Nitrosotalea devaniterrae gen. nov., sp. nov. and Nitrosotalea sinensis sp. nov., two acidophilic ammonia oxidising archaea isolated from acidic soil, and proposal of the new order Nitrosotaleales ord. nov. within the class Nitrososphaeria of the phylum Nitrososphaerota.

Two obligately acidophilic, mesophilic and aerobic soil ammonia-oxidising archaea were isolated from a pH 4.5 arable sandy loam (UK) and pH 4.7 acidic sulphate paddy soil (PR China) and designated strains Nd1T and Nd2T, respectively. The strains shared more than 99 % 16S rRNA gene sequence identity and their genomes were both less than 2 Mb in length, sharing 79 % average nucleotide identity, 81 % average amino acid identity and a DNA G+C content of approximately 37 mol%. Both strains were chemolithotrophs that fixed carbon dioxide and gained energy by oxidising ammonia to nitrite, with no evidence of mixotrophic growth. Neither strain was capable of using urea as a source of ammonia. Both strains were non-motile in culture, although Nd1T does possess genes encoding flagella components and therefore may be motile under certain conditions. Cells of Nd1T were small angular rods 0.5-1 µm in length and grew at pH 4.2-5.6 and at 20-30 °C. Cells of Nd1T were small angular rods 0.5-1 µm in length and grew at pH 4.0-6.1 and at 20-42 °C. Nd1T and Nd2T are distinct with respect to genomic and physiological features and are assigned as the type strains for the species Nitrosotalea devaniterrae sp. nov. (type strain, Nd1T=NCIMB 15248T=DSM 110862T) and Nitrosotalea sinensis sp. nov. (type strain, Nd2T=NCIMB 15249T=DSM 110863T), respectively, within the genus Nitrosotalea gen. nov. The family Nitrosotaleaceae fam. nov. and order Nitrosotaleales ord. nov. are also proposed officially.

Acidithiobacillus acidisediminis sp. nov., an acidophilic sulphur-oxidizing chemolithotroph isolated from acid mine drainage sediment.

Strain S30A2T, isolated from the acid mine drainage sediment of Mengzi Copper Mine, Yunnan, is proposed to represent a novel species of the sulphur-oxidizing genus Acidithiobacillus. Cells were Gram-stain-negative, non-endospore forming, highly motile with one or two monopolar flagella and rod-shaped. The strain was mesophilic, growing at 30-50 °C (optimum, 38 °C), acidophilic, growing at pH 2.0-4.5 (optimum, pH 2.5), and tolerant of 0-4 % (w/v; 684 mol l-1) NaCl. The 16S rRNA gene-based sequence analysis showed that strain S30A2T belongs to the genus Acidithiobacillus and shows the largest similarity of 96.6 % to the type strain Acidithiobacillus caldus KUT. The genomic DNA G+C content of strain S30A2T was 59.25 mol%. The average nucleotide identity ANIb and ANIm values between strain S30A2T and A. caldus KUT were 70.95 and 89.78 %, respectively and the digital DNA-DNA hybridization value was 24.9 %. Strain S30A2T was strictly aerobic and could utilize elementary sulphur and tetrathionate to support chemolithotrophic growth. The major cellular fatty acid of S30A2T was C19 : 1ω7c. The respiratory quinones were ubiquinone-8 and ubiquinone-7. Based upon its phylogenetic, genetic, phenotypic, physiologic and chemotaxonomic characteristics, strain S30A2T is considered to represent a novel species of the genus Acidithiobacillus, for which the name Acidithiobacillus acidisediminis sp. nov. is proposed. The type strain is S30A2T (=CGMCC 1.17059T=KCTC 72580T).

The Expanding Diversity of Viruses from Extreme Environments.

Viruses are nonliving biological entities whose host range encompasses all known forms of life. They are deceptively simple in description (a protein shell surrounding genetic material with an occasional lipid envelope) and yet can infect all known forms of life. Recently, due to technological advancements, viruses from more extreme environments can be studied through both culture-dependent and independent means. Viruses with thermophilic, halophilic, psychrophilic, and barophilic properties are highlighted in this paper with an emphasis on the properties that allow them to exist in said environments. Unfortunately, much of this field is extremely novel and thus, not much is yet known about these viruses or the microbes they infect when compared to non-extremophilic host-virus systems. With this review, we hope to shed some light on these relatively new studies and highlight their intrinsic value.

