The combined actions of the copper‐responsive repressor CsoR and copper‐metallochaperone CopZ modulate CopA‐mediated copper efflux in the intracellular pathogen Listeria monocytogenes

Abstract

Summary We have characterized the csoR‐copA‐copZ copper resistance operon of the important human intracellular pathogen Listeria monocytogenes. Transcription of the operon is specifically induced by copper, and mutants lacking the P1‐type ATPase CopA have reduced copper tolerance and over‐accumulate copper relative to wild type. The copper‐responsive repressor CsoR autoregulates transcription by binding to a single 32 bp site spanning the −10 and −35 elements of the promoter. Copper co‐ordination by CsoR derepresses transcription of the operon and alters CsoR:DNA complex assembly as determined by DNase I footprinting and electrophoretic mobility shift assays, with some DNA‐binding capacity being retained in the presence of 2 mole equivalents of copper. Analysis of the CsoR copper sensory site demonstrated that substitution of Cys42 with Ala generated a CsoR variant that was unresponsive to copper. Importantly, in the absence of CopZ, copper responsiveness of csoR‐copA‐copZ expression is substantially increased, implying that CopZ reduces the access of CsoR to copper. Furthermore, CopZ is shown to confer copper resistance in mutants lacking copper‐inducible csoR‐copA‐copZ expression, thus providing protection from the deleterious effects of copper within the cytoplasm.

Cu(I)-mediated Allosteric Switching in a Copper-sensing Operon Repressor (CsoR)

“…We have recently developed a CsoR from Geobacillus thermodenitrificans (Gt) as a model system for this purpose (28). We show here that Gt CsoR is representative of CsoRs from other mesophilic bacilli, including B. subtilis (16) and the pathogenic bacteria L. monocytogenes (19) and Bacillus anthracis, and is more distantly related to other CsoRs characterized previously (14,18,20). The x-ray crystallographic structure of Cu(I)-bound Gt CsoR and companion NMR and small angle x-ray scattering (SAXS) experiments provide new insights into the Cu(I)-dependent conformational switching associated with allosteric negative regulation of DNA binding by Cu(I).…”

“…Our phylogenetic analysis could be interpreted to suggest that Cu(I)-sensing CsoRs arose multiple times during the course of evolution within CsoR/ RcnR family repressors, although this cannot be stated with certainty, given the relatively small data set. Gt CsoR is representative of one of these groups and is most closely related to other CsoRs from mesophilic bacilli, including those of B. subtilis (15,16) and the human pathogens B. anthracis and L. monocytogenes (19). The observed distribution of functionally characterized proteins and their uncharacterized homologs as well as the presence of highly conserved clade-specific residues figure prominently in the structure of the Gt CsoR and provide support for the hypothesis that features that distinguish one Cu(I)-sensing CsoR group from another derive from these clade-specific residues outside the X-Y-Z motif (Cys 50 , His 75 , and Cys 79 ) and previously characterized second coordination residues (Tyr 49 and Glu 95 ) (Fig.…”

“…The copper-sensitive operon repressor (CsoR) 2 was discovered in M. tuberculosis (14) as the first representative of the DUF156 family; other CsoRs were subsequently studied in Bacillus subtilis, Staphylococcus aureus, Thermus thermophilus, L. monocytogenes, and Streptomyces lividans (15)(16)(17)(18)(19)(20). A second M. tuberculosis CsoR paralog, RicR, was shown to be involved in the regulation of several genes only found in pathogenic mycobacteria in response to copper stress (2).…”

The Role of Copper and Zinc Toxicity in Innate Immune Defense against Bacterial Pathogens

“…The copA mutant strain of N. gonorrhoeae also had impaired survival within cervical epithelial cell host (33). By contrast, the copA mutants of Listeria monocytogenes (61,62), Vibrio cholerae (63), and S. Typhimurium (56) displayed no loss of pathogenic fitness in animal or macrophage infection models.…”

Two Zinc Uptake Systems Contribute to the Full Virulence of Listeria monocytogenes during Growth In Vitro and In Vivo

“…1]) was achieved in a similar fashion, except that lacZ was inserted into the BamHI site between the zur flanking regions in plasmid pAUL-A⌬zur, followed by transformation into wildtype L. monocytogenes and standard integration and double recombination as described above. ␤-Galactosidase assays were performed as described previously (7). A single-copy complementation of the ⌬zurAM ⌬zinA strain (Fig.…”