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Metabolic sensing is a crucial prerequisite for cells to adjust their physiology to rapidly changing environments. In bacteria, the response to intra- and extracellular ligands is primarily controlled by transcriptional regulators, which activate or repress gene expression to ensure metabolic acclimation. Translational control, such as ribosomal stalling, can also contribute to cellular acclimation and has been shown to mediate responses to changing intracellular molecules. In the current study, we demonstrate that the cotranslational export of the Rhodobacter capsulatus protein CutF regulates the translation of the downstream cutO -encoded multicopper oxidase CutO in response to extracellular copper (Cu). Our data show that CutF, acting as a Cu sensor, is cotranslationally exported by the signal recognition particle pathway. The binding of Cu to the periplasmically exposed Cu-binding motif of CutF delays its cotranslational export via its C-terminal ribosome stalling-like motif. This allows for the unfolding of an mRNA stem-loop sequence that shields the ribosome-binding site of cutO , which favors its subsequent translation. Bioinformatic analyses reveal that CutF-like proteins are widely distributed in bacteria and are often located upstream of genes involved in transition metal homeostasis. Our overall findings illustrate a highly conserved control mechanism using the cotranslational export of a protein acting as a sensor to integrate the changing availability of extracellular nutrients into metabolic acclimation. IMPORTANCE Metabolite sensing is a fundamental biological process, and the perception of dynamic changes in the extracellular environment is of paramount importance for the survival of organisms. Bacteria usually adjust their metabolisms to changing environments via transcriptional regulation. Here, using Rhodobacter capsulatus, we describe an alternative translational mechanism that controls the bacterial response to the presence of copper, a toxic micronutrient. This mechanism involves a cotranslationally secreted protein that, in the presence of copper, undergoes a process resembling ribosomal stalling. This allows for the unfolding of a downstream mRNA stem-loop and enables the translation of the adjacent Cu-detoxifying multicopper oxidase. Bioinformatic analyses reveal that such proteins are widespread, suggesting that metabolic sensing using ribosome-arrested nascent secreted proteins acting as sensors may be a common strategy for the integration of environmental signals into metabolic adaptations.

CcoA belongs to the widely distributed bacterial copper (Cu) importer subfamily CalT ( C co A - l ike T ransporters) of the Major Facilitator Superfamily (MFS) and provides cytoplasmic Cu needed for cbb 3 -type cytochrome c oxidase ( cbb 3 -Cox) biogenesis. Earlier studies have supported a 12-transmembrane helix (TMH) topology of CcoA with the well-conserved Met 233 xxxMet 237 and His 261 xxxMet 265 motifs in its TMH7 and TMH8, respectively. Of these residues, Met 233 and His 261 are essential for Cu uptake and cbb 3 -Cox production, whereas Met 237 and Met 265 contribute partly to these processes. CcoA also contains five Cys residues of unknown role and, remarkably, its structural models predict that three of these are exposed to the highly oxidizing periplasm. Here, we first demonstrate that elimination of both Met 237 and Met 265 completely abolishes Cu uptake and cbb 3 -Cox production, indicating that CcoA requires at least one of these two Met residues for activity. Second, using scanning mutagenesis to probe plausible metal-interacting Met, His, and Cys residues of CcoA, we found that the periplasm-exposed Cys 49 located at the end of TMH2, the Cys 247 on a surface loop between TMH7 and THM8, and the C 367 located at the end of TMH11 are important for CcoA function. Analyses of the single and double Cys mutants revealed the occurrence of a disulfide bond in CcoA in vivo , possibly related to conformational changes it undergoes during Cu import as MFS-type transporter. Our overall findings suggest a model linking Cu import for cbb 3 -Cox biogenesis with a thiol:disulfide oxidoreduction step, advancing our understanding of the mechanisms of CcoA function. IMPORTANCE Copper (Cu) is a redox-active micronutrient that is both essential and toxic. Its cellular homeostasis is critical for supporting cuproprotein maturation while avoiding excessive oxidative stress. The Cu importer CcoA is the prototype of the widespread CalT subfamily of the MFS-type transporters. Hence, understanding its molecular mechanism of function is significant. Here, we show that CcoA undergoes a thiol:disulfide oxidoreduction cycle, which is important for its Cu import activity.