Insights into the complementation potential of the extreme acidophile's orthologue in replacing Escherichia coli hfq gene-particularly in bacterial resistance to environmental stress.

Acidithiobacillus caldus is a typical extreme acidophile widely used in the biohydrometallurgical industry, which often experiences extreme environmental stress in its natural habitat. Hfq, an RNA-binding protein, typically functions as a global regulator involved in various cellular physiological processes. Yet, the biological functions of Hfq derived from such extreme acidophile have not been extensively investigated. In this study, the recombinant strain Δhfq/Achfq, constructed by CRISPR/Cas9-mediated chromosome integration, fully or partially restored the phenotypic defects caused by hfq deletion in Escherichia coli, including impaired growth performance, abnormal cell morphology, impaired swarming motility, decreased stress resistance, decreased intracellular ATP and free amino acid levels, and attenuated biofilm formation. Particularly noteworthy, the intracellular ATP level and biofilm production of the recombinant strain were increased by 12.2% and 7.0%, respectively, compared to the Δhfq mutant. Transcriptomic analysis revealed that even under heterologous expression, AcHfq exerted global regulatory effects on multiple cellular processes, including metabolism, environmental signal processing, and motility. Finally, we established a potential working model to illustrate the regulatory mechanism of AcHfq in bacterial resistance to environmental stress.

Identification and characterization of an abundant lipoprotein from Methylacidiphilum fumariolicum SolV.

Bacterial lipoproteins are characterized by the presence of a conserved N-terminal lipid-modified cysteine residue that allows the hydrophilic protein to anchor into bacterial cell membranes. These lipoproteins play essential roles in a wide variety of physiological processes. Based on transcriptome analysis of the verrucomicrobial methanotroph Methylacidiphilum fumariolicum SolV, we identified a highly expressed lipoprotein, WP_009060351 (139 amino acids), in its genome. The first 86 amino acids are specific for the methanotrophic genera Methylacidiphilum and Methylacidmicrobium, while the last 53 amino acids are present only in lipoproteins of members from the phylum Verrucomicrobiota (Hedlund). Heterologous expression of WP_009060351 in Escherichia coli revealed a 25-kDa dimeric protein and a 60-kDa tetrameric protein. Immunoblotting showed that WP_009060351 was present in the total membrane protein and peptidoglycan fractions of M. fumariolicum SolV. The results suggest an involvement of lipoprotein WP_009060351 in the linkage between the outer membrane and the peptidoglycan.

Methylacidiphilum kamchatkense gen. nov., sp. nov., an extremely acidophilic and moderately thermophilic methanotroph belonging to the phylum Verrucomicrobiota.

The thermo-acidophilic aerobic methanotrophic Verrucomicrobia bacterium, designated strain Kam1T was isolated from an acidic geothermal mud spring in Kamchatka, Russia. Kam1T is Gram-stain-negative, with non-motile cells and non-spore-forming rods, and a diameter of 0.45-0.65 µm and length of 0.8-1.0 µm. Its growth is optimal at the temperature of 55 °C (range, 37-60 °C) and pH of 2.5 (range, pH 1-6), and its maximal growth rate is ~0.11 h-1 (doubling time ~6.3 h). Its cell wall contains peptidoglycan with meso-diaminopimelic acid. In addition to growing on methane and methanol, strain Kam1T grows on acetone and 2-propanol. Phylogenetically, it forms a distinct group together with other Methylacidiphilum strains and with the candidate genus Methylacidimicrobium as a sister group. These findings support the classification of the strain Kam1T as a representative of a novel species and genus of the phylum Verrucomicrobiota. For this strain, we propose the name Methylacidiphilum kamchatkense sp. nov. as the type species within Methylacidiphilum gen. nov. Strain Kam1T (JCM 30608T=KCTC 4682T) is the type strain.

Production of poly (3-hydroxybutyrate) and extracellular polymeric substances from glycerol by the acidophile Acidiphilium cryptum.

