CtpA, a copper-translocating P-type ATPase involved in the biogenesis of multiple copper-requiring enzymes

Abstract

The ctpA (ccoI) gene product, a putative inner membrane copper-translocating P1B-type ATPase present in many bacteria, has been shown to be involved only in the cbb(3) assembly in Rhodobacter capsulatus and Bradyrhizobium japonicum. ctpA was disrupted in Rubrivivax gelatinosus, and the mutants showed a drastic decrease in both cbb(3) and caa(3) oxidase activities. Inactivation of ctpA results also in a decrease in the amount of the nitrous oxide reductase, NosZ. This pleiotropic phenotype could be partially rescued by excess copper in the medium, indicating that CtpA is likely a copper transporter that supplies copper-requiring proteins in the membrane with this metal. Although CtpA shares significant sequence homologies with the homeostasis copper efflux P1B-type ATPases including the bacterial CopA and the human ATP7A and ATP7B, disruption of ctpA did not result in any sensitivity to excess copper. This indicates that the CtpA is not crucial for copper tolerance but is involved in the assembly of membrane and periplasmic copper enzymes in this bacterium. The potential roles of CtpA in bacteria in comparison with CopA are discussed.

FIGURE 1.

Genetic organization of the cbb₃ encoding operon ccoNOQP and the flanking regions in R. gelatinosus, R. capsulatus, T. denitrificans (ATCC 25259), and H. pylori (G27). Note that ctpA (ccoI) homologues are not present in these regions in T. denitrificans and H. pylori. ccoH is absent in T. denitrificans and H. pylori genomes. However, homologues of ccoG, ctpA, and ccoS were found in H. pylori (GPG27_1432), HPG27–356 copA, and HGP27_1107, respectively). In T. denitrificans, homologues of ctpA and ccoS are denoted Tbd_2044 and Tbd_2045, respectively.

FIGURE 2.

BN-PAGE of solubilized membranes from the wt, ctpA⁻ and ccoO⁻ mutants. A, shown is cbb₃ oxidase in-gel activity assay on the same gradient BN-PAGE. B, wt, ctpA⁻, and ccoO⁻ mutants were grown semi-aerobically in malate medium (1.6 μm CuSO₄). wt and ctpA⁻ were also grown in malate supplemented with 20 μm CuSO₄ (second and fourth lanes). Inactivation of ctpA resulted in a significant decrease of cbb₃ oxidase activity. The cbb₃ complex (brownish bands) is revealed in the membrane of the wild type and of the ctpA⁻ mutant grown with excess copper but not in the cbb₃ null mutant ccoO⁻. The upper part of the gel was deleted for better viewing.

FIGURE 3.

SDS-PAGE and 3,3′,5,5′-tetramethylbenzidine (TMBZ) analyses of c-type cytochromes. Comparable amounts of membrane proteins (∼120 μg) from wt and the ctpA⁻ mutant grown under microaerobic conditions were loaded on a 12% SDS-PAGE gel. The gel was first stained for covalently bound heme with 3,3′,5,5′-tetramethylbenzidine (upper) then with Coomassie Blue (lower). The band at 43 kDa present in the WT and in the mutant corresponds to the reaction center-attached tetraheme cytochrome c (PufC). Both CcoP and CcoO are drastically reduced in the ctpA⁻ membranes compared with the wild type. These TMBZ bands turn green in the Coomassie Blue-stained gel (B).

FIGURE 4.

Measurement of Cox activity in whole cells. wt, ctpA⁻, and ccoO⁻ mutants were grown in malate medium with increasing copper concentrations; the control growth medium contained 1.6 μm of CuSO₄. The activity was measured spectrophotometrically by following the oxidation of TMPD as absorption increases at 562 nm. Data are the mean values of triplicates. The ctpA⁻ mutant displayed a dramatic decrease in cbb₃ activity relative to that observed in the wild type. The activity increased, however, in the mutant with increasing concentrations of copper.

FIGURE 5.

X-band EPR spectra of oxidized membrane fragments from wt and ctpA⁻ mutant strains in the spectral range were paramagnetic copper species are typically observed. g = 2.6 to g = 2.0). The spectra were normalized to corresponding protein contents in both types of membranes. Instrument settings: microwave frequency, 9.43 GHz; temperature, 15 K; microwave power, 6.3 milliwatts; modulation amplitude, 3.2 millitesla.

FIGURE 6.

caa₃ oxidase in-gel activity assay on gradient BN-PAGE. Res2 and Res2- ctpA⁻ mutants were grown semi-aerobically in malate medium (1.6 μm CuSO₄) or in malate supplemented with CuSO₄ (20 μm). Inactivation of ctpA resulted in a significant decrease of caa₃ oxidase activity. The addition of 20 μm of CuSO₄ to the growth medium of ctpA⁻ mutant can also partially restore the caa₃ oxidase activity in the mutant.

FIGURE 7.

cbb₃ oxidase in-gel activity assay on gradient BN-PAGE. wt and ctpA⁻ mutant were grown semi-aerobically in malate medium. LH-RC, light harvesting reaction center. A drastic decrease in the amount of the high molecular blue-violet band (containing the nitrous oxide reductase NosZ) observed in the wild type was decreased in the ctpA⁻ mutant.

FIGURE 8.

Analysis of ctpA⁻ mutant membranes compared with wild type on gradient BN-PAGE. Cells were grown semi-aerobically in malate medium supplemented or not with 20 or 100 μm CuSO₄. In-gel cbb₃ activity in these membranes is shown. The addition of 20 or 100 μm CuSO₄ to the growth medium of ctpA⁻ mutant can also restore the NosZ accumulation in the membranes in the mutant. LH-RC, light harvesting reaction center.