Bacteria execute a variety of protein transport systems for maintaining the proper composition of their different cellular compartments. The SecYEG translocon serves as primary transport channel and is engaged in transporting two different substrate types. Inner membrane proteins are cotranslationally inserted into the membrane after their targeting by the signal recognition particle (SRP). In contrast, secretory proteins are posttranslationally translocated by the ATPase SecA. Recent data indicate that SecA can also bind to ribosomes close to the tunnel exit. We have mapped the interaction of SecA with translating and nontranslating ribosomes and demonstrate that the N terminus and the helical linker domain of SecA bind to an acidic patch on the surface of the ribosomal protein uL23. Intriguingly, both also insert deeply into the ribosomal tunnel to contact the intratunnel loop of uL23, which serves as a nascent chain sensor. This binding pattern is remarkably similar to that of SRP and indicates an identical interaction mode of the two targeting factors with ribosomes. In the presence of a nascent chain, SecA retracts from the tunnel but maintains contact with the surface of uL23. Our data further demonstrate that ribosome and membrane binding of SecA are mutually exclusive, as both events depend on the N terminus of SecA. Our study highlights the enormous plasticity of bacterial protein transport systems and reveals that the discrimination between SRP and SecA substrates is already initiated at the ribosome. IMPORTANCE Bacterial protein transport via the conserved SecYEG translocon is generally classified as either cotranslational, i.e., when transport is coupled to translation, or posttranslational, when translation and transport are separated. We show here that the ATPase SecA, which is considered to bind its substrates posttranslationally, already scans the ribosomal tunnel for potential substrates. In the presence of a nascent chain, SecA retracts from the tunnel but maintains contact with the ribosomal surface. This is remarkably similar to the ribosome-binding mode of the signal recognition particle, which mediates cotranslational transport. Our data reveal a striking plasticity of protein transport pathways, which likely enable bacteria to efficiently recognize and transport a large number of highly different substrates within their short generation time., (Copyright © 2019 Knüpffer et al.)

Cytochrome c oxidases are members of the heme-copper oxidase superfamily. These enzymes have different subunits, cofactors, and primary electron acceptors, yet they all contain identical heme-copper (Cu B ) binuclear centers within their catalytic subunits. The uptake and delivery pathways of the Cu B atom incorporated into this active site, where oxygen is reduced to water, are not well understood. Our previous work with the facultative phototrophic bacterium Rhodobacter capsulatus indicated that the copper atom needed for the Cu B site of cbb 3 -type cytochrome c oxidase ( cbb 3 -Cox) is imported to the cytoplasm by a major facilitator superfamily-type transporter, CcoA. In this study, a comparative genomic analysis of CcoA orthologs in alphaproteobacterial genomes showed that CcoA is widespread among organisms and frequently co-occurs with cytochrome c oxidases. To define the specificity of CcoA activity, we investigated its function in Rhodobacter sphaeroides , a close relative of R. capsulatus that contains both cbb 3 - and aa 3 -Cox. Phenotypic, genetic, and biochemical characterization of mutants lacking CcoA showed that in its absence, or even in the presence of its bypass suppressors, only the production of cbb 3 -Cox and not that of aa 3 -Cox was affected. We therefore concluded that CcoA is dedicated solely to cbb 3 -Cox biogenesis, establishing that distinct copper uptake systems provide the Cu B atoms to the catalytic sites of these two similar cytochrome c oxidases. These findings illustrate the large variety of strategies that organisms employ to ensure homeostasis and fine control of copper trafficking and delivery to the target cuproproteins under different physiological conditions. IMPORTANCE The cbb 3 - and aa 3 -type cytochrome c oxidases belong to the widespread heme-copper oxidase superfamily. They are membrane-integral cuproproteins that catalyze oxygen reduction to water under hypoxic and normoxic growth conditions. These enzymes diverge in terms of subunit and cofactor composition, yet they all share a conserved heme-copper binuclear site within their catalytic subunit. In this study, we show that the copper atoms of the catalytic center of two similar cytochrome c oxidases from this superfamily are provided by different copper uptake systems during their biogenesis. This finding illustrates different strategies by which organisms fine-tune the trafficking of copper, which is an essential but toxic micronutrient., (Copyright © 2018 Khalfaoui-Hassani et al.)

Unlabelled: Uptake and trafficking of metals and their delivery to their respective metalloproteins are important processes. Cells need precise control of each step to avoid exposure to excessive metal concentrations and their harmful consequences. Copper (Cu) is a required micronutrient used as a cofactor in proteins. However, in large amounts, it can induce oxidative damage; hence, Cu homeostasis is indispensable for cell survival. Biogenesis of respiratory heme-Cu oxygen (HCO) reductases includes insertion of Cu into their catalytic subunits to form heme-Cu binuclear centers. Previously, we had shown that CcoA is a major facilitator superfamily (MFS)-type bacterial Cu importer required for biogenesis of cbb3-type cytochrome c oxidase (cbb3-Cox). Here, using Rhodobacter capsulatus, we focused on the import and delivery of Cu to cbb3-Cox. By comparing the CcoA amino acid sequence with its homologues from other bacterial species, we located several well-conserved Met, His, and Tyr residues that might be important for Cu transport. We determined the topology of the transmembrane helices that carry these residues to establish that they are membrane embedded, and substituted for them amino acids that do not ligand metal atoms. Characterization of these mutants for their uptake of radioactive (64)Cu and cbb3-Cox activities demonstrated that Met233 and His261 of CcoA are essential and Met237 and Met265 are important, whereas Tyr230 has no role for Cu uptake or cbb3-Cox biogenesis. These findings show for the first time that CcoA-mediated Cu import relies on conserved Met and His residues that could act as metal ligands at the membrane-embedded Cu binding domain of this transporter., Importance: Cu is a micronutrient that is both essential and toxic; hence, its cellular homeostasis is crucial. Respiratory cbb3-type cytochrome c oxidases (cbb3-Cox) are Cu-containing energy-transducing enzymes that are important for many microaerophilic processes, including photosynthesis, respiration, and bacterial pathogenesis. How Cu is incorporated into cbb3-Cox enzymes is not well known. So far, CcoA is the only known major facilitator superfamily (MFS)-type transporter required for Cu import into the bacterial cytoplasm and for cbb3-Cox biogenesis. This study shows that the membrane-embedded, universally conserved Met and His residues of CcoA are essential for its Cu import function and also for its role in cbb3-Cox biogenesis, shedding light on the mechanism of function of this bacterial prototypical Cu importer., (Copyright © 2016 Khalfaoui-Hassani et al.)

