Genomic Islands Confer Heavy Metal Resistance in Mucilaginibacter kameinonensis and Mucilaginibacter rubeus Isolated from a Gold/Copper Mine

Abstract

Heavy metals (HMs) are compounds that can be hazardous and impair growth of living organisms. Bacteria have evolved the capability not only to cope with heavy metals but also to detoxify polluted environments. Three heavy metal-resistant strains of Mucilaginibacer rubeus and one of Mucilaginibacter kameinonensis were isolated from the gold/copper Zijin mining site, Longyan, Fujian, China. These strains were shown to exhibit high resistance to heavy metals with minimal inhibitory concentration reaching up to 3.5 mM Cu^(II), 21 mM Zn^(II), 1.2 mM Cd^(II), and 10.0 mM As^(III). Genomes of the four strains were sequenced by Illumina. Sequence analyses revealed the presence of a high abundance of heavy metal resistance (HMR) determinants. One of the strain, M. rubeus P2, carried genes encoding 6 putative P_IB-1-ATPase, 5 putative P_IB-3-ATPase, 4 putative Zn^(II)/Cd^(II) P_IB-4 type ATPase, and 16 putative resistance-nodulation-division (RND)-type metal transporter systems. Moreover, the four genomes contained a high abundance of genes coding for putative metal binding chaperones. Analysis of the close vicinity of these HMR determinants uncovered the presence of clusters of genes potentially associated with mobile genetic elements. These loci included genes coding for tyrosine recombinases (integrases) and subunits of mating pore (type 4 secretion system), respectively allowing integration/excision and conjugative transfer of numerous genomic islands. Further in silico analyses revealed that their genetic organization and gene products resemble the Bacteroides integrative and conjugative element CTnDOT. These results highlight the pivotal role of genomic islands in the acquisition and dissemination of adaptive traits, allowing for rapid adaption of bacteria and colonization of hostile environments.

Keywords: CTnDOT; Mucilaginibacer rubeus; Mucilaginibacter kameinonensis; draft genome sequence; evolution; genomic island; heavy metal resistance.

Figure 1

Neighbour-joining phylogenetic tree constructed based on the 16S ribosomal RNA (rRNA) gene sequences from the draft genome sequence showing the phylogenetic relationships between strains Mucilaginibacter rubeus P1, P2 and P3 and Mucilaginibacter kameinonensis P4 and other species in the genus Mucilaginibacter. Values indicate percentages of identical branching in 1000 bootstrappings. The sequence of Pedobacter koreensis WPCB189T was used as an out-group. Bar, 0.01 substitutions per nucleotide position.

Figure 2

Representative putative genomic island carrying genes encoding HM determinants in contig 26 of M. Rubeus P2. Genomic analysis was performed via RAST (http://rast.nmpdr.org/). Genes encoding determinants related to metalloid (Arsenical and Antimony) resistance are highlighted in maroon, metal (Copper/Cobalt/Cadmium/Zinc/Lead/Mercury) resistance in blue, and the genes encoding putative transfer functions in red. Genes encoding hypothetical proteins and unknown functions are highlighted in gray.

Figure 3

Molecular phylogenetic analysis of TraG proteins of putative conjugative genomic islands (GIs) of Mucilaginibacter. The evolutionary history was inferred by using the Maximum Likelihood method based on the JTT matrix-based model [32]. The percentage of trees in which the associated taxa clustered together is shown next to the branches. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. Evolutionary analyses were conducted in MEGA6 [28]. Initial alignment of sequences was performed using Muscle for the presented tree. An identical tree with minor changes in bootstrap values was obtained using ClustalW for alignment. The VirB4 subunit of MPFB T4SS is named TraG [41]. For convenience and consistence in TraG protein identification, nomenclature is as follows: TraGPX-Y, where X is the strain number and Y the contig carrying the gene coding for TraG. TraG CTnDOT (TraGDOT) accession number: AAG17832.1.