Isolation and characterization of five genotypic mutants of chlorate-resistant cyanobacteria unable to utilize nitrate

Abstract

Spontaneous mutants of the cyanobacteriumSynechococcus PCC 7002 resistant to chlorate were isolated. Either 40mM or 400mM Na₂ClO₃ was used as the selective agent. Putative Chl^r colonies were picked onto medium containing ammonia as the sole N source, then replicaplated to media containing either NH₄ ⁺, NO₂ ⁻ as N sources. Of 252 putative mutants, 106 were able to use either NH₄Cl or NaNO₂ but not NaNO₃ as their sole source of nitrogen. All of the mutant isolates had generation times similar to wild-type 7002 when grown on either ammonium (3.8–4.1 h/generation) or nitrite (4.5–4.7 h/generation). None had detectable methyl viologensupported nitrate reductase activity and are thus phenotypically NRase⁻. The Chl^r mutants had photomediated O₂ production and dark O₂ uptake rates similar to the wild type and responded similarly to selected metabolic inhibitors. They expressed increased levels of phycocyanin (PC) synthesis under normal, nitrogen-replete growth conditions, but rapidly lapsed into a chlorotic state upon a shift to either medium containing nitrate or to N-free medium. Genetic analysis of the Chl⁴ mutants indicated that each could be rescued by direct transformation with chromosomally derived DNA from the wild-type strain. Frequencies of transformation for the mutants were characteristic for single genetic lesions in this cyanobacterium. On the basis of marker rescue by a cosmid library of wild-type DNA, the NRase⁻ mutants could be grouped into five distinctive genotypic families.