Acidiphilium cryptum is an acidophilic, heterotrophic, and metallotolerant bacteria able to use dissolved oxygen or Fe(III) as an electron sink. The ability of this extremophile to accumulate poly(3-hydroxybutyrate) (PHB) and secrete extracellular polymeric substances (EPS) has also been reported. Hence, the aim of this work is to characterize the production of PHB and EPS by the wild strain DSM2389 using glycerol in shaken flasks and bioreactor. Results showed that maximum PHB accumulation (37-42% w/w) was obtained using glycerol concentrations of 9 and 15 g L-1, where maximum dry cell weight titers reached 3.6 and 3.9 g L-1, respectively. The culture in the bioreactor showed that PHB accumulation takes place under oxygen limitation, while the redox potential of the culture medium could be used for online monitoring of the PHB production. Recovered EPS was analyzed by Fourier-transform infrared spectroscopy and subjected to gas chromatography-mass spectrometry after cleavage and derivatization steps. These analyses showed the presence of sugars which were identified as mannose, rhamnose and glucose, in a proportion near to 3.2:2.3:1, respectively. Since glycerol had not been used in previous works, these findings suggest the potential of A. cryptum to produce biopolymers from this compound at a large scale with a low risk of microbial contamination due to the low pH of the fermentation process.

Enhancement of acid tolerance of Escherichia coli by introduction of molecule chaperone CbpA from extremophile.

Molecular chaperone CbpA from extreme acidophile Acidithiobacillus caldus was applied to improve acid tolerance of Escherichia coli via CRISPR/Cas9. Cell growth and viability of plasmid complementary strain indicated the importance of cbpAAc for bacteria acid tolerance. With in situ gene replacement by CRISPR/Cas9 system, colony formation unit (CFU) of genome recombinant strain BL21-ΔcbpA/AccbpA showed 7.7 times higher cell viability than deficient strain BL21-ΔcbpA and 2.3 times higher than wild type. Cell morphology observation using Field Emission Scanning Electron Microscopy (FESEM) revealed cell breakage of BL21-ΔcbpA and significant recovery of BL21-ΔcbpA/AccbpA. The intracellular ATP level of all strains gradually decreased along with the increased stress time. Particularly, the value of recombinant strain was 56.0% lower than that of deficient strain after 5 h, indicating that the recombinant strain consumed a lot of energy to resist acid stress. The arginine concentration in BL21-ΔcbpA/AccbpA was double that of BL21-ΔcbpA, while the aspartate and glutamate contents were 14.8% and 6.2% higher, respectively, compared to that of wild type. Moreover, RNA-Seq analysis examined 93 genes down-regulated in BL21-ΔcbpA compared to wild type strain, while 123 genes were up-regulated in BL21-ΔcbpA/AccbpA compared to BL21-ΔcbpA, with an emphasis on energy metabolism, transport, and cell components. Finally, the working model in response to acid stress of cbpA from A. caldus was developed. This study constructed a recombinant strain resistant to acid stress and also provided a reference for enhancing microorganisms' robustness to various conditions.

Alicyclobacillus curvatus sp. nov. and Alicyclobacillus mengziensis sp. nov., two acidophilic bacteria isolated from acid mine drainage.

Two acidophilic strains, designated as ALEF1T and S30H14T, were isolated from acid mine drainage sediment. Cells of both strains were Gram-stain-positive, aerobic, endospore-forming rods. Strains ALEF1T and S30H14T were acidophilic and mesophilic, the former grew at 20-40 °C (optimum, 30 °C) and pH 2.5-4.5 (optimum, pH 3.5), while the latter grew at 20-45 °C (optimum, 30 °C) and pH 2.0-5.5 (optimum, pH 4.5). The 16S rRNA gene-based sequence analysis revealed that strains ALEF1T and S30H14T belonged to the genus Alicyclobacillus, and were phylogenetically close to Alicyclobacillus ferrooxydans TC-34T with 97.1 and 97.4% similarity, respectively. The similarity between the two novel strains was 98.6 %. The average nucleotide identity value between the genome sequences of ALEF1T and S30H14T was 79.5 %, and that between each of the two isolates and A. ferrooxydans TC-34T were 72.0 and 74.3 %. In addition, the digital DNA-DNA hybridization value between ALEF1T and S30H14T was 24.9 %, between strain ALEF1T and A. ferrooxydans TC-34T was 21.7 %, and between S30H14T and A. ferrooxydans TC-34T was 26.3 %, far below the interspecies threshold. Both strains could utilize diverse carbon sources for heterotrophic growth; strain ALEF1T could utilize ferrous iron as the energy source for autotrophic growth. Menaquinone 7 was the only quinone detected in either strain. Both strains contained anteiso-C15 : 0 and anteiso-C17 : 0, while ω-alicyclic fatty acids were not detected. Based on their phylogenetic positions, as well as phenotypic and genomic data, it is considered that strains ALEF1T and S30H14T represent two novel species within the genus Alicyclobacillus, for which the names Alicyclobacillus curvatus sp. nov. (type strain ALEF1T=CGMCC 1.17055T=KCTC 43124T) and Alicyclobacillus mengziensis sp. nov. (S30H14T=CGMCC 1.17050T=KCTC 43125T) are proposed.

