Sulfoacidibacillus ferrooxidans, gen. nov., sp. nov., Sulfoacidibacillus thermotolerans, gen. nov., sp. nov., and Ferroacidibacillus organovorans, gen. nov., sp. nov.: Extremely acidophilic chemolitho-heterotrophic Firmicutes.

Ten strains of extremely acidophilic bacteria, isolated from different environments form a distinct monophyletic clade within the phylum Firmicutes. Comparison of complete genomes of the proposed type strains confirm that they comprise two genera (proposed names Sulfoacidibacillus and Ferroacidibacillus), and at least three species (Sulfoacidibacillus ferrooxidans, Sulfoacidibacillus thermotolerans and Ferroacidibacillus organovorans). The bacterial strains share some physiological traits, including catalysing the dissimilatory oxidation and reduction of iron, and in being obligately heterotrophic. Both species of Sulfoacidibacillus are also able to oxidise elemental sulfur and tetrathionate. Both S. ferrooxidans and Ferroacidibacillus spp. are mesophilic, while S. thermotolerans isolates are moderate thermophiles. The isolates display different degrees of acid-tolerance: Ferroacidibacillus spp. are the most acid-sensitive while the type strain of S. ferrooxidans grows at pH 0.9. MK7 was detected as the sole menaquinone present in all three nominated type strains, and their peptidoglycans all contain meso-2,6 diaminopimelic acid type A1γ. The chromosomal DNA of the strains examined contain between 44 and 52 mol% G + C. The nominated type strains of the new species are S. ferrooxidans S0ABT (= DSM 105355T = JCM 33225T); S. thermotolerans Y002T (= ATCC TSD-104T = JCM 31946T); F. organovorans SLC66T (= ATCC TSD-103T = JCM 31945T).

Genome-guided prediction of acid resistance mechanisms in acidophilic methanotrophs of phylogenetically deep-rooted Verrucomicrobia isolated from geothermal environments.

Verrucomicrobia are a group of microorganisms that have been proposed to be deeply rooted in the Tree of Life. Some are methanotrophs that oxidize the potent greenhouse gas methane and are thus important in decreasing atmospheric concentrations of the gas, potentially ameliorating climate change. They are widespread in various environments including soil and fresh or marine waters. Recently, a clade of extremely acidophilic Verrucomicrobia, flourishing at pH < 3, were described from high-temperature geothermal ecosystems. This novel group could be of interest for studies about the emergence of life on Earth and to astrobiologists as homologs for possible extraterrestrial life. In this paper, we describe predicted mechanisms for survival of this clade at low pH and suggest its possible evolutionary trajectory from an inferred neutrophilic ancestor. Extreme acidophiles are defined as organisms that thrive in extremely low pH environments (≤ pH 3). Many are polyextremophiles facing high temperatures and high salt as well as low pH. They are important to study for both providing fundamental insights into biological mechanisms of survival and evolution in such extreme environments and for understanding their roles in biotechnological applications such as industrial mineral recovery (bioleaching) and mitigation of acid mine drainage. They are also, potentially, a rich source of novel genes and pathways for the genetic engineering of microbial strains. Acidophiles of the Verrucomicrobia phylum are unique as they are the only known aerobic methanotrophs that can grow optimally under acidic (pH 2-3) and moderately thermophilic conditions (50-60°C). Three moderately thermophilic genera, namely Methylacidiphilum, Methylacidimicrobium, and Ca. Methylacidithermus, have been described in geothermal environments. Most of the investigations of these organisms have focused on their methane oxidizing capabilities (methanotrophy) and use of lanthanides as a protein cofactor, with no extensive study that sheds light on the mechanisms that they use to flourish at extremely low pH. In this paper, we extend the phylogenetic description of this group of acidophiles using whole genome information and we identify several mechanisms, potentially involved in acid resistance, including "first line of defense" mechanisms that impede the entry of protons into the cell. These include the presence of membrane-associated hopanoids, multiple copies of the outer membrane protein (Slp), and inner membrane potassium channels (kup, kdp) that generate a reversed membrane potential repelling the intrusion of protons. Acidophilic Verrucomicrobia also display a wide array of proteins potentially involved in the "second line of defense" where protons that evaded the first line of defense and entered the cell are expelled or neutralized, such as the glutamate decarboxylation (gadAB) and phosphate-uptake systems. An exclusive N-type ATPase F0-F1 was identified only in acidophiles of Verrucomicrobia and is predicted to be a specific adaptation in these organisms. Phylogenetic analyses suggest that many predicted mechanisms are evolutionarily conserved and most likely entered the acidophilic lineage of Verrucomicrobia by vertical descent from a common ancestor. However, it is likely that some defense mechanisms such as gadA and kup entered the acidophilic Verrucomicrobia lineage by horizontal gene transfer.

Draft Genome Sequence of Firmicutes Strain S0AB, a Heterotrophic Iron/Sulfur-Oxidizing Extreme Acidophile.

The draft whole-genome sequence of the extremely acidophilic and novel Firmicutes strain S0AB is reported. The genome comprises 3.3 Mbp and has a GC content of 43.72%. In total, 3,240 protein-coding genes, 56 tRNA genes, and 11 rRNA genes were predicted.

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

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.

