Copper intoxication in group B Streptococcus triggers transcriptional activation of the cop operon that contributes to enhanced virulence during acute infection

Bacteria can utilize Copper (Cu) as a trace element to support cellular processes; however, excess Cu can intoxicate bacteria. Here, we characterize the cop operon in group B streptococcus (GBS), and establish its role in evasion of Cu intoxication and the response to Cu stress on virulence. Growth of GBS mutants deficient in either the copA Cu exporter, or the copY repressor, were severely compromised in Cu-stress conditions. GBS survival of Cu stress reflected a mechanism of CopY de-repression of the CopA efflux system. However, neither mutant was attenuated for intracellular survival in macrophages. Analysis of global transcriptional responses to Cu by RNA-sequencing revealed a stress signature encompassing homeostasis of multiple metals. Genes induced by Cu stress included putative metal transporters for manganese import, whereas a system for iron export was repressed. In addition, copA promoted the ability of GBS to colonize the blood, liver and spleen of mice following disseminated infection. Together, these findings show that GBS copA mediates resistance to Cu intoxication, via regulation by the Cu-sensing transcriptional repressor, copY . Cu stress responses in GBS reflect a transcriptional signature that heightens virulence and represents an important part of the bacteria’s ability to survive in different environments. Importance Understanding how bacteria manage cellular levels of metal ions, such as copper, helps to explain how microbial cells can survive in different stressful environments. We show how the opportunistic pathogen group B Streptococcus (GBS) achieves homeostasis of intracellular copper through the activities of the genes that comprise the cop operon, and describe how this helps GBS survive in stressful environments, including in the mammalian host during systemic disseminated infection.

Copper intoxication in group B Streptococcus triggers transcriptional activation of the cop operon that contributes to enhanced virulence during acute infection

Bacteria require Copper (Cu) as an essential trace element to support cell processes; however, excess Cu can intoxicate bacteria. Here, we characterize the cop operon in group B Streptococcus (GBS), and establish its role in evasion of Cu intoxication and the response to Cu stress on virulence. Growth of GBS mutants deficient in either the copA Cu exporter, or the copY repressor, were severely compromised in Cu-stress conditions. GBS survival of Cu stress reflected a mechanism of CopY activation of the CopA efflux system. However, neither mutant was attenuated for intracellular survival in macrophages. Analysis of global transcriptional responses to Cu by RNA-sequencing revealed a stress signature encompassing homeostasis of multiple metals. Genes induced by Cu stress included putative metal transporters for manganese import, whereas a system for iron export was repressed. In addition, copA promoted the ability of GBS to colonize the blood, liver and spleen of mice following disseminated infection. Together, these findings show that GBS copA mediates resistance to Cu intoxication, via regulation by the Cu-sensing transcriptional repressor, copY. Cu stress responses in GBS reflect a transcriptional signature that heightens virulence and represents an important part of the bacteria’s ability to survive in different environments.

Functional and Expression Analyses of the cop Operon, Required for Copper Resistance in Agrobacterium tumefaciens

ABSTRACT The copper resistance determinant copARZ, which encodes a CPx-type copper ATPase efflux protein, a transcriptional regulator, and a putative intracellular copper chaperone, was functionally characterized for the phytopathogenic bacterium Agrobacterium tumefaciens. These genes are transcribed as an operon, and their expression is induced in response to increasing copper and silver ion concentrations in a copR-dependent fashion. Analysis of the copARZ promoter revealed a putative CopR binding box located within the spacer of the −35 and −10 promoter motifs. In vitro, purified CopR could specifically bind to the box. The inactivation of the copARZ operon or copZ reduces the level of resistance to copper but not to other metal ions. Also, the copARZ operon mutant shows increased sensitivity to the superoxide generators menadione and plumbagin. In addition, the loss of functional copZ does not affect the ability of copper ions to induce the copARZ promoter, indicating that CopZ is not involved in the copper-sensing mechanism of CopR. Altogether, the results demonstrate a crucial role for the copARZ operon as a component of the copper resistance machinery in A. tumefaciens.

Characterization of copABCD operon from a copper-sensitive Pseudomonas putida strain

We describe an operon, copABCD, that encodes copper-binding and sequestering proteins for copper homeostasis in the copper-sensitive strain Pseudomonas putida PNL-MK25. This is the second operon characterized as being involved in copper homeostasis, in addition to a P1-type ATPase encoded by cueAR, which was previously shown to be active in the same strain. In this study, 3 copper-responsive mutants were obtained through mini-Tn5::gfp mutagenesis and were found to exhibit reduced tolerance to copper. Sequencing analysis of the transposon-tagged region in the 3 mutants revealed insertions in 2 genes of an operon homologous to the copABCD of P. syringae and pcoABCD of Escherichia coli. Gene expression studies demonstrated that the P. putida copABCD is inducible starting from 3 µmol/L copper levels. Copper-sensitivity studies revealed that the tolerance of the mutant strains was reduced only marginally (only 0.16-fold) in comparison to a 6-fold reduced tolerance of the cueAR mutant. Thus, the cop operon in this strain has a minimal role when compared with its role both in other copper-resistant strains, such as P. syringae pv. syringae, and in the cueAR operon of the same strain. We propose that the reduced function of the copABCD operon is likely to be due to the presence of fewer metal-binding domains in the encoded proteins.Key words: cop operon, copper-binding proteins, mini-Tn5::gfp mutagenesis, transition metal.

Role of proteolysis in copper homoeostasis

The cop operon of Enterococcus hirae controls cytoplasmic copper levels. It encodes two copper ATPases, a repressor, and the CopZ metallo-chaperone. Transcription of these genes is induced by copper. However, at higher copper concentrations, CopZ is degraded by a copper-activated proteolytic activity. This specific proteolysis of CopZ can also be demonstrated in vitro with E. hirae extracts. Growth of the cells in copper increases the copper-inducible proteolytic activity in extracts. Zymography reveals the presence of a copper-dependent protease in crude cell lysates. Copper-stimulated proteolysis of CopZ appears to play an important role in copper homoeostasis by E. hirae.