Isolation and Characterization of High CO 2 -Requiring-Mutants of the Cyanobacterium Synechococcus PCC7942

Abstract

A total of 24 high CO2-requiring-mutants of the cyanobacterium Synechococcus PCC7942 have been isolated and partially characterized. These chemically induced mutants are able to grow at 1% CO2, on agar media, but are incapable of growth at air levels of CO2. All the mutants were able to accumulate inorganic carbon (Ci) to levels similar to or higher than wild type cells, but were apparently unable to generate intracellular CO2. On the basis of the rate of Ci release following a light (5 minutes) → dark transition two extreme phenotypes (fast and slow release mutants) and a number of `intermediate' mutants (normal release) were identified. Compared to wild-type cells, Type I mutants had the following characteristics: fast Ci release, normal internal Ci pool, normal carbonic anhydrase (CA) activity in crude extracts, reduced internal exchange of 18O from 18O-labeled CO2, 1% CO2 requirement for growth in liquid media, normal affinity of carboxylase for CO2, and long, rod-like carboxysomes. Type II mutants had the following characteristics: slow Ci release, increased internal Ci pool, normal CA activity in crude extracts, normal internal 18O exchange, a 3% CO2 requirement for growth in liquid media, high carboxylase activity, normal affinity of carboxylase for CO2, and normal carboxysome structure but increased in numbers per cell. Both mutant phenotypes appear to have genetic lesions that result in an inability to convert intracellular HCO3 − to CO2 inside the carboxysome. The features of the type I mutants are consistent with a scenario where carboxysomal CA has been mistargeted to the cytosol. The characteristics of the type II phenotype appear to be most consistent with a scenario where CA activity is totally missing from the cell except for the fact that cell extracts have normal CA activity. Alternatively the type II mutants may have a lesion in their capacity for H+ import during photosynthesis. Images: Figure 6: