Mechanism of regulation of the formate‐hydrogenlyase pathway by oxygen, nitrate, and pH: definition of the formate regulon

Abstract

The products of a minimum of 15 genes are required for the synthesis of an active formate-hydrogenlyase (FHL) system in Escherichia coli. All are co-ordinately regulated in response to variations in the oxygen and nitrate concentration and the pH of the culture medium. Formate is obligately required for transcriptional activation of these genes. Analysis of the transcription of one of these genes, hycB linked to the lacZ reporter gene, revealed that oxygen and nitrate repression of transcription could be relieved completely, or partially in the case of nitrate, either by the addition of formate to the medium or by increasing the copy number of the gene encoding the transcriptional activator (fhlA) of this regulon. These studies uncovered a further level of regulation in which the transcription of hycB was reduced in cells grown on glucose. This effect was most clearly seen in aerobically grown cells when formate was added externally. Addition of cAMP overcame this glucose repression, which could be shown to be mediated by the cAMP receptor protein. These results would be consistent with the transport of formate being regulated by catabolite repression. Moreover, the repression of transcription through high pH also could be partially overcome by addition of increasing concentrations of formate to the medium, again being consistent with regulation at the level of formate import and export. Taken together, all these observations indicate that it is the intracellular level of formate that determines the transcription of the genes of the formate regulon by FhlA. This represents a novel positive feedback mechanism in which the activator of a regulon induces its own synthesis in response to increases in the concentration of the catabolic substrate, and this in turn is governed by the relative affinities of FhlA and the three formate dehydrogenase isoenzymes for formate.

Analysis of hydrogenase 1 levels reveals an intimate link between carbon and hydrogen metabolism in Escherichia coli K-12

“…Expression of hyc with fructose was threefold higher than with glucose; however, this does not reflect what was observed with the total hydrogenase activities. Because expression of hyc is dependent on the availability of formate (Rossmann et al, 1991;Schlensog et al, 1994), our results, therefore, clearly show that fructose must be metabolized to formate in MC4100. In addition, gas production in Durham tubes (Varenne et al, 1975) could also be observed after growth with fructose, while no dihydrogen production was seen when cells were grown in TYEP without additional carbon source (data not shown).…”

“…3a). This indicated that the strong increase in hydrogenase activity probably resulted from increased levels of Hyd-3, presumably through increased intracellular formate concentrations (Rossmann et al, 1991), while Hyd-1 levels remained unaffected by the mutation.…”

“…Hyd-3 synthesis is induced when the intracellular formate concentration is high (Rossmann et al, 1991). Western blot analysis of the levels of pyruvate formate-lyase (PflB), which generates formate from the cleavage of pyruvate (Knappe & Sawers, 1990), revealed that PflB levels (Fig.…”

Transcriptional regulation in response to oxygen and nitrate of the operons encoding the [NiFe] hydrogenases 1 and 2 of Escherichia coli

“…Thus, the FHL complex offsets the potentially deleterious effects of formate accumulation on fermentation by maintaining pH homeostasis Rossmann et al, 1991 ;Sauter et al, 1992).…”

“…An analysis of the transcriptional regulation of the operons encoding both enzymes would resolve this question unequivocally and at the same time provide key information on the control of enzyme synthesis. Such an approach has greatly enhanced our understanding of the regulation and function of hydrogenase isoenzyme 3 (Schlensog & Bo$ ck, 1990 ;Lutz et al, 1990 ;Rossmann et al, 1991 ;Schlensog et al, 1994 ;Hopper et al, 1996).…”