Dispersion of sulfur creates a valuable new growth medium formulation that enables earlier sulfur oxidation in relation to iron oxidation in Acidithiobacillus ferrooxidans cultures.

Acidithiobacillus ferrooxidans is an acidophilic chemolithoautotroph that is commonly reported to exhibit diauxic population growth behavior where ferrous iron is oxidized before elemental sulfur when both are available, despite the higher energy content of sulfur. We have discovered sulfur dispersion formulations that enables sulfur oxidation before ferrous iron oxidation. The oxidation of dispersed sulfur can lower the culture pH within days below the range where aerobic ferrous iron oxidation can occur. Thus, ferric iron reduction can be observed quickly which had previously been reported over extended incubation periods with untreated sulfur. Therefore, we demonstrate that this substrate utilization pattern is strongly dependent on the cell loading in relation to sulfur concentration, sulfur surface hydrophobicity, and the pH of the culture. Our dispersed sulfur formulation, lig-sulfur, can be used to support the rapid antibiotic selection of plasmid-transformed cells, which is not possible in liquid cultures where ferrous iron is the main source of energy for these acidophiles. Furthermore, we find that media containing lig-sulfur supports higher production of green fluorescent protein compared to media containing ferrous iron. The use of dispersed sulfur is a valuable new tool for the development of engineered A. ferrooxidans strains and it provides a new method to control iron and sulfur oxidation behaviors.

Acidiferrimicrobium australe gen. nov., sp. nov., an acidophilic and obligately heterotrophic, member of the Actinobacteria that catalyses dissimilatory oxido-reduction of iron isolated from metal-rich acidic water in Chile.

A novel acidophilic member of the phylum Actinobacteria was isolated from an acidic, metal-contaminated stream draining from an abandoned underground coal mine (Trongol mine), situated close to Curanilahue, Biobío Region, Chile. The isolate (USS-CCA1T) was demonstrated to be a heterotroph that catalysed under aerobic conditions the oxidation of ferrous iron and the reduction of ferric iron under anaerobic conditions, but not the oxidation of sulfur nor hydrogen. USS-CCA1T is a Gram-positive, motile, short rod-shaped, mesophilic bacterium with a temperature growth optimum at 30 °C (range 20-39 °C). It was categorized as an extreme acidophile growing between 1.7 and 4.5 and optimally at pH 3.0. The G+C content of the chromosomal DNA of the isolate was 74.1 mol%, which is highly related to Aciditerrimonas ferrireducens IC-180T , (the most closely related genus; 94.4 % 16S rRNA gene identity), and higher than other acidophilic actinobacteria. The isolate (USS-CCA1T) was shown to form a distinct 16S rRNA clade from characterized acidophilic actinobacteria, well separated from the genera Acidimicrobium, Ferrimicrobium, Ferrithrix, 'Acidithrix' and Aciditerrimonas. Genomic indexes (ANIb, DDH, AAI, POCP) derived from the USS-CCA1T draft genome sequence (deposited at DDBJ/ENA/GenBank under the accession WJHE00000000) support assignment of the isolate to a new species and a new genus within the Acidimicrobiaceae family. Isolate USS-CCA1T is the designated type strain of the novel species Acidiferrimicrobium australe (=DSM 106828T,=RGM 2506T).

Genome-based classification of Acidihalobacter prosperus F5 (=DSM 105917=JCM 32255) as Acidihalobacter yilgarnensis sp. nov.

