No abstract

Bacterial periplasmic nickel-containing hydrogenases are composed of a small subunit containing a twin-arginine signal sequence and a large subunit devoid of an export signal. To understand how the large subunit is translocated into the periplasm, we cloned the hyb operon encoding the hydrogenase 2 of Escherichia coli, constructed a deletion mutant, and studied the mechanism of translocation of hydrogenase 2. The small subunit (HybO) or the large subunit (HybC) accumulated in the cytoplasm as a precursor when either of them was expressed in the absence of the other subunit. Therefore, contrary to most classical secretory proteins, the signal sequence of the small subunit itself is not sufficient for membrane targeting and translocation if the large subunit is missing. On the other hand, the small subunit was required not only for membrane targeting of the large subunit, but also for the acquisition of nickel by the large subunit. Most interestingly, the signal sequence of the small subunit determines whether the large subunit follows the Sec or the twinarginine translocation pathway. Taken together, these results provide for the first time compelling evidence for a naturally occurring hitchhiker co-translocation mechanism in bacteria.

We report here on the isolation and primary characterization of the yohM gene of Escherichia coli. We show that yohM encodes a membrane-bound polypeptide conferring increased nickel and cobalt resistance in E. coli. yohM was specifically induced by nickel or cobalt but not by cadmium, zinc, or copper. Mutation of yohM increased the accumulation of nickel inside the cell, whereas cells harboring yohM in multicopy displayed reduced intracellular nickel content. Our data support the hypothesis that YohM is the first described efflux system for nickel and cobalt in E. coli. We propose rcnA (resistance to cobalt and nickel) as the new denomination of yohM.Nickel and cobalt are both required as trace elements in prokaryotes to fulfill a variety of metabolic functions, but high intracellular concentrations of these transition metals are toxic. One of the strategies evolved by bacteria to prevent damage is to export excess metal by efflux systems. Plasmid-borne determinants responsible for nickel and/or cobalt resistance have been described for the heavy-metal-resistant bacterium Ralstonia metallidurans (11,15), among which are members of the resistance-nodulation-cell division superfamily: the best-characterized CzcCBA (cobalt-zinc-cadmium) three-component cation antiporter (14) and the homologous CnrCBA (cobaltnickel resistance) (10) and NccCBA (nickel-cobalt-cadmium resistance) (18) efflux systems. Moreover, cobalt can be extruded from the cytoplasm by the cation diffusion facilitator CzcD of R. metallidurans at the expense of the proton motive force or a potassium gradient (15). Cobalt may also be a substrate of Zn-CPx-type ATPases, as in Helicobacter pylori (8). There is no evidence for the transport of nickel by one of these two modes of efflux. Instead, this metal can be transported outside the cytoplasm by NreB from R. metallidurans (7) or NrsD from Synechocystis sp. strain PCC 6803 (6), which are members of the major facilitator superfamily and which each exhibit 12 putative transmembrane helices and a histidine-rich carboxy terminus contributing to nickel resistance.In Escherichia coli, anaerobic hydrogenase isoenzymes and urease (in ureolytic strains) require incorporation of nickel to become active (12). Complex assembly processes involve accessory proteins, namely, HypB, implicated in nickel insertion into hydrogenase, and UreE, which delivers nickel to urease. HypB and UreE are well conserved among bacteria apart from a terminal histidine-rich stretch whose function would be nickel storage and which is absent in E. coli proteins (3, 5). In order to gain insights into nickel trafficking and, more precisely, to find proteins that would be involved in nickel resistance, we searched the E. coli genome database with a query based on a consensus alignment of the UreE and HypB histidine-rich variants. The best returned hit was yohM, whose product bears a histidine-rich domain in its center. The aim of the present work is to demonstrate the implication of yohM in nickel and cobalt trafficking in E. coli.Inactivati...