Extremophiles

Eukaryotic organisms of continental hydrothermal systems Abstract Continental hydrothermal systems are a dynamic component of global thermal and geochemical cycles, exerting a pronounced impact on water chemistry and heat storage. As such, these environments are commonly classified by temperature, thermal fluid ionic concentration, and pH. Terrestrial hydrothermal systems are a refuge for extremophilic organisms, as extremes in temperature, metal concentration, and pH profoundly impact microorganism assemblage composition. While numerous studies focus on Bacteria and Archaea in these environments, few focus on Eukarya—likely due to lower temperature tolerances and because they are not model organisms for understanding the evolution of early life. However, where present, eukaryotic organisms are significant members of continental hydrothermal microorganism communities. Thus, this manuscript focuses on the eukaryotic occupants of terrestrial hydrothermal systems and provides a review of the current status of research, including microbe–eukaryote interactions and suggestions for future directions.

High-quality draft genome sequence of Pseudomonas aeruginosa san ai, an environmental isolate resistant to heavy metals Abstract The strain Pseudomonas aeruginosa san ai, isolated from an extreme environment (industrial mineral cutting oil, pH 10), is able to survive and persist in the presence of a variety of pollutants such as heavy metals and organic chemicals. The genome of P. aeruginosa san ai is 6.98 Mbp long with a GC content of 66.08% and 6485 protein encoding genes. A large number of genes associated with proteins, responsible for microbial resistance to heavy metal ions and involved in catabolism of toxic aromatic organic compounds were identified. P. aeruginosa san ai is a highly cadmium-resistant strain. Proteome analysis of biomass after cadmium exposal confirmed a high tolerance to sublethal concentrations of cadmium (100 mg/L), based on: extracellular biosorption, bioaccumulation, biofilm formation, controlled siderophore production and a pronounced metalloprotein synthesis. Proteins responsible for survival in osmostress conditions during exposure to elevated concentrations of cadmium (200 mg/L) demonstrate a strong genetic potential of P. aeruginosa san ai for survival and adaptation. Sequencing of P. aeruginosa san ai genome provides valuable insights into the evolution and adaptation of this microbe to environmental extremes at the whole-genome level, as well as how to optimally use the strain in bioremediation of chemically polluted sites.

Toward a rapid method for the study of biodiversity in cold environments: the characterization of psychrophilic yeasts by MALDI-TOF mass spectrometry Abstract To investigate the potential of matrix-assisted laser desorption ionization mass spectrometry (MALDI-TOF/MS) as a platform to support biodiversity and phylogenetic studies of psychrophilic yeasts in cold environments, the technique was employed to rapidly characterize and distinguish three psychrophilic yeasts (Rhodotorula mucilaginosa, Naganishia vishniacii, and Dioszegia cryoxerica) from three mesophilic counterparts (Saccharomyces cerevisiae Cry Havoc, S. cerevisiae California V Ale, and S. pastorianus). A detailed workflow for providing reproducible mass spectral fingerprints of low molecular weight protein/peptide features specific to the organisms studied is presented. The potential of this approach as a tool in the study of biodiversity, systematics, and phylogeny of psychrophilic microorganisms is highlighted.

Protein acetylation on 2-isopropylmalate synthase from Thermus thermophilus HB27 Abstract Protein lysine Nε-acetylation is one of the important factors regulating cellular metabolism. We performed a proteomic analysis to identify acetylated proteins in the extremely thermophilic bacterium, Thermus thermophilusHB27. A total of 335 unique acetylated lysine residues, including many metabolic enzymes and ribosomal proteins, were identified in 208 proteins. Enzymes involved in amino acid metabolism were the most abundant among acetylated metabolic proteins. 2-Isopropylmalate synthase (IPMS), which catalyzes the first step in leucine biosynthesis, was acetylated at four lysine residues. Acetylation-mimicking mutations at Lys332 markedly decreased IPMS activity in vitro, suggesting that Lys332, which is located in subdomain II, plays a regulatory role in IPMS activity. We also investigated the acetylation-deacetylation mechanism of IPMS and revealed that it was acetylated non-enzymatically by acetyl-CoA and deacetylated enzymatically by TT_C0104. The present results suggest that leucine biosynthesis is regulated by post-translational protein modifications, in addition to feedback inhibition/repression, and that metabolic enzymes are regulated by protein acetylation in T. thermophilus.

