Genetic regulation of formate hydrogenlyase of Escherichia coli: role of the fhlA gene product as a transcriptional activator for a new regulatory gene, fhlB

Abstract

A new gene whose product is required for the production of formate hydrogenlyase (FHL) has been identified in Escherichia coli. This gene, termedjhlB, maps between thefrdA (94.4 min) and argl (96.6 min) genes on the E. coli chromosome and is transcribed in a clockwise direction toward argI. Biochemical analysis of an FhIBmutant, strain SE-2011 [(I(fldB-lacZ+)], revealed that the mutant lacks formate dehydrogenase activity associated with FHL (FDH-H) and hydrogenase activity. As a result of these defects, fermentative hydrogen production and hydrogen uptake reactions were undetectable in strain SE-2011. Fumarate reductase activity of this mutant was also reduced to about 15% of the levels of the parent (strain MC4100), and strain SE-2011 did not produce succinate as a fermentation end product. Regulation of expression of theJflB gene, studied as production of ,-galactosidase activity by strain SE-2011, revealed that the operon is expressed at low levels under aerobic conditions. Under anaerobic growth conditions, this activity increased by two-to threefold. Addition of formate enhanced the differential rate of synthesis of thefluB gene product to as high as 130 U of ,B-galactosidase specific activity per ,ug of cell protein, but only under anaerobic conditions. Formatedependent expression of I(ffllB-lacZ+) required the ar-subunit of RNA polymerase and theflUA gene product. Under anaerobic growth conditions, Escherichia coli ferments glucose to yield acetate, ethanol, succinate, lactate, and formate (9). Formate is further metabolized by formate hydrogenlyase (FHL) to dihydrogen and carbon dioxide (13). Various components of the FHL complex, a unique formate dehydrogenase isoenzyme (FDH-H) and a specific hydrogenase isoenzyme (HYD3), have been identified, and the genes coding for these proteins (fdhF and hyd-17, respectively) have been described (10,24,32,41,45). These genes are transcribed only under anaerobic conditions and require formate as an inducer (4,25,26,42,44). Several other genes whose products are essential for production of one or both of the enzyme activities have also been described. These include the hyd, fllA, chl, mol, and rpoN genes (3, 6, 18, 23, 25, 28-31, 33, 37, 38, 40, 43). Among these genes, mutations in the fhlA and rpoN genes affect only the production of FHL' activity, and these gene products are needed for transcription of the fdhF and hyd-17 genes (3,28,29,33). This report presents evidence of an additional gene, ffhlB, which is also needed for production of FHL activity of E. coli. Transcription of this gene requires the FhIA protein.The role of the FhlA protein as a putative transcriptional activator in regulation of theflzlB gene was deduced from the * Corresponding author.

A conserved region in the σ54‐dependent activator DctD is involved in both binding to RNA polymerase and coupling ATP hydrolysis to activation

“…In the designations to the left of the amino acid sequences, the first three letters indicate the organism and the last four letters indicate the acronym for the activator. The organisms and activators are: R. meliloti (Rme) DctD (Jiang et al, 1989); S. typhimurium (Sty) NtrC (Flashner et al, 1995); Agrobacterium tumefaciens (Atu) NtrC (Wardhan et al, 1989); R. capsulatus (Rca) NifA (Masepohl et al, 1988); R. capsulatus NtrC (Jones and Haselkorn, 1989); Klebsiella pneumoniae (Kpn) NifA (Drummond et al, 1986); E. coli (Eco) FhlA (Maupin and Shanmugam, 1990); E. coli HydG (Stoker et al, 1989); E. coli TyrR (Cornish et al, 1986); Pseudomonas aeruginosa (Pae) PilR (Ishimoto and Lory, 1992); P. aeruginosa AlgB (Wozniak and Ohman, 1991); Pseudomonas putida (Ppu) XylR (Inouye et al, 1988); P. putida DehR (Topping et al, 1995); Pseudomonas sp. strain CF600 (Psp) DmpR (Shingler et al, 1993); Caulobacter crescentus (Ccr) FlbD (Ramakrishnan and Newton, 1990); Alcaligenes eutrophus (Aeu) HoxA (Eberz and Friedrich, 1991); A .…”

Mutations in trans that affect formate dehydrogenase (fdhF) gene expression in Salmonella typhimurium

“…frhIB has been suggested as a third potential regulator of fdhF, since it is required for the formate dehydrogenase activity associated with formate hydrogenlyase and since mutations in fhIB pleiotropically affect all three hydrogenase isoenzymes (21). FhlB is maximally expressed under anaerobic conditions in the presence of formate, and it has been proposed that formate interacts with the FhlA protein to initiate transcription of fhIlB (21). Thus, the picture of how fdhF is regulated in E. coli is far from clear.…”

“…What is the function of FhlA, and what factor in turn regulates its activity? FhlA has recently been implicated in the regulation of the expression of frilB (21), which is located in the 95-min region of the E. coli chromosome. frhIB has been suggested as a third potential regulator of fdhF, since it is required for the formate dehydrogenase activity associated with formate hydrogenlyase and since mutations in fhIB pleiotropically affect all three hydrogenase isoenzymes (21).…”

“…FhlA has recently been implicated in the regulation of the expression of frilB (21), which is located in the 95-min region of the E. coli chromosome. frhIB has been suggested as a third potential regulator of fdhF, since it is required for the formate dehydrogenase activity associated with formate hydrogenlyase and since mutations in fhIB pleiotropically affect all three hydrogenase isoenzymes (21). FhlB is maximally expressed under anaerobic conditions in the presence of formate, and it has been proposed that formate interacts with the FhlA protein to initiate transcription of fhIlB (21).…”

The Escherichia coli fdv gene probably encodes mutS and is located at minute 58.8 adjacent to the hyc-hyp gene cluster

“…The last gene of the hyp transcriptional unit (6), IhIA, codes for a regulatory protein which activates the expression of the structural genes of the FHL complex under fermentative conditions (11). The product of the gene followingJIhA in the same transcriptional direction (termed fdv) has been proposed to also play a role in the FHL system (7,9).…”