Cra-controlled antisense RNA-downregulation of isocitrate dehydrogenase in Escherichia coli

Catabolite repressor activator (Cra) protein (formerly called FruR) found in E. coli is known to regulate the expression of many genes positively and negatively in response to the intracellular levels of fructose-1-phosphate (F-1-P) and fructose-1,6-bisphopahate (F-1,6-bisP). In this paper, we report synthesis and characterization of a conditionally expressed antisense RNA corresponding to 101 bp of isocitrate dehydrogenase (icd) gene (as-icd) under Cra (FruR) responsive promoter fruB (P_fruB as-icd construct denoted as pVS2K3) in E. coli K-12 derivative (DH5α) and E. coli B derivative (BL21) strains. Previous studies have shown that ICD mutants accumulated citrate intracellularly but failed to grow on glucose in absence of glutamate. Hence, a conditional downregulation of icd gene could be helpful in overcoming this lethality and also aid in understanding the flux towards citrate accumulation. Effect of pVS2K3 construct was monitored in E. coli DH5α and E. coli BL21 during growth on carbon sources wherein the fruB promoter is active (glucose) or repressed (glycerol). A 3-to 4-fold decrease in ICDH activity was observed in E. coli DH5α expressing pVS2K3 on glucose but no change in ICDH activity was observed in E. coli BL21 expressing pVS2K3 on glucose. This alteration could be attributed to the anomalous Cra regulation seen in E. coli B strain which could be a crucial factor while choosing P_fruB promoter for expression studies.