Periplasmic response upon disruption of transmembrane Cu transport in Pseudomonas aeruginosa

Abstract

Pseudomonas aeruginosa, an opportunistic pathogen, has two transmembrane Cu(+) transport ATPases, CopA1 and CopA2. Both proteins export cytoplasmic Cu(+) into the periplasm and mutation of either gene leads to attenuation of virulence. CopA1 is required for maintaining cytoplasmic copper levels, while CopA2 provides copper for cytochrome c oxidase assembly. We hypothesized that transported Cu(+) ions would be directed to their destination via specific periplasmic partners and disruption of transport should affect the periplasmic copper homeostasis. Supporting this, mutation of either ATPase gene led to large increments in periplasmic cuproprotein levels. Toward identifying the proteins participating in this cellular response the periplasmic metalloproteome was resolved in non-denaturing bidimensional gel electrophoresis, followed by X-ray fluorescence visualization and identification by mass-spectrometry. A single spot containing the electron shuttle protein azurin was responsible for the observed increments in cuproprotein contents. In agreement, lack of either Cu(+)-ATPase induced an increase in azu transcription. This is associated with an increase in the expression of anr and rpoS oxidative stress response regulators, rather than cueR, a copper sensing regulator. We propose that azurin overexpression and accumulation in the periplasm is part of the cellular response to cytoplasmic oxidative stress in P. aeruginosa.

Mechanisms of copper homeostasis in bacteria

“…In this organism, high Cu + induces temporal changes on transcription rates of two Cu + -chaperones and one Cu + -transporting ATPase through the activation of a Mer-like transcription factor. As observed previously in P. aeruginosa , the Cu + responsive metalloregulator is not up-regulated by Cu + (Teitzel et al, 2006; Raimunda et al, 2013). This, plus the fact that pools of small Cu + sequestering molecules like glutathione do not compete with the metalloregulator for Cu + (Changela et al, 2003), implies that a regulatory feedback involving physical interaction and Cu + transfer between the chaperones and the regulator might exist.…”

“…For instance, mutation of P. aeruginosa cinA , an azurin/plastocyanin-like protein, leads to increased sensitivity to Cu 2+ in the media (Elguindi et al, 2009). Deletion of either P. aeruginosa Cu + -ATPase, CopA1, or CopA2, induces an increase in azurin transcription as a cellular response to Cu + -derived oxidative stress (Raimunda et al, 2013). However, whether azurins are metallated by the transporters or participate in Cu + oxidation has not been established.…”

“…The presence of different cuproenzymes such as azurin (Raimunda et al, 2013), CueO (Roberts et al, 2002), Cu/Zn-Sod (Gort et al, 1999), laccases (Claus, 2003), cytochrome c oxidases (Richter and Ludwig, 2003), and tyrosinases (Claus and Decker, 2006) among other cuproproteins, exemplifies the vast diversity of periplasmic Cu + -binding proteins in different bacterial species (Figure 1). Although the function of these enzymes is fairly understood, the mechanisms underlying their metallation and Cu + traffic in this compartment require further studies.…”

Differential expression of zinc transporters accompanies the differentiation of C2C12 myoblasts

“…Then, samples were acid digested in concentrated HNO 3 (trace metal grade) for 1 h at 80 °C and overnight at 20 °C. Digestions were concluded by adding 30 μl H 2 O 2 [94–97]. The resulting solution was measured by triplicate using atomic absorption spectroscopy (AAS) equipped with a graphite furnace (PerkinElmer, Aanalyst 800), which provides an ideal limit of detection (LOD) of Zn from currently available techniques [98].…”

Comparative Analyses of Transport Proteins Encoded within the Genomes of Bdellovibrio bacteriovorus HD100 and Bdellovibrio exovorus JSS

“…Each BALO has another P-ATPase (TC 3.A.3.27) encoded in an operon with 5 or 6 other genes, where 3 of them encode proteins of the cytochrome oxidase complex (e.g., cytochrome oxidase maturation protein, cytochrome C oxidase, and an iron-sulfur cluster protein). The function of this ATPase is probably the insertion of copper into cytochrome oxidase [Gonzalez-Guerrero et al, 2010; Raimunda et al, 2013]. There are 2 and 3 more P-ATPases in Bex and Bba, respectively, all of them encoded in separate operons.…”