N-terminal truncations in the FhlA protein result in formate- and MoeA-independent expression of the hyc (formate hydrogenlyase) operon of Escherichia coli

The formate hydrogenlyase complex of Escherichia coli catalyses the cleavage of formate to CO2 and H-2 and consists of a molybdoenzyme formate dehydrogenase-H, hydrogenase 3 and intermediate electron carriers. The structural genes of this enzyme complex are activated by the FhIA protein in the presence of both formate and molybdate; ModE-Mo serves as a secondary activator. Mutational analysis of the FhIA protein established that the unique N-terminal region of this protein was responsible for formate- and molybdenum-dependent transcriptional control of the hyc operon. Analysis of the N-terminal sequence of the FhIA protein revealed a unique motif (amino acids 7-37), which is also found in ATPases associated with several members of the ABC-type transporter family. A deletion derivative of FhIA lacking these amino acids (FhIA9-2) failed to activate the hyc operon in vivo, although the FhIA9-2 did bind to hyc promoter DNA in vitro. The ATPase activity of the FhIA9-2-DNA-formate complex was at least three times higher than that of the native protein-DNA-formate complex, and this degree of activity was achieved at a lower formate level. Extending the deletion to amino acid 117 (FhIA167) not only reversed the FhIA(-) phenotype of FhIA9-2, but also led to both molybdenum- and formate-independence. Deleting the entire N-terminal domain (between amino acids 5 and 374 of the 692 amino acid protein) also led to an effector-independent transcriptional activator (FhIA165), which had a twofold higher level of hyc operon expression than the native protein. Both FhIA165 and FhIA167 still required ModE-Mo as a secondary activator for an optimal level of hyc-lac expression. The FhIA165 protein also had a twofold higher affinity to hyc promoter DNA than the native FhIA protein, while the FhIA167 protein had a significantly lower affinity for hyc promoter DNA in vitro. Although the ATPase activity of the native protein was increased by formate, the ATPase activity of neither FhIA165 or FhIA167 responded to formate. Removal of the first 117 amino acids of the FhIA protein appears to result in a constitutive, effector-independent activation of transcription of the genes encoding the components of the formate hydrogenlyase complex. The sequence similarity to ABC-ATPases, combined with the properties of the FhIA deletion proteins, led to the proposal that the N-terminal region of the native FhIA protein interacts with formate transport proteins, both as a formate transport facilitator and as a cytoplasmic acceptor.