Fluoroacetamide resistance mutations in Chlamydomonas reinhardtii

Acetamide, a nitrogen and carbon source for Chlamydomonas reinhardtii, is hydrolyzed by acetamidase to ammonium and acetate. It also induces urea pathway activities. Fluoroacetamide (F-acetamide) is toxic to wildtype through conversion to F-citrate, a respiratory inhibitor. Resistant mutants were selected on plates of F-acetamide plus urea. When tested on acetamide plates two mutant classes were obtained, acm+ (utilized acetamide as sole N source) and acm-. All acm+ isolates had acetamidase activity and were obligate phototrophs (i.e. "dark-diers"). Acm- isolates had either normal urea assimilation (ure+) or lacked all urea pathway activities, namely transport, urea carbooxylase and allophanate hydrolase (ure-). Inheritance patterns for both types indicated single nuclear gene mutations. The acm- ure+ type presumably resulted from a defective acetamidase gene, and the acm- ure- strains might be regulatory gene mutants. Temperature conditional F-acetamide tolerant mutants were also obtained. Acetamidase extracted from one such strain was more thermolabile than the wildtype enzyme, indicating a mutation in the coding region. The hypothesis that acetamidase is involved in urea assimilation was not supported by the genetic and biochemical evidence.