Complex Transcriptional Control Links NikABCDE-Dependent Nickel Transport with Hydrogenase Expression in Escherichia coli

Abstract

Escherichia coli requires nickel under anaerobic growth conditions for the synthesis of catalytically active NiFe hydrogenases. Transcription of the NikABCDE nickel transporter, which is required for NiFe hydrogenase synthesis, was previously shown to be upregulated by FNR (fumarate-nit rate regulator) in the absence of oxygen and repressed by the NikR repressor in the presence of high extracellular nickel levels. We present here a detailed analysis of nikABCDE transcriptional regulation and show that it closely correlates with hydrogenase expression levels. We identify a nitrate-dependent mechanism for nikABCDE repression that is linked to the NarLX two-component system. NikR is functional under all nickel conditions tested, but its activity is modulated by the total nickel concentration present as well as by one or more components of the hydrogenase assembly pathway. Unexpectedly, NikR function is independent of NikABCDE function, suggesting that NikABCDE is a hydrogenase-specific nickel transporter, consistent with its original identification as a hydrogenase (hyd) mutant. Further, the results suggest that the hydrogenase assembly pathway is sequestered within the cell. A second nickel import pathway in E. coli is implicated in NikR function.Several energetically difficult reactions, such as nitrogen or carbon fixation, are catalyzed by enzymes with complex metal cofactors (26). A striking feature of the synthesis of these enzymes is the requirement of intricate assembly pathways that utilize several protein cofactors to ensure the fidelity of catalytic-site assembly (18). Metalloenzyme expression levels can be tightly regulated in response to changes in environmental conditions; for example, the nitrogenase operon is induced by nitrogen availability but repressed in the presence of oxygen (14). This shifting metabolism, combined with the biosynthetic cost of making these enzymes, means that the transcriptional regulation of these pathways is both necessary and complex. Cells are unlikely to synthesize large quantities of apoenzyme in the absence of the required cofactor(s), just as they are unlikely to expend the energy necessary to synthesize the transporter and accessory proteins necessary for cofactor assembly when the apoenzyme is not being expressed.Escherichia coli exhibits a complex transcriptional response to growth conditions at low oxygen tensions (38). Respiration still occurs, but at lower energetic yield, and it requires the presence of an alternative electron acceptor, such as nitrate, dimethyl sulfoxide (DMSO), trimethylamine oxide (TMAO), or fumarate, and a corresponding terminal reductase (2,16,32,42). E. coli can also ferment carbon sources in the absence of a suitable electron acceptor. E. coli expresses NiFe hydrogenases under anaerobic growth conditions (1, 5, 27, 29, 38) when energetic yields are low, for example, during fermentation or with low-energy-yield electron acceptors such as fumarate. Hydrogenases 1 and 2 (expressed by hya and hyb, respectively) oxidize H 2 in the presence of ...

Selenium Is Involved in Regulation of Periplasmic Hydrogenase Gene Expression in Desulfovibrio vulgaris Hildenborough

“…A nickeldependent regulator, NikR, has been identified in the genomes of D. vulgaris Hildenborough and Desulfovibrio desulfuricans G20, and putative binding sites for this regulator were identified upstream of genes encoding Ni transport systems (28 1 Hase synthesis between the H 2 /S medium and the other conditions suggests that hydrogen may induce a high-affinity uptake system for Ni, which is able to scavenge the low levels of Ni in the Fe-only medium in order to support a high expression level of this Hase, and that this system is not present in the other conditions. In Escherichia coli, it was recently shown that transcription of the nickel transport system NikABCDE is regulated in order to match the Hase expression level of the cell (32). Regarding the effect of Ni on the [NiFeSe] Hase, this protein is not detected, by activity-stained gel or Western blotting, in cells grown in media containing FeϩNi.…”

“…Interestingly, the genes coding for the [ Gene regulation by both Fe and Ni in prokaryotes is well documented (2,12,19). In the particular case of Hases, Ni is required for complete maturation of [NiFe] Hases (32), and transcriptional regulation by Ni has been shown, for example, in Bradyrhizobium japonicum (22) and in Methanothermobacter marburgensis. In the latter organism, growth in Ni-limited conditions leads to increased expression of the Ni-free Hase H 2 -forming methylenetetrahydromethanopterin dehydrogenase (Hmd) and strongly decreased expression of the [NiFe] Hase F 420 -reducing hydrogenase (Frh) (1).…”

Metal Selectivity of the Escherichia coli Nickel Metallochaperone, SlyD

“…Although deletion of the genes for the Nik importer abrogates hydrogenase activity (43), it does not affect the response of NikR to exogenous Ni(II), suggesting that metallochaperones sequester Ni(II) for hydrogenase maturation at the transporter (42,53). Given that disruption of slyD impacts the activity of NikR as well as hydrogenase maturation, it is likely that its role is not just as a source of nickel for the hydrogenase enzymes.…”

“…Under anoxic conditions the Nik transporter is expressed to establish a supply of nickel that meets the demands of the [NiFe]-hydrogenase enzymes (1,53). Deletion of slyD caused a down-regulation of nikA transcription, with or without Ni(II) supplementation of the media.…”

Histidine 416 of the periplasmic binding protein NikA is essential for nickel uptake in Escherichia coli

“…Once in E. coli's periplasm, nickel import through the inner membrane proceeds via a unique ATP-Binding Cassette (ABC)-type transporter called Nik-ABCDE encoded by the nik operon [7]. It is suggested to be a hydrogenase-specific nickel transporter [8] and insertion mutations in the nik operon completely abolish [NiFe] hydrogenase activity [9]. Its expression is under the positive control of the oxygen sensor Fumarate Nitrate reductase Regulator (FNR) [10], which forms a DNA-binding dimer during anaerobic growth, and is negatively controlled by the transcriptional metalloregulator NikR in the presence of excess nickel [11].…”