The genus Acidihalobacter has three validated species, Acidihalobacter ferrooxydans, Acidihalobacter prosperus and Acidihalobacter aeolinanus, all of which were isolated from Vulcano island, Italy. They are obligately chemolithotrophic, aerobic, acidophilic and halophilic in nature and use either ferrous iron or reduced sulphur as electron donors. Recently, a novel strain was isolated from an acidic, saline drain in the Yilgarn region of Western Australia. Strain F5T has an absolute requirement for sodium chloride (>5 mM) and is osmophilic, growing in elevated concentrations (>1 M) of magnesium sulphate. A defining feature of its physiology is its ability to catalyse the oxidative dissolution of the most abundant copper mineral, chalcopyrite, suggesting a potential role in biomining. Originally categorized as a strain of A. prosperus, 16S rRNA gene phylogeny and multiprotein phylogenies derived from clusters of orthologous proteins (COGS) of ribosomal protein families and universal protein families unambiguously demonstrate that strain F5T forms a well-supported separate branch as a sister clade to A. prosperus and is clearly distinguishable from A. ferrooxydans DSM 14175T and A. aeolinanus DSM14174T. Results of comparisons between strain F5T and the other Acidihalobacter species, using genome-based average nucleotide identity, average amino acid identity, correlation indices of tetra-nucleotide signatures (Tetra) and genome-to-genome distance (digital DNA-DNA hybridization), support the contention that strain F5T represents a novel species of the genus Acidihalobacter. It is proposed that strain F5T should be formally reclassified as Acidihalobacter yilgarnenesis F5T (=DSM 105917T=JCM 32255T).

Genome-based classification of two halotolerant extreme acidophiles, Acidihalobacter prosperus V6 (=DSM 14174 =JCM 32253) and 'Acidihalobacter ferrooxidans' V8 (=DSM 14175 =JCM 32254) as two new species, Acidihalobacter aeolianus sp. nov. and Acidihalobacter ferrooxydans sp. nov., respectively.

Phylogenomic analysis of recently released high-quality draft genome sequences of the halotolerant acidophiles, Acidihalobacter prosperus V6 (=DSM 14174=JCM 32253) and 'Acidihalobacter ferrooxidans' V8 (=DSM 14175=JCM 32254), was undertaken in order to clarify their taxonomic relationship. Sequence based phylogenomic approaches included 16S rRNA gene phylogeny, multi-gene phylogeny from the concatenated alignment of nine selected housekeeping genes and multiprotein phylogeny using clusters of orthologous groups of proteins from ribosomal protein families as well as those from complete sets of markers based on concatenated alignments of universal protein families. Non-sequence based approaches for species circumscription were based on analyses of average nucleotide identity, which was further reinforced by the correlation indices of tetra-nucleotide signatures as well as genome-to-genome distance (digital DNA-DNA hybridization) calculations. The different approaches undertaken in this study for species tree reconstruction resulted in a tree that was phylogenetically congruent, revealing that both micro-organisms are members of separate species of the genus Acidihalobacter. In accordance, it is proposed that A. prosperus V6T (=DSM 14174 T=JCM 32253 T) be formally classified as Acidihalobacter aeolianus sp. nov., and 'Acidihalobacter ferrooxidans' V8T (=DSM 14175 T=JCM 32254 T) as Acidihalobacter ferrooxydans sp. nov., and that both represent the type strains of their respective species.

Acidibrevibacterium fodinaquatile gen. nov., sp. nov., isolated from acidic mine drainage.

