MULTIPLE RESPIRATORY BURSTS AS A RESPONSE TO VERATRATE STRESS IN RHODOCOCCUS ERYTHROPOLIS CELLS

Abstract

Although Rhodococcus spp. strains are able to degrade methoxyphenols by enzymatic means, the contact with veratric acid (3, 4-dimethoxybenzoic acid, hereafter called veratrate) is very stressful for the cells of Rhodococcus erythropolis DSM 1069 (Rh). Within 5 min of contact veratrate in phosphate buffer, the emergence of many vacuoles was observed in the cell body and respiratory bursts, with violent endogenous oxygen uptake, took place several times during the 24 h incubation. During these peaks (where the cells were in their MAX states), increased activity of NADH oxidase was noted, accompanied by maximal accumulation of vanillic and isovanillic acids (3-methoxy-4-hydroxybenzoic acid and 4-hydroxy-3-methoxybenzoic acid respectively, hereafter called vanillates) in the incubation medium, which appeared to be products of veratrate demethylation. At the troughs (cell in their MIN state), the vacuoles disappeared from the cell body, oxygen uptake was normal, and the pool of vanillates decreased while the veratrate level in the medium increased. The cells from MAX and MIN states reacted in opposite ways in the presence of either formaldehyde and GSH, or paraquate and cAMP. The NADH oxidase activity, measured as oxygen uptake against NADH in the membrane pellets of MAX and MIN stage cells, differed in their response to the exogenous presence of FAD, ATP, cAMP, catalase, GSH, H(2)O(2)and methoxyphenolic substrates. The periodic character of these events is described here. Co-operation between two multiprotein membrane complexes (NAD(P)H oxidase and 3-O/4-O-demethylases) in Rhodococcus erythropolis cells and their competition for two common substrates-NAD(P)H and O(2)-is proposed as an explanation for rhythmical nature of these reactions.

Nonlinear changes in the activity of the oxygen-dependent demethylase system in Rhodococcus erythropolis cells in the presence of low and very low doses of formaldehyde

“…Cells of Rhodococcus erythropolis (previously Nocardia sp DSM 1069) were cultivated on a liquid medium according to [17]. Cultures were grown in 250 ml conical flasks filled with 100 ml medium on a rotary shaker at 30°C.…”

“…In 1998, in a study of transformations of methoxyphenols in fungi and ligninolytic bacteria [16], we described the cyclic process of demethylation/methylation of veratric acid and vanillic acids in Rh. erythropolis [17], coupled with the activity of membrane bound NADH oxidase [18] and periodic reconstruction of the pool of veratric acid as a result of alternate activation of methylase and demethylase activities in these bacteria combined with changes in the levels of oxygen uptake. Figure 1 shows a hypothetical mechanism for these processes.…”

“… The hypothetical mechanism of veratrate demethylation to vanillate by Rhodococcus erythropolis cells, dependent on activated state of formaldehyde (Malarczyk and Pazdzioch-Czochra, 2000) [17]. Reproduced with permission of the Biochemical Society.…”

A Cycle of Enzymatic Reactions that Behaves like a Neuronal Circuit

“…It was found (Malarczyk, 1989;Malarczyk & Kochmanska-Rdest, 1990;Malarczyk & Pazdzioch-Czochra, 2000) that the bacterium Rhodococcus erythropolis can utilize veratric acid as a carbon source. The bacterium was grown on agar medium supplemented with 0.04% guaiacol and stored on 2% agar slants according to Malarczyk (1984).…”

“…Almost identical optical densities of the bacterial suspension before and after incubation suggested that bacteria suspended in a bu!er with 0.02% veratric acid grew very slowly. Instead, oscillations in the concentrations of veratric acid and its demethylation products with the period of 8}10 hr have been observed (Malarczyk, 1989;Malarczyk & Kochmanska}Rdest, 1990;Malarczyk & Pazdzioch} Czochra, 1990, 2000. Two demethylation steps transformed veratric acid into protocatechuic acid.…”