9.B.105 The Peptidase/Phosphatase/Lead Resistance Fusion Protein (PPLR) Family

Proteome and transcriptome analysis, combined with mutagenesis, were used to better understand the response of Ralstonia (Cupriavidus) metallidurans CH34 to Pb²⁺ (Taghavi et al., 2009). Structural Pb²⁺-resistance genes of the pMOL30-encoded pbrUTRABCD operon form the major line of defense to Pb²⁺ (Taghavi et al., 2009). The expression of the pbrR(2) cadA pbrC(2) operon of the CMGI-1 region and the chromosomal zntA gene were clearly induced in the presence of Pb²⁺. After inactivation of the pbrA, pbrB or pbrD gene, expression of the pbrR(2) cadA pbrC(2) operon went up considerably. This points towards synergistic interactions between pbrUTRABCD and pbrR(2) cadA pbrC(2) to maintain a low intracellular Pb²⁺concentration, where pbrR(2) cadA pbrC(2) gene functions can complement and compensate for mutations in the pbrA and pbrD genes. This role of zntA and cadA to complement the loss of pbrA was further confirmed by mutation analysis (Taghavi et al., 2009). The pbrB::Tn(Km2) mutation resulted in the most significant decrease of Pb²⁺resistance, indicating that Pb²⁺ sequestration, avoiding re-entry of this toxic metal ion, forms a critical step in pbr-encoded Pb²⁺resistance. 

Residues 1-190 ( TMSs 1-6) in PbrB/C code for a PAP2-type protein in a family of phosphatases and haloperoxidases. These homologues are found only in bacteria and may be integral membrane phospholipid phosphatases. The C-terminal residues (residues 200-340; TMSs 7-10) may code for a signal peptidase II (pfam01252). In fact, several member of this family are annotated as lipoprotein signal peptidases, suggesting that these integral membrane proteins may be proteases. Other members (see subfamilies 4 - 6; subfamilies 5 and 6 were formerly subfamilies 9.B.196.1 and 2, respectively) appear to belong to the Phosphodiester PA Phosphatase (PAP2) Superfamily (Sigal et al. 2005).

The PAP2 superfamily consists of numerous integral membrane proteins, annotated in Pfam and NCBI as phosphodiesterases. Phosphatidylcholine (PC) hydrolysis generates two second messengers: phosphatidic acid (PA) and diacylglycerol (DAG). Phospholipase D (PLD) and phosphatidate phosphohydrolase (PAPase) are involved in their generation and therefore are key enzymes in signal transduction. Specific isoforms of these enzymes are activated by receptor occupancy in the brain. Phosphatidylinositol 4,5-bisphosphate-dependent PLD (PIP2-PLD) and N-ethylmaleimide-insensitive PAPase (PAP2) have been suggested to act in series to generate the biologically active lipids PA and DAG (Salvador et al. 2002).

PbrBC may be a phosphatase. While PbrA non-specifically exported Pb²⁺, Zn²⁺ and Cd²⁺, a specific increase in lead resistance is observed when PbrA and PbrB are coexpressed. Possibly Pb²⁺ is exported from the cytoplasm by PbrA and then sequestered as a phosphate salt with the inorganic phosphate produced by PbrB. Similar operons containing genes for heavy metal translocating ATPases and phosphatases can be found in many different bacterial species, suggesting that lead detoxification through active efflux and sequestration is a common lead-resistance mechanism (Hynninen et al. 2009).