Creaghan and Guest isolated and characterized amber mutants of Escherichia coli deficient in α-ketoglutarate dehydrogenase complex (α-kgdhc), a key tricarboxylic acid cycle enzyme. Of 21 suc mutants screened, five were identified as amber (nonsense) mutants through sensitivity to amber suppressors. These mutants lacked activity for the overall complex and its E1 component (α-ketoglutarate dehydrogenase), had little or no E2 activity (dihydrolipoyl transuccinylase), and showed 20-30% reduced E3 activity (dihydrolipoyl dehydrogenase). Fine-structure mapping via P1 transduction established that amber sites mapped within the sucA gene, indicating polarized expression from sucA to sucB. Double amber mutants carrying lesions in both pyruvate dehydrogenase (aceE) and α-ketoglutarate dehydrogenase (sucA) genes retained at least 30% of parental E3 activity, suggesting separate genes for the E3 component in each complex. The authors further mapped tricarboxylic acid cycle genes, establishing the order: gltA...sdh...sucA.sucB...tolI1...gal.

Key findings

• Five of twenty-one suc mutants were identified as amber (nonsense) mutations, suppressed by amber suppressors at sucA locus sites
• Amber mutants showed pleiotropic effects: absent E1 and E2 activities, and 20-30% reduced E3 activity compared to wild-type
• Gene expression in the suc region shows polarity from sucA to sucB, with E1 synthesis preceding E2 synthesis
• Double amber mutants (aceE and sucA) retained ≥30% E3 activity, suggesting separate dihydrolipoyl dehydrogenase genes for pyruvate and α-ketoglutarate dehydrogenase complexes
• Genetic mapping established the chromosomal order of tricarboxylic acid cycle genes: gltA...sdh...sucA.sucB...tolI1...gal