Biochemical, genomic and structural characteristics of the Acr3 pump in Exiguobacterium strains isolated from arsenic-rich Salar de Huasco sediments

Abstract

Arsenic is a highly toxic metalloid of major concern for public safety. However, microorganisms have several resistance mechanisms, particularly the expression of arsenic pumps is a critical component for bacterial ability to expel it and decrease intracellular toxicity. In this study, we aimed to characterize the biochemical, structural, and genomic characteristics of the Acr3 pump among a group of Exiguobacterium strains isolated from different sites of the arsenic-rich Salar de Huasco (SH) ecosystem. We also determined whether the differences in As(III) resistance levels presented by the strains could be attributed to changes in the sequence or structure of this protein. In this context, we found that based on acr3 sequences the strains isolated from the SH grouped together phylogenetically, even though clustering based on gene sequence identity did not reflect the strain's geographical origin. Furthermore, we determined the genetic context of the acr3 sequences and found that there are two versions of the organization of acr3 gene clusters, that do not reflect the strain's origin nor arsenic resistance level. We also contribute to the knowledge regarding structure of the Acr3 protein and its possible implications on the functionality of the pump, finding that although important and conserved components of this family of proteins are present, there are several changes in the amino acidic sequences that may affect the interactions among amino acids in the 3D model, which in fact are evidenced as changes in the structure and residues contacts. Finally, we demonstrated through heterologous expression that the Exiguobacterium Acr3 pump does indeed improve the organisms As resistance level, as evidenced in the complemented E. coli strains. The understanding of arsenic detoxification processes in prokaryotes has vast biotechnological potential and it can also provide a lot of information to understand the processes of evolutionary adaptation.

Keywords: ACR3; Exiguobacterium; arsenic; efflux pumps; resistance.

Figure 1

Schematic representation of the phylogenetic relationships between the arsenic efflux pumps found in Exiguobacterium and their function inside the cell to face arsenic.

Figure 2

Genomic relationships among the studied acr3 sequences. (A) Phylogenetic reconstruction for all compared gene sequences (Exiguobacterium strains and references) and (B) MDS ordination using sequence identities as distance (for the Exiguobacterium strains and close related references).

Figure 3

Comparison of the acr3 gene cluster organization in the compared strains. Blue and orange arrows display the acr3 and arsK genes respectively; green arrows show the conserved flanking genes in the acr3 gene context and gray arrows display the uncommon genes. Shaded color connections represent gene conservation.

Figure 4

Exiguobacterium Acr3 protein model. (A) Topology of the Acr3 protein. The ten transmembrane helices are shown, the eight continuous helices in light blue and discontinuous helices in purple and green. (B) Acr3 protein model from the SH0S1 strain, showing the three-dimensional conformation and the spatial orientation of the ten transmembrane helices; color corresponds to the modeling confidence level and green spheres represent the non-conserved amino acids.

Figure 5

Non-conserved amino acids among the Acr3 protein sequences of Exiguobacterium strains. The amino acid occurring in each variable position identified is shown for every sequence, as well as the amino acidic classification according to the side chain and how many variable positions each one has, with respect to the consensus sequence. The topological location of each position variable is also shown (E, M, C). The strains are sorted according to their As(III) MIC value.

Figure 6

Contact analysis comparing selected Acr3 proteins from the Exiguobacterium strains. Contact maps: panels (A1–A3) shows the inter-cluster comparisons. The upper diagonal represents the pairwise comparison of the two contact maps: pink for the contacts that are unique to the first Acr3 protein of A1: E. sp. SH0S1, A2: E. sp. SH0S7, and A3: E. sp. SH0S1, and green the contacts unique to the second Acr3 protein of A1: E. sp. SH0S7, A2: E. sp. SH3S2, and A3: E. sp. SH3S2. The lower diagonal shows a heat-map of differences between the protein contacts, where red represents differences and blue represent similarities. The arrows indicate the positions of high variability and the amino acids for each case. (B) The position and localization of the eight residues with greatest variation are depicted by different colors on the structural model of the Acr3 protein.

Figure 7

Distribution and possible effects of relevant residues in the Exiguobacterium Acr3 model. The location of non-conserved amino acids in the protein structure are shown (A) in a longitudinal view and (B) in a top-down view. Panel (C) shows the number of H-bonds that the non-conserved residues as well as the key Cys-107 and Glu-294 can form with proteins from the three clusters or exclusively with some of them.

Figure 8

Growth curves of the studied strains, under the tested conditions: control, As(III) and As(V) at different concentrations. OD₆₀₀ readings were recorded every hour during 16 h. Data represents an average of three independent experiments with three technical replicates each.

Figure 9

Viable cell recovery after arsenic treatment [control, As(III) and As(V)] of the studied strains. Bars represents an average of three independent experiments with three technical replicates each. * p<0.05; ** p<0.01; *** p<0.001.