Regulation of the Hydrogenase-4 Operon of Escherichia coli by the σ ⁵⁴ -Dependent Transcriptional Activators FhlA and HyfR

Abstract

The hyf locus (hyfABCDEFGHIJ-hyfR-focB) of Escherichia coli encodes a putative 10-subunit hydrogenase complex (hydrogenase-4 [Hyf]); a potential 54 -dependent transcriptional activator, HyfR (related to FhlA); and a putative formate transporter, FocB (related to FocA). In order to gain insight into the physiological role of the Hyf system, we investigated hyf expression by using a hyfA-lacZ transcriptional fusion. This work revealed that hyf is induced under fermentative conditions by formate at a low pH and in an FhlA-dependent fashion. Expression was 54 dependent and was inhibited by HycA, the negative transcriptional regulator of the formate regulon. Thus, hyf expression resembles that of the hyc operon. Primer extension analysis identified a transcriptional start site 30 bp upstream of the hyfA structural gene, with appropriately located ؊24 and ؊12 boxes indicative of a 54 -dependent promoter. No reverse transcriptase PCR product could be detected for hyfJ-hyfR, suggesting that hyfR-focB may be independently transcribed from the rest of the hyf operon. Expression of hyf was strongly induced (ϳ1,000-fold) in the presence of a multicopy plasmid expressing hyfR from a heterologous promoter. This induction was dependent on low pH, anaerobiosis, and postexponential growth and was weakly enhanced by formate. The hyfR-expressing plasmid increased fdhF-lacZ transcription just twofold but did not influence the expression of hycB-lacZ. Interestingly, inactivation of the chromosomal hyfR gene had no effect on hyfA-lacZ expression. Purified HyfR was found to specifically interact with the hyf promoter/operator region. Inactivation of the hyf operon had no discernible effect on growth under the range of conditions tested. No Hyf-derived hydrogenase or formate dehydrogenase activity could be detected, and no Ni-containing protein corresponding to HyfG was observed.Escherichia coli is capable of three alternative modes of energy generation: aerobic respiration, anaerobic respiration and fermentation. In the absence of the appropriate electron acceptors (O 2 , NO 3 Ϫ , NO 2 Ϫ , sulfur or nitrogen oxides, or fumarate), E. coli resorts to mixed-acid fermentation, yielding a maximum of just 3 mol of ATP per mol of glucose consumed (12). During fermentative growth, glycolytic carbon sources are converted to pyruvate that is in turn converted to acetyl coenzyme A and formate by pyruvate formatelyase (22) The formate produced may then be either excreted or further metabolized to H 2 and CO 2 by a membrane-associated, nonenergy-conserving formate hydrogenlyase (Fhl-1) system consisting of formate dehydrogenase H (Fdh-H) and the hydrogenase-3 complex (Hyc) (12,17,37).The genes required for the synthesis of Fhl-1 form the formate regulon, which includes three transcriptional units, namely, the hyc and hyp-fhlA operons and the fdhF gene (13,26,28,37,49). The hyc operon (hycABCDEFGHI) encodes the Hyc complex of Fhl-1 (encoded by hycBCDEFGH), as well as a negative-transcriptional regulator (HycA) of the formate regulon and a protease ...

Nitrogen recycling and nutritional provisioning byBlattabacterium, the cockroach endosymbiont

“…Blattabacterium does encode one RpoN enhancer-activator, FhlA, which, in E. coli, senses environmental formate and stimulates the transcription of genes involved in anaerobic ATP synthesis, but it lacks known FhlAregulated genes (37,42). Genomic evidence also indicates that Blattabacterium generates energy via aerobic respiration.…”

The dual-function chaperone HycH improves assembly of the formate hydrogenlyase complex

“…However, H 2 production by this putative complex has yet to be unambiguously demonstrated and little is known about specific maturation requirements of this complex. The hyf operon is not transcribed at a significant level under the conditions where FHL is active [11,12]. Genetic analysis suggests that the hyf operon encodes 12 proteins, but no gene for a specific endoprotease equivalent to HycI is present.…”

Membrane Potential as a Regulation Mechanism of Periplasmic Nitrite Reductase Activity: A Mathematical Model

“…Особенности структурной организации Hyd-4 гидрогеназы, входящей в состав FHL-2 комплекса, позволяют предположить наличие у нее прямой протон-транслоцирующей активности [14,27,29]. Причем, согласно данным [30,31], в кислых рН активность Hyd-4 гидрогеназы в направлении продукции молекулярного водорода существенно снижена по сравнению с таковой для Hyd-3, что в условиях хемостата создает условия для реализации респираторного сценария.…”