A Large-Scale Genome-Based Survey of Acidophilic Bacteria Suggests That Genome Streamlining Is an Adaption for Life at Low pH.

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.

Integrative Genomics Sheds Light on Evolutionary Forces Shaping the Acidithiobacillia Class Acidophilic Lifestyle.

Extreme acidophiles thrive in environments rich in protons (pH values <3) and often high levels of dissolved heavy metals. They are distributed across the three domains of the Tree of Life including members of the Proteobacteria. The Acidithiobacillia class is formed by the neutrophilic genus Thermithiobacillus along with the extremely acidophilic genera Fervidacidithiobacillus, Igneacidithiobacillus, Ambacidithiobacillus, and Acidithiobacillus. Phylogenomic reconstruction revealed a division in the Acidithiobacillia class correlating with the different pH optima that suggested that the acidophilic genera evolved from an ancestral neutrophile within the Acidithiobacillia. Genes and mechanisms denominated as "first line of defense" were key to explaining the Acidithiobacillia acidophilic lifestyle including preventing proton influx that allows the cell to maintain a near-neutral cytoplasmic pH and differ from the neutrophilic Acidithiobacillia ancestors that lacked these systems. Additional differences between the neutrophilic and acidophilic Acidithiobacillia included the higher number of gene copies in the acidophilic genera coding for "second line of defense" systems that neutralize and/or expel protons from cell. Gain of genes such as hopanoid biosynthesis involved in membrane stabilization at low pH and the functional redundancy for generating an internal positive membrane potential revealed the transition from neutrophilic properties to a new acidophilic lifestyle by shaping the Acidithiobacillaceae genomic structure. The presence of a pool of accessory genes with functional redundancy provides the opportunity to "hedge bet" in rapidly changing acidic environments. Although a core of mechanisms for acid resistance was inherited vertically from an inferred neutrophilic ancestor, the majority of mechanisms, especially those potentially involved in resistance to extremely low pH, were obtained from other extreme acidophiles by horizontal gene transfer (HGT) events.

A Large-Scale Multiple Genome Comparison of Acidophilic Archaea (pH ≤ 5.0) Extends Our Understanding of Oxidative Stress Responses in Polyextreme Environments.

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.

Evolution of Predicted Acid Resistance Mechanisms in the Extremely Acidophilic Leptospirillum Genus.

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.

Identification of copper-induced genes in the marine alga Ulva compressa (Chlorophyta).

In order to identify genes/proteins involved in copper tolerance, the marine alga Ulva compressa was cultivated with 10 µM copper for 3 days. The activities of antioxidant enzymes ascorbate peroxidase (AP), peroxiredoxin (PRX), thioredoxin (TRX), and glutathione-S-transferase (GST) and the level of lipoperoxides were determined in the alga cultivated with and without copper addition. Antioxidant enzyme activities and lipoperoxides level increased in response to copper excess, indicating that the alga was under oxidative stress. A cDNA library was prepared using U. compressa cultivated with 10 µM copper for 3 days. A total of 3 × 10(4) clones were isolated and 480 clones were sequenced, resulting in 235 non-redundant ESTs, of which 104 encode proteins with known functions. Among them, we identified proteins involved in (1) antioxidant metabolism such as AP, PRX, TRX, GST, and metalothionein (MET), (2) signal transduction, such as calmodulin (CAM), (3) calcium-dependent protein kinase (CDPK) and nucleoside diphosphate kinase (NDK), (4) gene expression, (5) protein synthesis and degradation, and (6) chloroplast and mitochondria electron transport chains. Half of the identified proteins are potentially localized in organelles. The relative level of 18 genes, including those coding for AP, PRX, TRX, GST, MET, CAM, CDPK, and NDK were determined by quantitative RT-PCR in the alga cultivated with 10 µM copper for 0 to 7 days. Transcript levels increased in response to copper stress and most of them reached a maximum at days 3 and 5. Thus, the selected genes are induced by copper stress and they are probably involved in copper acclimation and tolerance.

Copper-induced calcium release from ER involves the activation of ryanodine-sensitive and IP(3)-sensitive channels in Ulva compressa.

The marine alga Ulva compressa (Chlorophyta) showed a triphasic release of intracellular calcium with maximal levels at 2, 3 and 12 h and a biphasic accumulation of intracellular hydrogen peroxide with peaks at 3 and 12 h when cultivated with copper excess. Intracellular hydrogen peroxide originated exclusively in organelles. In this work, we analyzed the intracellular origin of calcium release and the type of calcium channels activated in response to copper excess. U. compressa was treated with thapsigargin, an inhibitor of endoplasmic reticulum (ER) calcium ATPase, ryanodine, an inhibitor of ryanodine-sensitive channels, and xestospongin C, an inhibitor of inositol 1, 4, 5-triphosphate (IP(3))-sensitive channels. Thapsigargin induced the depletion of calcium stored in ER at 75 min and completely inhibited calcium release at 2, 3 and 12 h of copper exposure indicating that calcium release originated in ER. In addition, ryanodine and xestospogin C inhibited calcium release at 2 and 3 h of copper exposure whereas the peak at 12 h was only inhibited by ryanodine. Thus, copper induced the activation of ryanodine-sensitive and IP(3)-sensitive calcium channels in ER of U. compressa.