Unlabelled: Copper is an essential micronutrient used as a metal cofactor by a variety of enzymes, including cytochrome c oxidase (Cox). In all organisms from bacteria to humans, cellular availability and insertion of copper into target proteins are tightly controlled due to its toxicity. The major subunit of Cox contains a copper atom that is required for its catalytic activity. Previously, we identified CcoA (a member of major facilitator superfamily transporters) as a component required for cbb3-type Cox production in the Gram-negative, facultative phototroph Rhodobacter capsulatus. Here, first we demonstrate that CcoA is a cytoplasmic copper importer. Second, we show that bypass suppressors of a ccoA deletion mutant suppress cbb3-Cox deficiency by increasing cellular copper content and sensitivity. Third, we establish that these suppressors are single-base-pair insertion/deletions located in copA, encoding the major P1B-type ATP-dependent copper exporter (CopA) responsible for copper detoxification. A copA deletion alone has no effect on cbb3-Cox biogenesis in an otherwise wild-type background, even though it rescues the cbb3-Cox defect in the absence of CcoA and renders cells sensitive to copper. We conclude that a hitherto unknown functional interplay between the copper importer CcoA and the copper exporter CopA controls intracellular copper homeostasis required for cbb3-Cox production in bacteria like R. capsulatus., Importance: Copper (Cu) is an essential micronutrient required for many processes in the cell. It is found as a cofactor for heme-copper containing cytochrome c oxidase enzymes at the terminus of the respiratory chains of aerobic organisms by catalyzing reduction of dioxygen (O2) to water. Defects in the biogenesis and copper insertion into cytochrome c oxidases lead to mitochondrial diseases in humans. This work shows that a previously identified Cu transporter (CcoA) is a Cu importer and illustrates the link between two Cu transporters, the importer CcoA and the exporter CopA, required for Cu homeostasis and Cu trafficking to cytochrome c oxidase in the cell.

Unlabelled: The acquisition, delivery, and incorporation of metals into their respective metalloproteins are important cellular processes. These processes are tightly controlled in order to prevent exposure of cells to free-metal concentrations that could yield oxidative damage. Copper (Cu) is one such metal that is required as a cofactor in a variety of proteins. However, when present in excessive amounts, Cu is toxic due to its oxidative capability. Cytochrome c oxidases (Coxs) are among the metalloproteins whose assembly and activity require the presence of Cu in their catalytic subunits. In this study, we focused on the acquisition of Cu for incorporation into the heme-Cu binuclear center of the cbb(3)-type Cox (cbb(3)-Cox) in the facultative phototroph Rhodobacter capsulatus. Genetic screens identified a cbb(3)-Cox defective mutant that requires Cu(2+) supplementation to produce an active cbb(3)-Cox. Complementation of this mutant using wild-type genomic libraries unveiled a novel gene (ccoA) required for cbb(3)-Cox biogenesis. In the absence of CcoA, the cellular Cu content decreases and cbb(3)-Cox assembly and activity become defective. CcoA shows homology to major facilitator superfamily (MFS)-type transporter proteins. Members of this family are known to transport small solutes or drugs, but so far, no MFS protein has been implicated in cbb(3)-Cox biogenesis. These findings provide novel insights into the maturation and assembly of membrane-integral metalloproteins and on a hitherto-unknown function(s) of MFS-type transporters in bacterial Cu acquisition., Importance: Biogenesis of energy-transducing membrane-integral enzymes, like the heme copper-containing cytochrome c oxidases, and the acquisition of transition metals, like copper, as their catalytic cofactors are vital processes for all cells. These widespread and well-controlled processes are poorly understood in all organisms, including bacteria. Defects in these processes lead to severe mitochondrial diseases in humans and poor crop yields in plants. In this study, using the facultative phototroph Rhodobacter capsulatus as a model organism, we report on the discovery of a novel major facilitator superfamily (MFS)-type transporter (CcoA) that affects cellular copper content and cbb(3)-type cytochrome c oxidase production in bacteria.