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Cell Biology International (2003) 27, 325–336 (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 A660=1 and a concentration of 0.02% veratrate. All the products of demethylation (i.e. vanillic and protocatechuic acids) were found to be present and correlated with the appearance of high levels of free radicals and formaldehyde after contact of the cells with veratrate. Demethylation was accompanied by oscillatory changes in the levels of endogenous oxygen uptake and phenolic products. Changes in veratrate concentration and cell density caused a disturbance in the demethylation process and also in the efficiency of phenolics, formaldehyde and reactive oxygen species.

Keywords: Rhodococcus erythropolis, Veratric acid, Free radicals, Formaldehyde, Demethylation.

*Corresponding author. Fax: +48-815-375-761.

1 Introduction

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 O2in the cytoplasm, which is important for bacterial growth (Arraset al., 1998). During the cultivation of Nocardia autotrophica (Malarczyk and Kochmańska-Rdest, 1990) and Rhodococcus erythropolis (Eggeling and Sahm, 1980; Malarczyk, 1984; Paździoch, Malarczyk and Sielewiesiuk, 1997; Malarczyk and Paździoch-Czochra,2000) with methoxyphenolic compounds, the concentration of products and substrates fluctuates. The proven oscillatory character of these fluctuations indicates the cooperation of many reactions that are dependent on the number of methoxyphenolic groups in demethylated compounds. The participation of reactive oxygen species in the oscillation phenomenon is also important. As a result of stress caused by the contact of cells with veratrate, there is an increase in H2O2and other reactive oxygen species (Malarczyk and Paździoch-Czochra, 2000).

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 O2, is responsible for the rhythmical nature of these reactions (Malarczyk and Paździoch-Czochra, 2000). The aim of this study was to establish the dependence of the dynamics of the demethylation process on veratric acid concentration and cell density in the incubation medium of R. erythropolis cultures.

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 A660=1 was incubated with 0.01, 0.02 or 0.04% veratric acid (3,4-dimethoxybenzoic acid) sodium salt, and used as a joint substrate for 3-O- and 4-O-demethylations, for 12h at 30°C. Two further cell densities, A660=0.5 and A660=2, were also incubated with 0.02% veratric acid. Thus, the following combinations of the two variables were used:


A660=1; 0.02% veratrate.


A660=1; 0.01% veratrate.


A660=1; 0.04% veratrate.


A660=0.5; 0.02% veratrate.


A660=2; 0.02% veratrate.

2.3 Preparation of cell homogenates

The cell suspension (10ml) was centrifuged and divided into supernatant (medium) and cell pellet. The cell pellet was later homogenized in a chilled mortar in 2.5ml of 0.06M phosphate buffer, pH 7.4, and centrifuged. The resulting supernatant contained the cellmaterial. The clear supernatant fluids (medium and cell material) were used for enzyme activity determination and measurement of phenolic compounds, formaldehyde, free radicals and hydrogen peroxide.

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 3ml of cell suspension. Each measurement continued for 15min per cell suspension and the oxygen uptake was calculated in nMO2/ml of suspended material, according toBernhardt et al. (1970).

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 0.2ml of each sample was mixed with 0.2ml of 2% sulphanilamide solutionin 10% hydrochloric acid followed by 0.2ml of 5% NaNO2. The mixture was stirred thoroughly, neutralized with 1ml of 20% Na2CO3, and absorbance at 400nm (for protocatechuic acid) and 500nm (for vanillic acids) was measured and compared with the calibration curve (y=18.25x−0.075, R2=0.0998 and y=6.85x−0.0218, R2=0.999, respectively).

2.6 Determination of formaldehyde concentration

Formaldehyde concentration was determined spectrophotometrically with a Merck (Darmstadt, Germany) test, correcting the volume of the sample to 1ml. Readings were taken at 560nm and compared with the calibration curve (y=0.0457x−0.0039, R2=0.999). The detection reaction was based on the condensation of aldehydes with 4-amino-3-hydrazino-5-mercapto-1,2,4-triazol (Dickinson and Jacobsen, 1970), which gave rise to purple-coloured tetrazine derivatives following atmospheric oxidation.

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 3ml of 0.05M NaOH containing 0.5μM NBT and 0.1ml of sample. After incubation at 25°C for 30min, the absorbance was monitored at 560nm. Using these conditions, the precipitation of dark-blue formazan was avoided (Malarczyk, Zińko, Nowak, Ziaja, Kochmońska-Rdest and Leonowicz, 1997)

2.8 Determination of hydrogen peroxide concentration

The concentration of H2O2was determined spectrophotometrically based on the absorbance at 240nm and obtained values were compared with a calibration curve (y=0.0282x+0.0286, R2=0.989).

