Plasmid pZM3H1 carries a large portion of this C. litoralis transposon (17 ORFs; orf8-orf23), RSL3 concentration although it lacks the 5.3 -kbterminal selleck screening library region of the element, which contains three genes coding for a putative NADP-specific glutamate dehydrogenase,
a conserved membrane protein and a transposase (Figure 1). This truncated transposon contains (i-ii) two heavy metal resistance cassettes – a Co/Zn/Cd efflux system (orf11, orf12) and mercury resistance determinants (orf16-orf22), (iii) an ORF encoding a protein of the metallo-beta-lactamase family (orf15), (iv) a site-specific resolution system (composed of two genes tnpS and tnpT, and a putative resolution site with a hairpin structure) homologous to the MRS system of Tn4651, as well as (v) four ORFs encoding hypothetical proteins with unknown functions (orf8, orf13, orf14 and orf23) (Figure 1). The putative efflux system (CZC CH5183284 mouse module; orf11, orf12) encodes a predicted CzcD metal transport membrane protein (a member of the cation diffusion facilitator
protein family), which mediates cobalt (Co2+), zinc (Zn2+) and cadmium (Cd2+) resistance (as shown in Cupriavidus metallidurans CH34 ). The mercury resistance module (MER) contains 7 ORFs (orf16-orf22) with significant levels of homology to the merRTPABDE genes, responsible for enzymatic detoxification of Hg2+ ions to the less toxic form
Hg0. The key enzymes in this mercury resistance system are (i) organomercurial lyase (MerB) – effectively performs hydrolysis of stable mercury-carbon bonds, and (ii) mercuric reductase (MerA) – reduces Hg2+ to Hg0 (metallic mercury) in a process that involves hydride transfer from the electron carrier NADPH to flavin. Three other important components are (i) two transcriptional regulatory proteins (MerR Morin Hydrate and MerD), (ii) two mercury ion transport proteins (MerT and MerP), and (iii) an accessory membrane protein (MerE)  (Figure 1 and Additional file 1: Table S1). To investigate whether the analyzed resistance cassettes are functional, plasmid pBBR-ZM3CZCMER was constructed by inserting the orf11-orf23 gene cluster of pZM3H1 (contains the CZC and MER modules) into broad-host-range (BHR) mobilizable vector pBBR-MCS2 (see Methods for details). Since we were unable to remove (by incompatibility) plasmid pZM3H1 from its natural host (Halomonas sp. ZM3), the obtained plasmid pBBR-ZM3CZCMER was introduced (by conjugation or transformation) into Pseudomonas spp. LM7R and LM12R (pZM3H1 was shown to replicate in both strains) and E. coli TG1 (three members of Gammaproteobacteria), as well as A. tumefaciens LBA288 (Alphaproteobacteria).