Phenazine biosynthesis by a pseudomonad.

The acidophilic bacterium Pseudomonas phenazinium (N.C.I.B. 11027), isolated from soil by virtue of its ability to grow on L-threonine as sole source of carbon and nitrogen (Bell et al., 1972), was observed to produce the water-insoluble purple pigment iodinin (phenazine-l,6-diol 5,lO-dioxide) during growth on L-threonine or glycine media (Bell & Turner, 1973). Evidence was obtained that iodinin was metabolically related to the yellow pigment phenazine-l,6-diol and a number of additional water-soluble pigments were separated and provisionally identified as phenazines. Although some information is available on the mode of assembly of the phenazine skeleton by bacteria, details of the biosynthetic route and individual enzyme-catalysed reactions are not known. Experiments in which specifically labelled ~-['~C]shikimic a id was fed to Pseudomonas aureo faciens, Pseudomonas aeruginosa and Chromobacterium iodinum led Hollstein & Marshall (1972) and Hollstein & McCamey (1973) to conclude that phenazine synthesis by these bacteria proceeds by the symmetrical condensation of two identically substituted chorismic acid molecules. Herbert et al. (1974) have confirmed these results and propose the same mode of assembly for the iodinin skeleton. Two groups have obtained evidence that chorismic acid is the branch point compound leading from the shikimic acid pathway to the biosynthesis of phenazines in pseudomonads (Longley et al., 1972; Calhoun et al., 1972). We now report the identity of a number of phenazines other than iodinin produced by Pseudomonas phenazinium and describe experiments on themode of regulation or aromatic biosynthesis in the bacterium. Our hypothesis is that under certain culture conditions regulation of the shikimate pathway is impaired and the over-production (or under-utilization) of chorismate leads to phenazine formation by a secondary metabolic route. The pseudomonad was grown on L-threonine (5g/litre) plus mineral salts medium (Bell &Turner, 1973) except where indicated to the contrary. After growth, cultures were centrifuged to remove cells and most of the relatively water-insoluble iodinin. The residual relatively water-soluble pigments were removed from culture supernatants by acidification with HCI to pH2 and extraction twice with equal volumes of chloroform. The chloroform extracts were concentrated by rotary evaporation under reduced pressure and pigments were fractionated by t.1.c. on silica gel G with chloroform-acetic acid (9:1, v/v) as the solvent system. In some cases cellulose acetate (40% acetylated) was used with tolueneethanol-water (4:17:1, by vol.) as the solvent. In other cases prior methylation of phenazines with diazomethane was necessary to effect their separation. Individual pigments were detected visually on thin-layer plates, bands were scraped off and phenazines were eluted with the appropriate solvent. Each pigment was charac-