Prediction and Inferred Evolution of Acid Tolerance Genes in the Biotechnologically Important Acidihalobacter Genus

Abstract

Acidihalobacter is a genus of acidophilic, gram-negative bacteria known for its ability to oxidize pyrite minerals in the presence of elevated chloride ions, a capability rare in other iron-sulfur oxidizing acidophiles. Previous research involving Acidihalobacter spp. has focused on their applicability in saline biomining operations and their genetic arsenal that allows them to cope with chloride, metal and oxidative stress. However, an understanding of the molecular adaptations that enable Acidihalobacter spp. to thrive under both acid and chloride stress is needed to provide a more comprehensive understanding of how this genus can thrive in such extreme biomining conditions. Currently, four genomes of the Acidihalobacter genus have been sequenced: Acidihalobacter prosperus DSM 5130^T, Acidihalobacter yilgarnensis DSM 105917^T, Acidihalobacter aeolianus DSM 14174^T, and Acidihalobacter ferrooxydans DSM 14175^T. Phylogenetic analysis shows that the Acidihalobacter genus roots to the Chromatiales class consisting of mostly halophilic microorganisms. In this study, we aim to advance our knowledge of the genetic repertoire of the Acidihalobacter genus that has enabled it to cope with acidic stress. We provide evidence of gene gain events that are hypothesized to help the Acidihalobacter genus cope with acid stress. Potential acid tolerance mechanisms that were found in the Acidihalobacter genomes include multiple potassium transporters, chloride/proton antiporters, glutamate decarboxylase system, arginine decarboxylase system, urease system, slp genes, squalene synthesis, and hopanoid synthesis. Some of these genes are hypothesized to have entered the Acidihalobacter via vertical decent from an inferred non-acidophilic ancestor, however, horizontal gene transfer (HGT) from other acidophilic lineages is probably responsible for the introduction of many acid resistance genes.

Keywords: acid resistance; chloride/proton antiporters; extreme acidophile; genome evolution; phylogenomics; polyextremophile; potassium transporters; urease.

FIGURE 1

Phylogenomic tree constructed, using 400 protein markers of 52 representative complete genomes of the Chromatiales order, using the Phylophlan3 program. Gray highlights slightly halophilic genomes (optimum growth 2–5% w/v NaCl), blue highlights moderately halophilic genomes (optimum growth 5–20% w/v NaCl), green highlights slightly halotolerant genomes (no NaCl required to grow however optimum growth in 2–8% w/v NaCl) and no highlight represents non-halophiles. A, acidophiles (optimum growth < pH 5); N, neutrophiles (optimum growth between pH 5 and 9); and ALK, alkaliphiles (optimum growth > pH 9). Red lines indicate the phylogenetic relationships between the acidophiles. The sequences were aligned with MAFFT and the concatenated alignment was used to construct the phylogenetic tree with IQTREE and LG+R3 as the best-suited evolutionary model. Scale bar indicates 0.2 substitutions per amino acid. See Supplementary Table 1 for the full information on the microorganisms used in the tree.

FIGURE 2

Spermidine biosynthesis pathway identified in Acidihalobacter. CPT, N-carbamoylputrescine; SAM, S-Adenosyl methionine; dSAM, decarboxylated-Adenosyl methionine; MTA, methylthioadenosine.

FIGURE 3

Hopanoid biosynthesis pathway identified in Acidihalobacter. Blue text indicates pathway identified in all Acidihalobacter genomes, black text indicates hopanoid biosynthesis pathway found in A. ferrooxydans. HSQ, hydroxysqualene; BHT, bacteriohopanetetrol.

FIGURE 4

Unrooted phylogenetic tree of the predicted Acidihalobacter Slp amino acid sequences and their best hits from the NCBI non-redundant database, with collapsed branches by genera when possible (showing in parenthesis the number of leaves inside each). Acidihalobacter Slp proteins are colored red, Chromatiales proteins are colored green and other acidophiles proteins are colored blue. The time scale bar represents 1 amino acid substitution per site.

FIGURE 5

Genomic context of the urease gene cluster in the Acidihalobacter spp. genomes. Gray background shows synteny between genomes; blue genes are found in all genomes; red shows genes hypothesized to be involved in acid and osmotic tolerance; genes in orange correspond to unique genes (only found in their respective genomes). Ziz-zag pattern indicates a frameshift mutation in the gene. Hyp1, hypothetical 1; Hyp2, hypothetical 2; cynS, cynanate hydratase; ycgL, protein YcgL; arsR, arsenic resistance transcriptional regulator; amrS, AmmeMemoRadiSam system radical SAM enzyme; amrB, AmmeMedoRadiSam system protein B; amrA, AmmeMedoRadiSam system protein A.

FIGURE 6

(A) The genomic context of clcA-1 in A. yilgarnensis, A. prosperus and A. aeolianus. (B) The genomic context of the clcA-1 genes in A. ferrooxydans. Genes in red; involved in acid tolerance, in green; involved in osmotolerance, in blue; others, in black; genes associated with HGT. Hyp, hypothetical proteins with no predicted putative domains; cysQ, 3′(2’),5′-bisphosphate nucleotidase; nudE, ADP compounds hydrolase; yggS, Pyridoxal phosphate homeostasis protein; pilT, type IV pilus twitching motility protein; pilU, type IV pilus ATPase; terB, tellurite resistance protein; tauE, sulfite exporter; DUF4426, domain of unknown function 4426; yggU, UPF0235 protein YggU; yggT, uncharacterized protein; proH, pyrroline-5-carboxylate reductase 1; sspB, stringent starvation protein B; sspA, stringent starvation protein A; priA, primosomal protein N′; pntB, NAD(P) transhydrogenase subunit beta.

FIGURE 7

The model of acid resistance in the Acidihalobacter spp. First line of defense mechanisms are displayed in blue and second line of defense mechanisms are displayed in other colors (green = ClcA; yellow = β-CA; purple = urease system; and orange = Gad system).

FIGURE 8

Inferred phylogenetic distribution of acid resistance genes. Green triangles represent gene gain events (the ’ in slp and clcA genes represent a duplication event) and red triangles represent gene loss events. Gene names in blue indicate hypothesized HGT events (* marks the uncertain origin for that gene) and the bacteria-icon represents the putative origin of those events. Orange arrow = predicted genes inherited by vertical descent.