Multiple respiratory bursts as a response to veratrate stress in Rhodococcus erythropolis cells.

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.

Relationships between demethylase activity, formaldehyde and oxygen during incubation of Rhodococcus erythropolis with veratrate.

Relationships between demethylase activity, formaldehyde and oxygen were investigated. Demethylase activity was measured against the following substrates: veratric, vanillic, and isovanillic acids, as well as in the presence of guaiacol. The influence of ATP and GTP on demethylase activity was also checked. Demethylase activity was found to be dependent on the capability of the cells for endogenous oxygen uptake. In some cases ATP produced the opposite effect: instead of being taken up, oxygen was released, which suggested a reversibility of the demethylation reaction. Curiously enough, GTP demonstrated the same effect. Changes in enzyme activity were correlated with those occurring in the level of formaldehyde. The latter increased after addition of ATP, but decreased after addition of GTP.