Non-thermodynamic factors affect competition between thermophilic chemolithoautotrophs from deep-sea hydrothermal vents.

Various environmental factors, including H2 availability, metabolic tradeoffs, optimal growth temperature, stochasticity, and hydrology, were examined to determine if they affect microbial competition between three autotrophic thermophiles. The thiosulfate reducer Desulfurobacterium thermolithotrophum (Topt72°C) was grown in mono- and coculture separately with the methanogens Methanocaldococcus jannaschii (Topt82°C) at 72°C and Methanothermococcus thermolithotrophicus (Topt65°C) at 65°C at high and low H2 concentrations. Both methanogens showed a metabolic tradeoff shifting from high growth rate-low cell yield at high H2 concentrations to low growth rate-high cell yield at low H2 concentrations and when grown in coculture with the thiosulfate reducer. In 1:1 initial ratios, D. thermolithotrophum outcompeted both methanogens at high and low H2, no H2S was detected on low H2, and it grew with only CO2 as the electron acceptor indicating a similar metabolic tradeoff with low H2. When the initial methanogen-to-thiosulfate reducer ratio varied from 1:1 to 104:1 with high H2, D. thermolithotrophum always outcompeted M. jannaschii at 72°C. However, M. thermolithotrophicus outcompeted D. thermolithotrophum at 65°C when the ratio was 103:1. A reactive transport model that mixed pure hydrothermal fluid with cold seawater showed that hyperthermophilic methanogens dominated in systems where the residence time of the mixed fluid above 72°C was sufficiently high. With shorter residence times, thermophilic thiosulfate reducers dominated. If residence times increased with decreasing fluid temperature along the flow path, then thermophilic methanogens could dominate. Thermophilic methanogen dominance spread to previously thiosulfate-reducer-dominated conditions if the initial ratio of thermophilic methanogen-to-thiosulfate reducer increased. IMPORTANCE: The deep subsurface is the largest reservoir of microbial biomass on Earth and serves as an analog for life on the early Earth and extraterrestrial environments. Methanogenesis and sulfur reduction are among the more common chemolithoautotrophic metabolisms found in hot anoxic hydrothermal vent environments. Competition between H2-oxidizing sulfur reducers and methanogens is primarily driven by the thermodynamic favorability of redox reactions with the former outcompeting methanogens. This study demonstrated that competition between the hydrothermal vent chemolithoautotrophs Methanocaldococcus jannaschii, Methanothermococcus thermolithotrophicus, and Desulfurobacterium thermolithotrophum is also influenced by other overlapping factors such as staggered optimal growth temperatures, stochasticity, and hydrology. By modeling all aspects of microbial competition coupled with field data, a better understanding is gained on how methanogens can outcompete thiosulfate reducers in hot anoxic environments and how the deep subsurface contributes to biogeochemical cycling.

Mycovorax composti gen. nov., sp. nov., a member of the family Chitinophagaceae isolated from button mushroom compost.

Two Gram-stain-negative, aerobic, rod-shaped, orange-coloured bacterial strains, designated strain C216T and strain M2295, were isolated from mature mushroom compost from composting facilities in Victoria and South Australia, Australia, respectively. External structures such as flagella or pili were not observed on the cells under scanning electron microscopy. Optimal growth was found to occur at 45 °C, at pH 7.25 and in the absence of NaCl on Emerson's 350 YpSs medium. The genome sequence of strain C216T was 3 342 126 bp long with a G+C content of 40.5 mol%. Functional analysis of the genome of strain C216T revealed genes encoding chitinolytic and hemi-cellulolytic functions, with 166 predicted genes associated with carbohydrate metabolism (8.9% of the predicted genes). These functions are important for survival in the mushroom compost environment, which is rich in hemicelluloses. No antibiotic resistance genes were found in the genome sequence. The major fatty acids of strain C216T were iso-C15 : 0 (56.7%), iso-C17 : 0 3-OH (15.6%), C16 : 1 ω7c/iso-C15 : 0 2-OH (7.3%) and iso-C15 : 1 G (6.1%). The only respiratory quinone was MK-7. The major polar lipid of strain C216T was phosphatidylethanolamine, but three unidentified phospholipids, four unidentified aminophospholipids/aminolipids and one unidentified glycolipid were also detected. Phylogenetic analysis based on proteins encoded by the core genome (bac120, 120 conserved bacterial genes) showed that strain C216T forms a distinct lineage in the family Chitinophagaceae and that the closest identified relative is Niabella soli (69.69% ANI). These data demonstrate that strain C216T represents a novel genus and novel species within the family Chitinophagaceae, for which we propose the name Mycovorax composti. The type strain is C216T (=DSM 114558T=LMG 32998T).

