Approach to recognition of regulatory mutants of cyanobacteria

Antimetabolite analogs of essential amino acids are useful as selective agents for isolation of regulatory mutants of cyanobacteria, although we observed striking microbiological differences from other widely used eubacterial systems. Regulatory mutants shown to overproduce and excrete tryptophan, phenylalanine, tyrosine, methionine, or arginine were isolated from four cyanobacteria: Anabaena sp. 29151, Synechococcus sp. 602, Synechococcus sp. AN Tx20, and Synechocystis sp. 29108. Surprisingly, regulatory-mutant colonies did not support a halo of cross-fed wild-type growth on selective medium. Since regulatory mutants were shown to excrete substantial levels of amino acids, it was deduced that poor cross-feeding must reflect a generally low nutritional responsiveness of the cyanobacterial background. This conclusion was confirmed by results which showed that regulatory-mutant cells of cyanobacteria dispersed among wild-type populations of Bacillus subtilis did produce halo colonies on solid analog-containing medium. Cross-feeding between one cyanobacterial pair (a phenylalanine excretor and a phenylalanine auxotroph) was successfully demonstrated in the absence of the analog under conditions in which relatively large masses of each cell population type were spread near one another on agar plates. These results suggest that amino acid excreted by regulatory mutants of cyanobacteria on analog-containing selective medium is transported into nearby wild-type cells too inefficiently to overcome the antimetabolite effects of the analog, thereby failing to generate halos of physiologically resistant background cells. Consistent with this interpretation was the finding that the pheA1 auxotroph from Synechococcus sp. 602 exhibited a linearly proportional dependence of growth rate upon exogenous concentration of l-phenylalanine (below 20 muM). Wild-type B. subtilis serves as a convenient and sensitive test lawn for screening obvious regulatory mutants from among collections of analog-resistant cyanobacterial mutants. Appropriate B. subtilis auxotrophs can be used as convenient indicator strains for the identification of regulatory mutants in cyanobacteria through the observation of syntrophic growth responses.