Production, characterization and antimicrobial activities of bio-pigments by Aquisalibacillus elongatus MB592, Salinicoccus sesuvii MB597, and Halomonas aquamarina MB598 isolated from Khewra Salt Range, Pakistan Abstract Hypersaline ecosystems offer unique habitats to microbial populations capable of withstanding extreme stress conditions and producing novel metabolites of commercial importance. Herein, we have characterized for the first time the production of bioactive pigments from newly isolated halophilic bacterial species. Halophilic bacteria were isolated from Khewra Salt Range of Pakistan. Three distinctly colored isolates were selected for pigment production. Selected colonies were identified as Aquisalibacillus elongatus MB592, Salinicoccus sesuvii MB597, and Halomonas aquamarina MB598 based on morphological, biochemical, and physiological evidences as well as 16S rRNA analysis. The optimum pigment production observed at mesophilic condition, nearly neutral pH, and moderate salinity was validated using response surface methodology. Different analytical techniques (UV spectroscopy, infrared spectroscopy, and HPLC) characterized these purified pigments as derivatives of bacterioruberin carotenoids. Antioxidant activity of pigments revealed up to 85% free-radical scavenging activity at the concentration of 30 µg ml−1. Pigments also showed significant antimicrobial activity against Bacillus subtilis, Bacillus pumilus, Enterococcus faecalis, Bacillus cereus, Klebsiella pneumoniae, Alcaligenes faecalis, Pseudomonas geniculata, Enterococcus faecium, Aspergillus fumigatus, Aspergillus flavus, Fusarium solani, and Mucor spp., suggesting potential biomedical applications.

Dual bloom of green algae and purple bacteria in an extremely shallow soda pan Abstract In April 2014, dual bloom of green algae and purple bacteria occurred in a shallow, alkaline soda pan (Kiskunság National Park, Hungary). The water was only 5 cm deep, in which an upper green layer was clearly separated from a near-sediment purple one. Based on microscopy and DNA-based identification, the upper was inhabited by a dense population of the planktonic green alga, Oocystis submarina Lagerheim, while the deeper layer was formed by purple, bacteriochlorophyll-containing bacteria, predominated by Thiorhodospira and Rhodobaca. Additional bacterial taxa with a presumed capability of anoxygenic phototrophic growth belonged to the genera Loktanella and Porphyrobacter. Comparing the bacterial community of the purple layer with a former blooming event in a nearby soda pan, similar functional but different taxonomic composition was revealed. Members from many dominant bacterial groups were successfully cultivated including potentially new species, which could be the result of the application of newly designed media.

Identification of a novel esterase from the thermophilic bacterium Geobacillus thermodenitrificans NG80-2 Abstract In the framework of the discovery of new thermophilic enzymes of potential biotechnological interest, we embarked in the characterization of a new thermophilic esterase from the thermophilic bacterium Geobacillus thermodenitrificans. The phylogenetic analysis of the GTNG_0744 esterase indicated that the sequence belongs to the enterochelin/enterobactin esterase group, which have never been recognized as a family in the lipases/esterase classification. These enzymes catalyze the last step in the acquisition of environmental Fe3+ through siderophore hydrolysis. In silico analysis revealed, for the first time, that the machinery for the uptake of siderophores is present in G. thermodenitrificans. The purified recombinant enzyme, EstGtA3, showed different substrate specificity from known enterochelin/enterobactin esterases, recognizing short chain esters with a higher specificity constant for 4-NP caprylate. The enzyme does not require cofactors for its activity, is active in the pH range 7.0–8.5, has highest activity at 60 °C and is 100% stable when incubated for 16 h at 55 °C. DTT, β-mercaptoethanol and Triton X-100 have an activating effect on the enzymatic activity. Organic solvents have in general a negative effect on the enzyme, but n-hexane is a strong activator up to 150, making EstGtA3 a good candidate for applications in biotechnology.

