczyk E, Sielewiesiuk J. Formaldehyde as a regulator of oscillations in oxygen-and photo-related process of demethylation in Rhodococcus erythropolis cell suspension. Collected abstracts of stress of life: stress and adaptation from molecules to man. International Congress, Budapest, Hungary, July 1–5, 1997. p. 187.
Ribbons DW. . Sardi É, Balla J. Effect of Fusarium infection on the formaldehyde cycle in Citrullus vulgaris L. Collected abstracts of stress of life: stress and adaptation from molecules to man. International Congress, Budapest, Hungary, July 1–5, 1997. p. 180.
Sielewiesiuk J, Czubla, A, Malarczyk, E, Paździoch, M. Kinetic model for oscillations in a cycle of enzymatic reactions related to methoxyphenols transformation in Rhodococcus erythropolis culture. Cell Mol Biol Lett 1999:14:1:131-46
Tyihak E, Kiraaly, Z, Gullner, G, Szarwas, T. Temperature-dependent formaldehyde metabolism in bean plants. The heat shock response. Plant Sci 1989:59:133-9
Received 6 December 2001/11 September 2002; accepted 8 October 2002doi:10.1016/S1065-6995(02)00282-2