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, 0.2ml of each sample was mixed with 0.1ml of 5mM pyrogallol and adjusted to a final volume of 3ml with 50mM Tris–HCl buffer, pH 8.5, containing 0.1mM EDTA. The auto-oxidation rate of pyrogallol was measured as absorbance changes at 315nm (Marlkund and Marklund, 1974) at 30°C. As suggested by Gilbert and Ferreira (2000), data were presented without error bars, as they may obscure the underlying curves, especially for high frequency rhythms.

All experiments were repeated three times. The relative standard deviation for three replicate determinations was 0.5%.

Fig. 1

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 A660=0.5; A660=1; A660=2.

Fig. 2

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 A660=0.5; A660=1; A660=2.

Fig. 3

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 A660=0.5; A660=1; A660=2.

3 Results

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 3h, and again between 5.5 and 7h of incubation for cells incubated with 0.02% veratrate (A660=1). The cells in the incubation medium of density A660=2 did not show oxygen burst events and the amount of oxygen uptake was below the level of solubility of oxygen in buffer. In experiments with three different concentrations of veratrate (0.01, 0.02, 0.04%) at density A660=1, the highest oxygen uptake was observed in cells incubated with 0.04% veratrate, but at the lowest concentration of veratrate (0.01%), the oxygen burst events occurred only during the first 5h of incubation.

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 (A660=1) (Figs. 2 and 3). We did not detect the presence of vanillic acids in the cells at density A660=0.5 or in the incubation medium at density A660=2. A lack of protocatechuic acid was seen with 0.01% veratrate, as well as density A660=2, in both the incubation medium and cells. The lowest density, A660=0.5, caused the accumulation of protocatechuic acid in the cells (Fig. 3 and Table 1). The concentration of protocatechuic acid fluctuated during the incubation period, particularly with 0.02% veratrate (A660=1). Formaldehyde was at its highest level at density A660=1 (0.02% veratrate). In the incubation medium with a density of A660=0.5, an accumulation of formaldehyde was observed during the incubation period, which peaked at 7h. In all other conditions, production of formaldehyde was distinctly lower (Fig. 4).

Table 1. Concentration of phenolic products during incubation of R. erythropolis cells with veratrate (0.01, 0.02, 0.04%)

Fig. 4

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 A660=0.5; A660=1; A660=2.


The cells were suspended in phosphate buffer at densities A660=0.5; A660=1; A660=2.

−, lack; +, presence; ±, small amounts; ++, accumulation.

Full-size image (34K) - Opens new windowFull-size image (34K)
a As sodium veratrate.

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.

Fig. 5

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 A660=0.5; A660=1; A660=2.

The production of superoxide radical anions was very violent in the culture with cell density A660=1 (0.02% veratrate) after just 30min of incubation (Fig. 6). The highest density of cells (A660=2) produced the highest levels of O2throughout the incubation period. In the case of density A660=0.5, as well as veratrate concentrations of 0.01 and 0.04%, only small numbers of free radicals were evident in the cells and incubation medium. Changes in the level of superoxide radical anions correlated with changes in SOD activity (Fig. 7). The highest activity was observed both in the cells and medium of cultures with a density of A660=0.5 and veratrate concentrations of 0.01 and 0.04%. In all cases, SOD activity showed periodic variations.

Fig. 6

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 A660=0.5; A660=1; A660=2.

Fig. 7

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 A660=0.5; A660=1; A660=2.

4 Discussion

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 A660=1 (0.02% veratrate) fulfilled these expectations in both the cells and incubation medium. In the other conditions tested, disturbances in the demethylation process were observed, particularly for A666=0.5 (only small amounts of protocatechuic acid in the medium) and A660=2 (the presence of phenolics in cells only). In comparison with A660=1 (0.02% veratrate), increasing (A660=2) and decreasing (A660=0.5) the cell density caused greater activity of SOD in both cells andmedium. The number of free radicals was lower than that observed for A660=1 (0.02% veratrate). This may be the reason why the demethylation of veratrate at densities A660=0.5 and A660=2 was not so effective. Our earlier study (Malarczyk and Paździoch-Czochra, 2000) showed that the presence of large amounts of free radicals at the beginning of the incubation period is necessary to activate the periodic demethylation process. The low levels of free radicals caused not only fewer oscillations, but also a lack of phenolics in the medium.

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 O2. Only in the presence of 0.02% veratrate (A660=1) was the full stress response of cells noted by the violent production of O2at the moment of cell contact with veratrate, and the production of stress HCHO—as a stress response. It is difficult to explain why, in the presence of 0.01 and 0.04% veratrate, the production of an extra pool of HCHO was not observed. It may be that the lowest concentration of veratrate was not toxic to the cells, but the highest (0.04%) may have inhibited the stress response and directed cell metabolism towards demethylation, which was not completed. A concentration of 0.02% veratrate and density A660=1 seem to be optimal conditions for the periodic demethylation process, because the production of both vanillic and protocatechuic acids was observed in both medium and cells.

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.


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Received 6 December 2001/11 September 2002; accepted 8 October 2002


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