Lipids and soluble carbohydrates in the mycelium and ascomata of alkaliphilic fungus Sodiomyces alkalinus Abstract Alkaliphilic fungi are fundamentally different from alkalitolerant ones in terms of mechanisms of adaptation. They accumulate trehalose in cytosol and phosphatidic acids (PA) in the membrane lipids, whereas alkalitolerants contain these compounds in low amounts. But it is unclear how the composition of osmolytes and lipids changes during cytodifferentiation. In this article the composition of lipids and soluble cytosol carbohydrates in the mycelium and fruit bodies of the alkaliphilic fungus Sodiomyces alkalinus was studied. In the mycelium, mannitol and trehalose dominated, while in fruit bodies only trehalose was predominant. Phosphatidylcholines (PC), PA and sterols were major membrane lipids of the mycelium, while PC and sterols were predominant in fruit bodies. The degree of fatty acids unsaturation of the main mycelium phospholipids (PC and PA) increased with age, while that of PC did not change regardless of the developmental stage. In young mycelium, storage lipids were represented mainly by free fatty acids, and in mature mycelium and fruit bodies—by triacylglycerols. Fruit bodies contained three times less membrane lipids and twice as many storage lipids as mycelium. Trehalose was the main cytosol carbohydrate in the mycelium and fruit bodies, which confirms its key value for alkaliphily.

Transcriptome analysis of a thermophilic and hydrogenogenic carboxydotroph Carboxydothermus pertinax Abstract A thermophilic and hydrogenogenic carboxydotroph, Carboxydothermus pertinax, performs hydrogenogenic CO metabolism in which CODH-II couples with distally encoded ECH. To enhance our knowledge of its hydrogenogenic CO metabolism, we performed whole transcriptome analysis of C. pertinax grown under 100% CO or 100% N2 using RNA sequencing. Of the 2577 genes, 36 and 64 genes were differentially expressed genes (DEGs) with false discovery rate adjusted P value < 0.05 when grown under 100% CO or 100% N2, respectively. Most of the DEGs were components of 23 gene clusters, suggesting switch between metabolisms via intensive expression changes in a relatively low number of gene clusters. Of the 9 significantly expressed gene clusters under 100% CO, CODH-II and ECH gene clusters were found. Only the ECH gene cluster was regulated by the CO-responsive transcriptional factor CooA, suggesting that others were separately regulated in the same transcriptional cascade as the ECH gene cluster. Of the 14 significantly expressed gene clusters under 100% N2, ferrous iron transport gene cluster involved in anaerobic respiration and prophage region were found. Considering that the expression of the temperate phage was strictly repressed under 100% CO, hydrogenogenic CO metabolism might be stable for C. pertinax.

Identification of a GntR family regulator BusR Tha and its regulatory mechanism in the glycine betaine ABC transport system of Tetragenococcus halophilus Abstract Glycine betaine is one of the most effective compatible solutes of the halophilic lactic acid bacterium Tetragenococcus halophilus, the transportation of which is essential for its survival under salinity stress condition. In the current study, we attempted to define a glycine betaine ABC transporter system of T. halophilus, busATha, which plays an important role in adapting to salinity condition. The expression of busATha enhanced the growth of the recombinant strain under high salinity. BusRTha, a transcription regulator that represses the expression of busATha, was characterized, and the repression was abrogated under high salinity. The binding of the regulator was demonstrated through electrophoretic mobility shift assays, and the binding sites were characterized as 5′-AAA(T/G)TGAC(C/A)(G/A)T(C/A)C-3′. This is the first studied transcription regulator of T. halophilus, and our findings provide insights into the molecular mechanism of halophilic life and tools for further application of halophiles as chassis in industrial biotechnology.