Regulation of flavoprotein synthesis studied in vivo in a riboflavin-requiring mutant of Arthrobacter oxidans

Abstract

The biosynthesis of two flavoproteins, 6-hydroxy-d-nicotine oxidase with covalently bound FAD and 6-hydroxy-l-nicotine oxidase containing non-covalently bound FAD, was studied in wild-type cells and in a riboflavin-requiring mutant of Arthrobacter oxidans. In the mutant cells, the rate of synthesis and the maximal activity level of both enzymes after induction by nicotine depended on the amount of added riboflavin. The low rate of synthesis in the presence of 2 μM riboflavin could be enhanced during the induction phase by further addition of riboflavin (33 μM). Inhibitors of translation (chloramphenicol or streptomycin) completely blocked the synthesis of both flavoproteins.

Inhibitors of transcription (rifamycin S or actinomycin D) stopped the synthesis of both enantiozymes in wild-type cells and in the mutant grown in the presence of a saturating supply of riboflavin (15 μM). Under conditions of restricted flavoprotein synthesis (2 μM riboflavin in the medium), however, the mutant cells continued to synthesize the enzyme for 2–3 h after the addition of the transcription inhibitors. It appears, that in these cells a rather stable m-RNA accumulated during riboflavin-limited flavoprotein synthesis.

The dependence of the effect of transcription inhibitors on the extracellular supply of riboflavin suggests that the regulation of the synthesis of both flavoproteins occurs not only by control of gene expression (induction by nicotine), but also at the level of translation through the availability of FAD.