Effects of small heat shock proteins from thermotolerant bacteria on the stress resistance of Escherichia coli to temperature, pH, and hyperosmolarity.

Small heat shock proteins (HSPs), such as HSP20, represent cellular thermal resistance mechanisms, to avoid protein aggregation at elevated temperatures. Recombinantly expressed HSP20s serve as a molecular tool for improving the tolerance of living cells to various physical and chemical stressors. Here, we aimed to heterologously express 18 HSP20s from 12 thermotolerant bacteria in Escherichia coli and evaluate their effects on various physical and chemical cellular stresses. Seventeen HSP20s were successfully expressed as soluble proteins. Recombinant E. coli cells were subjected to heat, cold, acidic, alkaline, and hyperosmolar stress to evaluate the effects of HSP20 proteins on stress resistance. Notably, the overexpression of 15 HSP20s enhanced the stress resistance of E. coli compared to that of the control strain. In particular, HSPs from Tepidimonas sediminis and Oceanithermus profundus improved the stress tolerance of E. coli under all tested conditions. In addition, E. coli harboring HSP20 from T. sediminis retained cell viability even after heat treatment at 52 °C for 5 days. To our knowledge, this is the first report of E. coli tolerance to prolonged (> 100 h) high-temperature stress. These findings indicate the potential of thermotolerant HSPs as molecular tools for improving stress tolerance in E. coli.

Extremophiles in a changing world.

Extremophiles and their products have been a major focus of research interest for over 40 years. Through this period, studies of these organisms have contributed hugely to many aspects of the fundamental and applied sciences, and to wider and more philosophical issues such as the origins of life and astrobiology. Our understanding of the cellular adaptations to extreme conditions (such as acid, temperature, pressure and more), of the mechanisms underpinning the stability of macromolecules, and of the subtleties, complexities and limits of fundamental biochemical processes has been informed by research on extremophiles. Extremophiles have also contributed numerous products and processes to the many fields of biotechnology, from diagnostics to bioremediation. Yet, after 40 years of dedicated research, there remains much to be discovered in this field. Fortunately, extremophiles remain an active and vibrant area of research. In the third decade of the twenty-first century, with decreasing global resources and a steadily increasing human population, the world's attention has turned with increasing urgency to issues of sustainability. These global concerns were encapsulated and formalized by the United Nations with the adoption of the 2030 Agenda for Sustainable Development and the presentation of the seventeen Sustainable Development Goals (SDGs) in 2015. In the run-up to 2030, we consider the contributions that extremophiles have made, and will in the future make, to the SDGs.

Thermostable in vitro transcription-translation compatible with microfluidic droplets.

BACKGROUND: In vitro expression involves the utilization of the cellular transcription and translation machinery in an acellular context to produce one or more proteins of interest and has found widespread application in synthetic biology and in pharmaceutical biomanufacturing. Most in vitro expression systems available are active at moderate temperatures, but to screen large libraries of natural or artificial genetic diversity for highly thermostable enzymes or enzyme variants, it is instrumental to enable protein synthesis at high temperatures. OBJECTIVES: Develop an in vitro expression system operating at high temperatures compatible with enzymatic assays and with technologies that enable ultrahigh-throughput protein expression in reduced volumes, such as microfluidic water-in-oil (w/o) droplets. RESULTS: We produced cell-free extracts from Thermus thermophilus for in vitro translation including thermostable enzymatic cascades for energy regeneration and a moderately thermostable RNA polymerase for transcription, which ultimately limited the temperature of protein synthesis. The yield was comparable or superior to other thermostable in vitro expression systems, while the preparation procedure is much simpler and can be suited to different Thermus thermophilus strains. Furthermore, these extracts have enabled in vitro expression in microfluidic droplets at high temperatures for the first time. CONCLUSIONS: Cell-free extracts from Thermus thermophilus represent a simpler alternative to heavily optimized or pure component thermostable in vitro expression systems. Moreover, due to their compatibility with droplet microfluidics and enzyme assays at high temperatures, the reported system represents a convenient gateway for enzyme screening at higher temperatures with ultrahigh-throughput.

Aspartate-phobia of thermophiles as a reaction to deleterious chemical transformations.

