Growth was determined by

measuring the OD600 Trichostatin A of the cultures. C. crescentus NA1000 and mutant strains carrying either the empty vector pNPT228XNE or the vector harbouring either czrA or nczA genes were grown in PYE-kanamycin at 30°C with agitation to an OD600 of 0.3. Samples of 10 μl were streaked on PYE-kanamycin plates containing 2% xylose and with or without addition of each of the following metal salts: 35 μM CdCl2, 130 μM ZnCl2, 50 μM CoCl2 and 280 μM NiCl2, and plates were incubated at 30°C for 3 days. Statistical treatment of the data was carried out using Student’s T-Test. Phylogenetic and protein structure analyses Amino acid sequences presenting more than 55% identity with CzrA and NczA were used as an imput for CLUSTALX [40]. The complete list of the protein sequences used is found in Additional file 1: Table S1. The phylogenetic tree was constructed by a neighbor-joining method with 1000 bootstrap replicates using the CLUSTALX program. The multiple sequence alignment was used to create the logo representation of the CzrA and NczA orthologous grups. The figure was generated using the WebLogo server [42] and the height of the residue symbol indicates the degree of conservation. The sequence numbering shown below the logo corresponds to the proteins from C. crescentus NA1000. Homology modeling of CzrA was performed using the PHYRE2 [44] using

as a three-dimensional PLX4032 mw structural template the chain A of E. coli CusA [PDB: 3 k07; [25]. CzrA and CusA share Hydroxychloroquine 33% sequence identity. The model generated has 100% confidence and 93% coverage. The result was analyzed with the PyMOL Molecular Graphics System, Version 1.5 Schrödinger, LLC [43]. Acknowledgements This work was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and by Fundação

de Amparo à Pesquisa do Estado de São Paulo (FAPESP). EYV was supported by doctoral fellowship from CNPq. VSB was supported by postdoctoral fellowship from FAPESP. MVM was partially supported by CNPq. Electronic supplementary material Additional file 1: Table S1: Protein sequences used for the phylogenetic analysis of the HME-RND orthologs. (PDF 150 KB) Additional file 2: Figure S1: Sequence conservation profile within the CzrA and NczA orthologous groups. (PDF 1006 KB) Additional file 3: Figure S2: Potential methionine pairs/clusters in CzrA model structure. (PDF 423 KB) References 1. Valls M, de Lorenzo V: Exploiting the genetic and biochemical capacities of bacteria for the remediation of heavy metal pollution. FEMS Microbiol Rev 2002, 26:327–338.PubMed 2. Mitra RS, Bernstein IA: Single-strand breakage in DNA of Escherichia coli exposed to Cd2 + . J Bacteriol 1978, 133:75–80.PubMed 3. Bruins MR, Kapil S, Oehme FW: Microbial resistance to metals in the environment. Ecotoxicol Environ Saf 2000, 45:198–207.PubMedCrossRef 4.