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The formate hydrogenlyase complex of Escherichia coli catalyses the cleavage of formate to CO2 and H2 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 FhlA protein in the presence of both formate and molybdate; ModE-Mo serves as a secondary activator. Mutational analysis of the FhlA 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 FhlA 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 FhlA lacking these amino acids (FhlA9-2) failed to activate the hyc operon in vivo, although the FhlA9-2 did bind to hyc promoter DNA in vitro. The ATPase activity of the FhlA9-2-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 (FhlA167) not only reversed the FhlA(-) phenotype of FhlA9-2, 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 (FhlA165), which had a twofold higher level of hyc operon expression than the native protein. Both FhlA165 and FhlA167 still required ModE-Mo as a secondary activator for an optimal level of hyc-lac expression. The FhlA165 protein also had a twofold higher affinity to hyc promoter DNA than the native FhlA protein, while the FhlA167 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 FhlA165 or FhlA167 responded to formate. Removal of the first 117 amino acids of the FhlA 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 FhlA deletion proteins, led to the proposal that the N-terminal region of the native FhlA protein interacts with formate transport proteins, both as a formate transport facilitator and as a cytoplasmic acceptor.

Summary: Escherichia coli growing under anaerobic conditions produces several molybdoenzymes, such as formate hydrogenlyase (formate to H2 and CO2; hyc and fdhF genes) and nitrate reductase (narGHJI genes). Synthesis of these molybdoenzymes, even in the presence of the cognate transcriptional activators and effectors, requires molybdate in the medium. Besides the need for molybdopterin cofactor synthesis, molybdate is also required for transcription of the genes encoding these molybdoenzymes. In E. coli, ModE was previously identified as a repressor controlling transcription of the operon encoding molybdate transport components (modABCD). In this work, the ModE protein was also found to be a required component in the activation of hyc-lacZ to an optimum level, but only in the presence of molybdate. Mutant ModE proteins which are molybdate-independent for repression of modA-lacZ also restored hyc-lacZ expression to the wild-type level even in the absence of molybdate. Nitrate-dependent enhancement of transcription of narX-iacZ was completely abolished in a modE mutant. Nitrate-response by narG-lacZ and nark-lacZ was reduced by about 50% in a modE mutant. DNase I footprinting experiments revealed that the ModE protein binds the hyc promoter DNA in the presence of molybdate. ModE-molybdate also protected DNA in the intergenic region between narXL and narK from DNase I hydrolysis. DNA sequences (5′ TAYAT 3′ and 5′ GTTA 3′) found in ModE-molybdate-protected modABCD operator DNA were also found in the ModE-molybdate-protected region of hyc promoter DNA (5′ GTTA-7 bp-CATAT 3′) and narX-narK intergenic region (5′ GTTA-7 bp-TACAT 3′). Based on these results, a working model is proposed in which ModE-molybdate serves as a secondary transcriptional activator of both the hyc and narXL operons which are activated primarily by the transcriptional activators, FhIA and NarL, respectively.