ArcA and AppY Antagonize IscR Repression of Hydrogenase-1 Expression under Anaerobic Conditions, Revealing a Novel Mode of O ₂ Regulation of Gene Expression in Escherichia coli

Abstract

Transcription of the Escherichia coli hydrogenase-1 operon (hyaABCDEF) is increased by the transcription factors ArcA and AppY under anaerobic growth conditions. However, IscR, which represses transcription of the hyaA promoter (P hyaA ) under aerobic conditions, was not known to repress transcription of this promoter under anaerobic conditions. Here, we report that ArcA and AppY increase P hyaA expression under anaerobic conditions by antagonizing IscR binding at P hyaA , since IscR repression is observed when either ArcA or AppY is eliminated. The ability of ArcA and AppY to act as antirepressors of IscR repression of P hyaA depended on IscR levels, suggesting that IscR competes with ArcA and/or AppY for binding. In support of this competition model, electrophoretic mobility shift assays and DNase I footprinting showed that the ArcA and IscR binding sites overlap and that binding of ArcA and IscR is mutually exclusive. Unexpectedly, IscR with a C92A mutation (IscR-C92A), which mimics the clusterless form of the protein that is present predominantly under aerobic conditions, was a better repressor under anaerobic conditions of both P hyaA and a constitutive promoter containing the IscR binding site from P hyaA than wild-type IscR, which is predominantly in the [2Fe-2S] form under anaerobic conditions. This observation could not be explained by differences in DNA binding affinities or IscR levels, so we conclude that [2Fe-2S]-IscR is a weaker repressor of P hyaA than clusterless IscR. In sum, a combination of ArcA and AppY antirepression of IscR function, lower levels of IscR, and weak repression by [2Fe-2S]-IscR leads to increased P hyaA expression under anaerobic conditions. H ydrogenases, which catalyze the reversible oxidation of H 2 , are widespread throughout the prokaryotic world (36). Despite a detailed biochemical and structural understanding of these enzymes (11, 15), the importance of individual isoforms of hydrogenases to cellular physiology is often not known. In particular, hydrogenase-1 (Hyd-1), encoded by hyaAB of the hyaABCDEF operon, is one of two hydrogenases that function in H 2 oxidation under anaerobic conditions in Escherichia coli (30). Recent studies have demonstrated that, unlike most hydrogenases, Hyd-1 is O 2 tolerant (20), raising the question of whether it might also have a role under O 2 -limiting conditions. Expression of E. coli hya is also subject to various physiological inputs (i.e., anaerobiosis, stationary phase, nitrate, and phosphate starvation) (2, 5, 31), suggesting that it could have specialized roles.It is well established that expression of the hya operon is increased under anaerobic fermentative conditions and decreased under aerobic or anaerobic respiratory conditions with nitrate, consistent with the proposal that Hyd-1 functions in fermentation, not respiration (30). NarL and NarP appear to be responsible for repression of hya expression in response to nitrate (5, 31), whereas the anaerobic transcription factors ArcA and AppY are required for the increased express...

Coordinate Regulation of the Suf and Isc Fe-S Cluster Biogenesis Pathways by IscR Is Essential for Viability of Escherichia coli

“…S2 in the supplemental material), [2Fe-2S]-IscR is an inefficient repressor of transcription from the hyaA promoter (see Fig. S3) (10,30). In contrast, IscR-C92A is a robust repressor of P hyaA , which, like P sufA , contains a type 2 IscR binding site (30).…”

“…S3) (10,30). In contrast, IscR-C92A is a robust repressor of P hyaA , which, like P sufA , contains a type 2 IscR binding site (30). Thus, to compare the abilities of holo-IscR and apo-IscR to activate suf, wild-type IscR or IscR-C92A was produced from P tac and P sufA -lacZ and P sufA (Fur*)-lacZ expression was measured.…”

What a difference a cluster makes: The multifaceted roles of IscR in gene regulation and DNA recognition

“…Induction of hyaA expression during anaerobic growth required override of IscR repression by ArcA and AppY, as well as low amounts of IscR combined with weak repression by holo-IscR [46]. Repression of these particular set of genes (hyaABCDEF and napFDAGHBC) suggests that repression by IscR is required to prevent the expression of the encoded anaerobic Fe/S proteins, whose degradation upon exposure to oxygen would by itself generate oxidative stress [6], and explains the need for an aerobic repressor (IscR) in addition to anaerobic activators (FNR, ArcA and AppY) [46]. Thus, IscR is a transcriptional regulator contributing to the control of the expression of anaerobic Fe/S proteins, as well as the expression of proteins responsible for Fe/S cluster biogenesis.…”

“…The impact of IscR in the hyaA operon expression was more thoroughly investigated revealing a novel mechanism for O 2 modulation of gene expression in E. coli [46]. During anaerobic respiration, the operon hyaABCDEF is controlled by two transcription factors, ArcA and AppY that were shown to antagonize IscR repression [46]. Binding of IscR and ArcA is mutually exclusive and apo-IscR was a stronger repressor of the hya operon than its holo-form.…”

“…The napF operon is activated by ModE in response to molybdate (binding at the upstream promoter) and by FNR and NarP in response to anaerobiosis (binding at the downstream promoter) [47]. The impact of IscR in the hyaA operon expression was more thoroughly investigated revealing a novel mechanism for O 2 modulation of gene expression in E. coli [46]. During anaerobic respiration, the operon hyaABCDEF is controlled by two transcription factors, ArcA and AppY that were shown to antagonize IscR repression [46].…”