Nies further subdivided the HME-RND proteins into sub-groups, according to the substrate they transport: HME1 (Zn2+, Co2+, Cd2+), HME2 (Co2+, Ni2+), HME3a NVP-BSK805 mw (divalent cations), HME3b (monovalent cations), HME4 (Cu+ ou Ag+) and HME5 (Ni2+) [14]. The cytoplasmic membrane RND proteins have 12 transmembrane alpha helices (TMH), among which TMH IV contains amino acid residues that are conserved in most RND proteins [17]. The HME1-RND and HME2-RND have the same motifs, DFG-DGA-VEN, present in proteins CzcA (HME1) or CnrA

and NccA (HME2) [14, 23]. Both aspartate residues and the glutamate residue in TMH IV of CzcA are required for proton/substrate-antiport, suggesting that they are probably involved in proton translocation [14, 23, 24]. A model for cation transport by an HME-RND was recently proposed for the copper transporter CusA, in which the metal ion moves along a pathway of methionine find more residues, causing significant conformational changes

in both the periplasmic and transmembrane domains [25]. These systems are proposed to promote the efflux of both cytoplasmic and periplasmic substrates, CP-690550 chemical structure transporting of the substrate either via the RND protein or in some cases via the membrane fusion protein with the aid of periplasmic metal chaperones [14, 24]. The best characterized RND heavy metal efflux systems are mainly those from Cupriavidus (previously called Ralstonia and Alcaligenes): CzcCBA (Cd2+, Zn2+, and Co2+ resistance) from Ralstonia metallidurans CH34 [26–28]; CnrCBA (Ni2+ and Co2+) from Ralstonia eutropha[29, 30];

NccCBA (Ni2+, Co2+ and Cd2+) from Alcaligenes xylosoxidans 31A Reverse transcriptase [31]. However, other systems such as Pseudomonas aeruginosa Czr (Cd2+ and Zn2+ resistance) [32]; and Helicobacter pylori Czn (Cd2+, Zn2+ and Ni2+ resistance) were also studied [33]. In order to better understand the role of the RND efflux systems in the export of divalent cations in other Proteobacteria, we investigated the role of two HME-RND systems present in the Alphaproteobacterium Caulobacter crescentus. A previous bioinformatics analysis made by Nies (2003) through comparison of the genomes of 63 prokaryotes (Archaea and Bacteria) with the genome of C. metallidurans, identified seven ORFs encoding putative RND proteins in C. crescentus CB15 of which two, CC2724 (corresponding to CCNA_02809 in the derivative strain NA1000; here called CzrA) and CC2390 (CCNA_02473; here called NczA), belong to the HME subgroup. Previous works from our group [34] identified that the czrCBA locus is involved in resistance to cadmium and zinc and is induced by these cations, and other reports [35] confirmed that this operon is induced by cadmium.