A heterotrophic and acidophilic bacterial strain, G45-3T, was isolated from acidic mine drainage sampled in Fujian Province, PR China. Cells of strain G45-3T were Gram-stain-negative, non-spore-forming, non-motile and rod-shaped. Catalase and oxidase activities were positive. Strain G45-3T grew aerobically at 20-45 °C (optimum, 37 °C) and at pH 2.5-5.0 (optimum, pH 4.0). Photosynthetic pigments were not produced. Analysis of 16S rRNA gene sequences showed that strain G45-3T was phylogenetically related to different members of the family Acetobacteraceae, and the sequence identities to Acidisphaera rubrifaciens JCM 10600T, Rhodovastum atsumiense G2-11T and Rhodopila globiformis ATCC 35887T were 95.9 , 95.3 and 95.3 %, respectively. Strain G45-3T contained ubiquinone-10 as its respiratory quinone. The major polar lipids were determined to be diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine, an unidentified aminophospholipid and an unidentified aminolipid. The predominant fatty acids were cyclo-C19 : 0ω8c, C18 : 1ω7c, C16 : 0 and C18 : 0. The genome of G45-3T consists of one chromosome (3 907 406 bp) and three plasmids (68 344, 45 771 and 16 090 bp), with an average G+C content of 65.9 mol%. Based on the results of phenotypic and genomic analyses, it is concluded that strain G45-3T represents a novel species of a new genus, for which the name Acidibrevibacterium fodinaquatile gen. nov., sp. nov. is proposed. A. fodinaquatile is nominated as type species and its type strain is G45-3T (=CGMCC 1.16069T=KCTC 62275T).

Acidithiobacillus sulfuriphilus sp. nov.: an extremely acidophilic sulfur-oxidizing chemolithotroph isolated from a neutral pH environment.

The genus Acidithiobacillus currently includes seven species with validly published names, which fall into two major groups, those that can oxidize ferrous iron and those that do not. All seven species can use zero-valent sulfur and reduced sulfur oxy-anions as electron donors, are obligately chemolithotrophic and acidophilic bacteria with pH growth optima below 3.0. The 16S rRNA gene of a novel strain (CJ-2T) isolated from circum-neutral pH mine drainage showed 95-97 % relatedness to members of the genus Acidithiobacillus. Digital DNA-DNA hybridization (dDDH) values between strains and whole-genome pairwise comparisons between the CJ-2T strain and the reference genomes available for members of the genus Acidithiobacillus confirmed that CJ-2Trepresents a novel species of this genus. CJ-2T is a strict aerobe, oxidizes zero-valent sulfur and reduced inorganic sulfur compounds but does not use ferrous iron or hydrogen as electron donors. The isolate is mesophilic (optimum growth temperature 25-28 °C) and extremely acidophilic (optimum growth pH 3.0), though its pH optimum and maximum were significantly higher than those of non-iron-oxidising acidithiobacilli with validly published names. The major fatty acids of CJ-2T were C18 : 1ω7c, C:16 : 1ω7c/iso-C15 : 0 2-OH, C16 : 0 and C19 : 0 cyclo ω8c and the major respiratory quinone present was Q8. The name Acidithiobacillussulfuriphilus sp. nov. is proposed, the type strain is CJ-2T (=DSM 105150T=KCTC 4683T).

Bioremediation of highly toxic arsenic via carbon-fiber-assisted indirect As(III) oxidation by moderately-thermophilic, acidophilic Fe-oxidizing bacteria.

OBJECTIVE: To enable removal of highly toxic As(III) from acidic waters by inducing indirect microbial As(III) oxidation by Fe-oxidizing bacteria via carbon-assisted redox-coupling between As(III) oxidation and Fe3+ reduction. RESULTS: Carbon-fiber (CF) was shown to function as an electron-mediator to catalyze chemical (abiotic) redox-coupling between As(III) oxidation and Fe3+ reduction. Accordingly, by taking advantage of Fe3+ regeneration by Fe-oxidizing bacteria, it was possible to promote oxidative removal of As(III) as ferric arsenate at moderate temperature. This reaction can be of use under the situation where a high-temperature treatment is not immediately available. Arsenic once concentrated as ferric arsenate on carbon-fibers can be collected to undergo phase-transformation to crystalline scorodite as the next re-solubilization/re-crystallization step at a higher temperature (70 °C). CONCLUSIONS: While extremely acidophilic Fe-oxidizing bacteria are widely found in nature, the As-oxidizing counterparts, especially those grown on moderately-thermophilic and mesophilic temperatures, are hardly known. In this regard, the finding of this study could make a possible introduction of the semi-passive, low-temperature As-treatment using readily available Fe-oxidizing bacteria.

Multi-omics Reveals the Lifestyle of the Acidophilic, Mineral-Oxidizing Model Species Leptospirillum ferriphilumT.

