Organisms that thrive in extremely acidic environments (≤pH 3.5) are of widespread importance in industrial applications, environmental issues, and evolutionary studies. Leptospirillum spp. constitute the only extremely acidophilic microbes in the phylogenetically deep-rooted bacterial phylum Nitrospirae. Leptospirilli are Gram-negative, obligatory chemolithoautotrophic, aerobic, ferrous iron oxidizers. This paper predicts genes that Leptospirilli use to survive at low pH and infers their evolutionary trajectory. Phylogenetic and other bioinformatic approaches suggest that these genes can be classified into (i) “first line of defense”, involved in the prevention of the entry of protons into the cell, and (ii) neutralization or expulsion of protons that enter the cell. The first line of defense includes potassium transporters, predicted to form an inside positive membrane potential, spermidines, hopanoids, and Slps (starvation-inducible outer membrane proteins). The “second line of defense“ includes proton pumps and enzymes that consume protons. Maximum parsimony, clustering methods, and gene alignments are used to infer the evolutionary trajectory that potentially enabled the ancestral Leptospirillum to transition from a postulated circum-neutral pH environment to an extremely acidic one. The hypothesized trajectory includes gene gains/loss events driven extensively by horizontal gene transfer, gene duplications, gene mutations, and genomic rearrangements.

Motivation There are about 600 available genome sequences of acidophilic organisms (grow at a pH < 5) from the three domains of the Tree of Life. Information about acidophiles is scattered over many heterogeneous sites making it extraordinarily difficult to link physiological traits with genomic data. We were motivated to generate a curated, searchable database to address this problem. Results AciDB 1.0 is a curated database of sequenced acidophiles that enables researchers to execute complex queries linking genomic features to growth data, environmental descriptions and taxonomic information. Availability AciDB 1.0 is freely available online at: http://AciDB.cl. The source code is released under an MIT license at: https://gitlab.com/Hawkline451/acidb/

The genome streamlining theory suggests that reduction of microbial genome size optimizes energy utilization in stressful environments. Although this hypothesis has been explored in several cases of low-nutrient (oligotrophic) and high-temperature environments, little work has been carried out on microorganisms from low-pH environments, and what has been reported is inconclusive. In this study, we performed a large-scale comparative genomics investigation of more than 260 bacterial high-quality genome sequences of acidophiles, together with genomes of their closest phylogenetic relatives that live at circum-neutral pH. A statistically supported correlation is reported between reduction of genome size and decreasing pH that we demonstrate is due to gene loss and reduced gene sizes. This trend is independent from other genome size constraints such as temperature and G + C content. Genome streamlining in the evolution of acidophilic bacteria is thus supported by our results. The analyses of predicted Clusters of Orthologous Genes (COG) categories and subcellular location predictions indicate that acidophiles have a lower representation of genes encoding extracellular proteins, signal transduction mechanisms, and proteins with unknown function but are enriched in inner membrane proteins, chaperones, basic metabolism, and core cellular functions. Contrary to other reports for genome streamlining, there was no significant change in paralog frequencies across pH. However, a detailed analysis of COG categories revealed a higher proportion of genes in acidophiles in the following categories: “replication and repair,” “amino acid transport,” and “intracellular trafficking”. This study brings increasing clarity regarding the genomic adaptations of acidophiles to life at low pH while putting elements, such as the reduction of average gene size, under the spotlight of streamlining theory.

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 5130T, Acidihalobacter yilgarnensis DSM 105917T, Acidihalobacter aeolianus DSM 14174T, and Acidihalobacter ferrooxydans DSM 14175T. 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.

Acidophilic archaea thrive in anaerobic and aerobic low pH environments (pH < 5) rich in dissolved heavy metals that exacerbate stress caused by the production of reactive oxygen species (ROS) such as hydrogen peroxide (H2O2), hydroxyl radical (OH) and superoxide (O2−). ROS react with lipids, proteins and nucleic acids causing oxidative stress and damage that can lead to cell death. Herein, genes and mechanisms potentially involved in ROS mitigation are predicted in over 200 genomes of acidophilic archaea with sequenced genomes. These organisms are often be subjected to simultaneous multiple stresses such as high temperature, high salinity, low pH and high heavy metal loads. Some of the topics addressed include: (1) the phylogenomic distribution of these genes and what this can tell us about the evolution of these mechanisms in acidophilic archaea; (2) key differences in genes and mechanisms used by acidophilic versus non-acidophilic archaea and between acidophilic archaea and acidophilic bacteria and (3) how comparative genomic analysis predicts novel genes or pathways involved in oxidative stress responses in archaea and likely horizontal gene transfer (HGT) events.