A cytosolic copper storage protein provides a second level of copper tolerance inStreptomyces lividans

Abstract

Streptomyces lividans has a distinct dependence on the bioavailability of copper for its morphological development. A cytosolic copper resistance system is operative in S. lividans that serves to preclude deleterious copper levels. This system comprises of several CopZ-like copper chaperones and P-type ATPases, predominantly under the transcriptional control of a metalloregulator from the copper sensitive operon repressor (CsoR) family. In the present study, we discover a new layer of cytosolic copper resistance in S. lividans that involves a protein belonging to the newly discovered family of copper storage proteins, which we have named Ccsp (cytosolic copper storage protein). From an evolutionary perspective, we find Ccsp homologues to be widespread in Bacteria and extend through into Archaea and Eukaryota. Under copper stress Ccsp is upregulated and consists of a homotetramer assembly capable of binding up to 80 cuprous ions (20 per protomer). X-ray crystallography reveals 18 cysteines, 3 histidines and 1 aspartate are involved in cuprous ion coordination. Loading of cuprous ions to Ccsp is a cooperative process with a Hill coefficient of 1.9 and a CopZ-like copper chaperone can transfer copper to Ccsp. A Δccsp mutant strain indicates that Ccsp is not required under initial copper stress in S. lividans, but as the CsoR/CopZ/ATPase efflux system becomes saturated, Ccsp facilitates a second level of copper tolerance.

Modified chrome azurol S method for detection and estimation of siderophores having affinity for metal ions other than iron

“…Growth of isolates at higher concentrations of various metal ions is due to a variety of reasons such as requirement of heavy metal ions for cell growth and metabolism; requirement of these metal ions as cofactors for various enzymes may be due to multi-dimensional heavy metal resistance mechanisms, and bioaccumulation or transformations of metal ions (Straw et al 2018). Zn 2+ and Cu…”

Bacterial copper storage proteins

“…Furthermore, BsCsp3-bound Cu(I) can be withheld from the efflux pump. It has also been found that in S. lividans Csp3 enables growth at higher Cu levels (75).…”

“…S1 in ref. 75) are identical to bacterial Csp sequences and are therefore most likely not from the organism indicated, but are due to contamination with bacterial DNA. This is not surprising given that the bacteria in question are either soil dwelling or widely distributed in the environment.…”

“…Approximately 140 MtCsp3 homologues are found in Archaea. It has recently been claimed (75) that Csps are present in eukaryotes. However, the three proposed eukaryotic Csps (two in plants and another in a soil-dwelling fungus according to Fig.…”

“…3, A and C), again with little evidence of disulfide bond formation (36) (Fig. 3, A and C) (36), although the average Cu(I) affinity appears to be an order of magnitude weaker (75). Crystal structures of the apo-Csp3s from Pseudomonas aeruginosa (3KAW) and Nitrosospira multiformis (3LMF) have been deposited by a structural genomics consortium.…”