Leptospirillum ferriphilum plays a major role in acidic, metal-rich environments, where it represents one of the most prevalent iron oxidizers. These milieus include acid rock and mine drainage as well as biomining operations. Despite its perceived importance, no complete genome sequence of the type strain of this model species is available, limiting the possibilities to investigate the strategies and adaptations that Leptospirillum ferriphilum DSM 14647T (here referred to as Leptospirillum ferriphilumT) applies to survive and compete in its niche. This study presents a complete, circular genome of Leptospirillum ferriphilumT obtained by PacBio single-molecule real-time (SMRT) long-read sequencing for use as a high-quality reference. Analysis of the functionally annotated genome, mRNA transcripts, and protein concentrations revealed a previously undiscovered nitrogenase cluster for atmospheric nitrogen fixation and elucidated metabolic systems taking part in energy conservation, carbon fixation, pH homeostasis, heavy metal tolerance, the oxidative stress response, chemotaxis and motility, quorum sensing, and biofilm formation. Additionally, mRNA transcript counts and protein concentrations were compared between cells grown in continuous culture using ferrous iron as the substrate and those grown in bioleaching cultures containing chalcopyrite (CuFeS2). Adaptations of Leptospirillum ferriphilumT to growth on chalcopyrite included the possibly enhanced production of reducing power, reduced carbon dioxide fixation, as well as elevated levels of RNA transcripts and proteins involved in heavy metal resistance, with special emphasis on copper efflux systems. Finally, the expression and translation of genes responsible for chemotaxis and motility were enhanced.IMPORTANCELeptospirillum ferriphilum is one of the most important iron oxidizers in the context of acidic and metal-rich environments during moderately thermophilic biomining. A high-quality circular genome of Leptospirillum ferriphilumT coupled with functional omics data provides new insights into its metabolic properties, such as the novel identification of genes for atmospheric nitrogen fixation, and represents an essential step for further accurate proteomic and transcriptomic investigation of this acidophile model species in the future. Additionally, light is shed on adaptation strategies of Leptospirillum ferriphilumT for growth on the copper mineral chalcopyrite. These data can be applied to deepen our understanding and optimization of bioleaching and biooxidation, techniques that present sustainable and environmentally friendly alternatives to many traditional methods for metal extraction.

Mechanisms of Mineral Substrate Acquisition in a Thermoacidophile.

The thermoacidophile Acidianus is widely distributed in Yellowstone National Park hot springs that span large gradients in pH (1.60 to 4.84), temperature (42 to 90°C), and mineralogical composition. To characterize the potential role of flexibility in mineral-dependent energy metabolism in contributing to the widespread ecological distribution of this organism, we characterized the spectrum of minerals capable of supporting metabolism and the mechanisms that it uses to access these minerals. The energy metabolism of Acidianus strain DS80 was supported by elemental sulfur (S0), a variety of iron (hydr)oxides, and arsenic sulfide. Strain DS80 reduced, oxidized, and disproportionated S0 Cells growing via S0 reduction and disproportionation did not require direct access to the mineral to reduce it, whereas cells growing via S0 oxidation did require direct access, observations that are attributable to the role of H2S produced by S0 reduction/disproportionation in solubilizing and increasing the bioavailability of S0 Cells growing via iron (hydr)oxide reduction did not require access to the mineral, suggesting that the cells reduce Fe(III) that is being leached by the acidic growth medium. Cells growing via oxidation of arsenic sulfide with Fe(III) did not require access to the mineral to grow. The stoichiometry of reactants to products indicates that cells oxidize soluble As(III) released from oxidation of arsenic sulfide by aqueous Fe(III). Taken together, these observations underscore the importance of feedbacks between abiotic and biotic reactions in influencing the bioavailability of mineral substrates and defining ecological niches capable of supporting microbial metabolism.IMPORTANCE Mineral sources of electron donor and acceptor that support microbial metabolism are abundant in the natural environment. However, the spectrum of minerals capable of supporting a given microbial strain and the mechanisms that are used to access these minerals in support of microbial energy metabolism are often unknown, in particular among thermoacidophiles. Here, we show that the thermoacidophile Acidianus strain DS80 is adapted to use a variety of iron (hydro)oxide minerals, elemental sulfur, and arsenic sulfide to support growth. Cells rely on a complex interplay of abiologically and biologically catalyzed reactions that increase the solubility or bioavailability of minerals, thereby enabling their use in microbial metabolism.

