Lead(II) resistance in Cupriavidus metallidurans CH34: interplay between plasmid and chromosomally-located functions

Abstract

Proteome and transcriptome analysis, combined with mutagenesis, were used to better understand the response of Cupriavidus metallidurans CH34 against lead(II). Structural Pb(II)-resistance genes of the pMOL30-encoded pbrUTRABCD operon formed the major line of defense against Pb(II). However, several general stress response mechanisms under the control of alternative sigma factors such as sigma24/rpoK, sigma32/rpoH and sigma28/fliA were also induced. In addition, the expression of the pbrR(2) cadA pbrC(2) operon of the CMGI-1 region and the chromosomally encoded zntA were clearly induced in the presence of Pb(II), although their respective gene products were not detected via proteomics. After inactivation of the pbrA, pbrB or pbrD genes, the 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 Pb(II) concentration, where pbrR(2) cadA pbrC(2) gene functions can complement and compensate for the mutations in the pbrA and pbrD genes. This role of zntA and cadA to complement for the loss of pbrA was further confirmed by mutation analysis. The pbrB:: colonsTn(Km2) mutation resulted in the most significant decrease of Pb(II) resistance, indicating that Pb(II) sequestration, avoiding re-entry of this toxic metal ion, forms a critical step in the pbr-encoded Pb(II) resistance mechanism.

Contributions of Five Secondary Metal Uptake Systems to Metal Homeostasis of Cupriavidus metallidurans CH34

“…S1 in the supplemental material). The three P IB2 -type ATPases (TC 3.A.3) ZntA, CadA, and PbrA remove Zn 2ϩ , Cd 2ϩ , and Pb 2ϩ , respectively, from the cytoplasm (4,7,24,49,51,56 , respectively, from the cytoplasm to the periplasm to feed additional substrate cations into the RND-driven efflux systems (2,3,51). Up to four P IB1 -type ATPases, two RNDdriven efflux systems, and a variety of other proteins maintain copper homeostasis (31) and may also be involved in silver and gold transformation (22,47).…”

Heavy metal resistance in Cupriavidus metallidurans CH34 is governed by an intricate transcriptional network

“…Its genome, made out of two chromosomes and two megaplasmids, was sequenced and thoroughly analyzed (Janssen et al 2010). While the study of metal resistance determinants in CH34 initially focused on the bacterium's two megaplasmids pMOL28 and pMOL30, the importance of chromosomal genes was acknowledged in later studies (Legatzki et al 2003;Grosse et al 2004;Munkelt et al 2004;Nies et al 2006;Taghavi et al 2008). It is noteworthy that many heavy metal resistance determinants are linked to genomic islands and transposons present on the bacterium's four replicons (Monchy et al 2007;Mergeay et al 2009;).…”

Transcriptional activation of MerR family promoters in Cupriavidus metallidurans CH34

“…We also found that there was only slight Pb(II)-inducible expression from PpbrA in AE104, which lacks both pMOL28 and pMOL30. This suggests that there may be a very low level of cross-regulation of pMOL30 PpbrA by PbrR 2 (R_met2302) and/or PbrR 3 (R_met3456) [(previously known as PbrR691 and PbrR710, respectively (Taghavi et al 2009)], but sequential deletion of PbrR family regulators in CH34 would be required to determine unambiguously which promoters previously identified as being targets for PbrR regulators are activated by them. Levels of Pb(II) required for induction of PpbrA are Fig.…”

“…There is a precedent for this model with MerD competing for PmerT with MerR (reviewed in Hobman et al 2005). Regulatory cross-talk between different metal-sensing regulators and promoters in C. metallidurans is one explanation of recent transcriptomic data that showed expression of diverse metal ion resistance genes in response to single metal ions, but RNA polymerase r 24 , r 28 and r 32 sigma factors are also postulated as being responsible for this effect (Monchy et al 2007;Taghavi et al 2009). …”

“…A second phylogenetic grouping of MerR family regulators in C. metallidurans include the Pb(II)-responsive PbrR from the pbr operon (pbrTRABCD) carried on pMOL30 (Borremans et al 2001), the PAGI-2(C)-encoded PbrR homologue, pbrR691 (PbrR 2 ) (Mergeay et al 2003;Chen et al 2005Chen et al , 2007Sarkar et al 2007) and the chromosomally encoded pbrR710 (PbrR 3 ) (Mergeay et al 2003;Taghavi et al 2009). Other predicted MerR family regulators include the predicted chromosomal copper efflux ATPase-associated regulator, CupR (Monchy et al 2006b), and two further chromosomal MerR regulators which are not physically linked to known metal resistance genes (Mergeay et al 2003).…”