STUDY OF CHLORATE-RESISTANT MUTANTS IN ESCHERICHIA COLI K 12 .I. RESTORATION IN VITRO OF NITRATE REDUCTASE ACTIVITY IN PARTICULAR

Study of chlorate-resistant mutants in Escherichia coli K 12. I. Restoration in vitro of nitrate reductase activity in particular. Using chlorate as a selective agent, we have isolated two genetically distinct types of chlorate-resistant mutants in Escherichia coli K 12: chl A15 and chl B24. Both of these types are defective in several enzymatic activities specific to anaerobic respiration, and in particular, in nitrate reductase (NADH: nitrate oxidoreductase, EC 1.6.6.1) and hydrogen lyase (formate:cytochrome b1 oxidorecductase. EC 1.2.2.1). By mixing the cell-free extracts of these two mutants under certain precise conditions, we achieve a restoration of the nitrate reductase activity lost by each of the two organisms. When this complementation is performed with high-speed supernatants of the extracts (170 000 × g for 90 min), particles having nitrate reductase activity are formed de novo. The behavior of these particles on sucrose gradients is the same as that of particles having nitrate reductase activity isolated from the wild strain. We describe the conditions for this type of complementation, the nature of the reaction, and the biochemical characteristics of the synthesized particles. The restoration of nitrate reductase activity by complementation occurs only in the absence of O2, in a narrow range of temperature and pH (32°, pH 7-7.6), and at certain protein concentrations. The kinetics of complementation were studied by following the increase of nitrate reductase activity. The reaction is complete after 2 h of incubation. The enzymatic activity thus reconstituted is equal to 19 units/mg of total protein and represents a tenth of the wild-type activity. In the course of complementation 15-20%, of the total soluble protein becomes particulate. This includes the cytochrome b1 which was present in the soluble state in the mutant extracts. From these results, we present an hypothesis as to the nature and role of the chl B gene product. © 1969.