The copper-sensitive operon repressor (CsoR) family is the main Cu(I)-sensing family, which is widely distributed, and regulates regulons involved in detoxification in response to extreme copper stress (a general range of ≥ 3 g/L copper ions). Here, we identified CsoR
Ac
in hyper-copper-resistant
Acidithiobacillus caldus
, a type strain used in the bioleaching process of copper ores. CsoR
Ac
possesses highly conserved Cu(I) ligands and structures within the CsoR family members. Transcriptional analysis assays indicated that the promoter (PIII) of
csoR
was active but weakly responsive to copper in
Escherichia coli
. Copper titration assays gave a stoichiometry of 0.8 mol Cu(I) per apo-CsoR
Ac
monomer
in vitro
combined with atomic absorption spectroscopy analysis. Cu
I
-CsoR
Ac
and apo-CsoR
Ac
share essentially identical secondary structures and assembly states, as demonstrated by circular dichroism spectra and size exclusion chromatography profiles. The average dissociation constants (
K
D
= 2.26 × 10
−18
M and 0.53 × 10
−15
M) and Cu(I) binding affinity of apo-CsoR
Ac
were estimated by bathocuproine disulfonate (BCS) and bicinchoninic acid (BCA) competition assays, respectively. Site-directed mutations of conserved Cu(I) ligands in CsoR
Ac
did not significantly alter the secondary structure or assembly state. Competition assays showed that mutants shared the same order of magnitude of Cu(I) binding affinity with apo-CsoR
Ac
. Moreover, apo-CsoR
Ac
could bind to the DNA fragment P08430
in vitro
, although with low affinity. Finally, a working model was proposed to illustrate putative copper resistance mechanisms in
A. caldus
.
Importance
Research on copper resistance among various species has attracted considerable interest. However, due to the lack of effective and reproducible genetic tools, few studies regarding copper resistance have been reported for
A. caldus
. Here, we characterized a major Cu(I)-sensing family protein, CsoR
Ac
, which binds Cu(I) with an attomolar affinity higher than that of the Cu(I)-specific chelator, bathocuproine disulfonate. In particular, CsoR family proteins were only identified in
A. caldus
, rather than
A. ferrooxidans
and
A. thiooxidans
, which are both type strains used for bioleaching. Meanwhile,
A. caldus
harbored more copper resistance determinants and a relatively full-scale regulatory system involved in copper homeostasis. These observations suggested that
A. caldus
may play an essential role in the application of engineered strains with higher copper resistance in the near future.