Sulfurisphaera javensis sp. nov., a hyperthermophilic and acidophilic archaeon isolated from Indonesian hot spring, and reclassification of Sulfolobus tokodaii Suzuki et al. 2002 as Sulfurisphaera tokodaii comb. nov.

A novel hyperthermophilic, acidophilic and facultatively anaerobic archaeon, strain KD-1T, was isolated from an acidic hot spring in Indonesia and characterized with the phylogenetically related species Sulfurisphaera ohwakuensis Kurosawa et al. 1998, Sulfolobus tokodaii Suzuki et al., 2002 and Sulfolobus yangmingensis Jan et al. 1999. Cells of KD-1T were irregular cocci with diameters of 0.9-1.3 µm. The strain grew at 60-90 °C (optimum 80-85 °C), pH 2.5-6.0 (optimum pH 3.5-4.0) and 0-1.0 % (w/v) NaCl concentration. KD-1T grew anaerobically in the presence of S0 (headspace: H2/CO2) and FeCl3 (headspace: N2). Under aerobic conditions, chemolithoautotrophic growth occurred on S0, pyrite, K2S4O6, Na2S2O3 and H2. This strain utilized various complex substrates, such as yeast extract, but did not grow on sugars and amino acids as the sole carbon source. The main core lipids were calditoglycerocaldarchaeol and caldarchaeol. The DNA G+C content was 30.6 mol%. Analyses of phylogenetic trees based on 16S rRNA and 23S rRNA genes indicated that KD-1T formed an independent lineage near Sulfurisphaera ohwakuensis TA-1T, Sulfolobus tokodaii 7T and Sulfolobus yangmingensis YM1T. On the basis of the results of morphological, physiological, chemotaxonomic and phylogenetic analyses, KD-1T represents a novel species of the genus Sulfurisphaera Kurosawa et al. 1998, for which the name Sulfurisphaera javensis sp. nov. is proposed. The type strain is KD-1T (=JCM 32117T=InaCC Ar81T). Based on the data, we also propose the reclassification of Sulfolobus tokodaii Suzuki et al., 2002 as Sulfurisphaera tokodaii comb. nov. (type strain 7T=JCM 10545T=DSM 16993T).

Saccharolobus caldissimus gen. nov., sp. nov., a facultatively anaerobic iron-reducing hyperthermophilic archaeon isolated from an acidic terrestrial hot spring, and reclassification of Sulfolobus solfataricus as Saccharolobus solfataricus comb. nov. and Sulfolobus shibatae as Saccharolobus shibatae comb. nov.

A novel hyperthermophilic archaeon of strain HS-3T, belonging to the family Sulfolobaceae, was isolated from an acidic terrestrial hot spring in Hakone Ohwaku-dani, Japan. Based on 16S rRNA gene sequence analysis, the closest phylogenetic relatives of strain HS-3T were, first, Sulfolobus solfataricus (96.4 %) and, second, Sulfolobus shibatae (96.2 %), indicating that the strain belongs to the genus Sulfolobus. However, the sequence similarity to the type species of the genus Sulfolobus (Sulfolobus acidocaldarius) was remarkably low (91.8 %). In order to determine whether strain HS-3T belongs to the genus Sulfolobus, its morphological, biochemical and physiological characteristics were examined in parallel with those of S. solfataricus and S. shibatae. Although there were some differences in chemolithotrophic growth between strain HS-3T, S. solfataricus and S. shibatae, their temperature, pH and facultatively anaerobic characteristics of growth, and their utilization of various sugars were almost identical. In contrast, the utilization of various sugars by S. acidocaldarius was quite different from that of HS-3T, S. solfataricus and S. shibatae. Phylogenetic evidence based on the 16S and the 23S rRNA gene sequences also clearly distinguished the monophyletic clade composed of strain HS-3T, S. solfataricus, and S. shibatae from S. acidocaldarius. Based on these results, we propose a new genus and species, Saccharolobus caldissimus gen. nov., sp. nov., for strain HS-3T, as well as two reclassifications, Saccharolobus solfataricus comb. nov. and Saccharolobus shibatae comb. nov. The type strain of Saccharolobus caldissimus is HS-3T (=JCM 32116T and InaCC Ar80T). The type species of the genus is Saccharolobus solfataricus.