Prokaryotes growing at high temperatures have a high proportion of charged residues in their proteins to stabilize their 3D structure. By mining 175 disparate bacterial and archaeal proteomes we found that, against the general trend for charged residues, the frequency of aspartic acid residues decreases strongly as natural growth temperature increases. In search of the explanation, we hypothesized that the reason for such unusual correlation is the deleterious consequences of spontaneous chemical transformations of aspartate at high temperatures. Our subsequent statistical analysis supported this hypothesis. This finding reveals that organisms have likely adapted to high temperatures by minimizing the harmful consequences of spontaneous chemical transformations.

The Undeniable Potential of Thermophiles in Industrial Processes.

Extremophilic microorganisms play a key role in understanding how life on Earth originated and evolved over centuries. Their ability to thrive in harsh environments relies on a plethora of mechanisms developed to survive at extreme temperatures, pressures, salinity, and pH values. From a biotechnological point of view, thermophiles are considered a robust tool for synthetic biology as well as a reliable starting material for the development of sustainable bioprocesses. This review discusses the current progress in the biomanufacturing of high-added bioproducts from thermophilic microorganisms and their industrial applications.

From waste management to circular economy: Leveraging thermophiles for sustainable growth and global resource optimization.

Waste of any origin is one of the most serious global and man-made concerns of our day. It causes climate change, environmental degradation, and human health problems. Proper waste management practices, including waste reduction, safe handling, and appropriate treatment, are essential to mitigate these consequences. It is thus essential to implement effective waste management strategies that reduce waste at the source, promote recycling and reuse, and safely dispose of waste. Transitioning to a circular economy with policies involving governments, industries, and individuals is essential for sustainable growth and waste management. The review focuses on diverse kinds of environmental waste sources around the world, such as residential, industrial, commercial, municipal services, electronic wastes, wastewater sewerage, and agricultural wastes, and their challenges in efficiently valorizing them into useful products. It highlights the need for rational waste management, circularity, and sustainable growth, and the potential of a circular economy to address these challenges. The article has explored the role of thermophilic microbes in the bioremediation of waste. Thermophiles known for their thermostability and thermostable enzymes, have emerged to have diverse applications in biotechnology and various industrial processes. Several approaches have been explored to unlock the potential of thermophiles in achieving the objective of establishing a zero-carbon sustainable bio-economy and minimizing waste generation. Various thermophiles have demonstrated substantial potential in addressing different waste challenges. The review findings affirm that thermophilic microbes have emerged as pivotal and indispensable candidates for harnessing and valorizing a range of environmental wastes into valuable products, thereby fostering the bio-circular economy.

Expression and characterization of a thermostable monoacylglycerol lipase from thermophilic Geobacillus kaustophilus.

Thermophilic Geobacillus kaustophilus HTA426 genome possesses a monoacylglycerol lipase (MAGL) gene. MAGLs can synthesize emulsifiers for use in the food and pharmaceutical industries from fatty acids and glycerol. They can also be used to analyze monoacylglycerol (MAG) levels in serum and food. The MAGL gene from strain HTA426 was artificially synthesized and heterologously expressed in Escherichia coli BL21(DE3). The recombinant His-tag fused MAGL (GkMAGL) was purified using a Ni2+-affinity column. The purified enzyme showed a temperature optimum at 65 °C and was stable up to 75 °C after 30 min incubation. In addition, the enzyme exhibited a pH optimum of 7.5 and was stable from pH 5.0 to 11.0. The enzyme hydrolyzed monoacylglycerols and showed the highest activity toward 1-monolauroylglycerol. The enzyme was stable in the presence of various organic solvents and detergents. The addition of Triton X-100 significantly increased GkMAGL activity. The thermal stability of the enzyme was higher than that of thermostable MAGL from Geobacillus sp. 12AMOR1 (12AMOR1_MAGL). Circular dichroism spectral analysis showed that the conformational stability of the GkMAGL was higher than that of 12AMOR1_MAGL at higher temperatures. These results indicate that the GkMAGL has useful features that can be used for various biotechnological applications.

Identification and characterization of sugar-regulated promoters in Chaetomium thermophilum.

The thermophilic fungus Chaetomium thermophilum has been used extensively for biochemical and high-resolution structural studies of protein complexes. However, subsequent functional analyses of these assemblies have been hindered owing to the lack of genetic tools compatible with this thermophile, which are typically suited to other mesophilic eukaryotic model organisms, in particular the yeast Saccharomyces cerevisiae. Hence, we aimed to find genes from C. thermophilum that are expressed under the control of different sugars and examine their associated 5' untranslated regions as promoters responsible for sugar-regulated gene expression. To identify sugar-regulated promoters in C. thermophilum, we performed comparative xylose- versus glucose-dependent gene expression studies, which uncovered a number of enzymes with induced expression in the presence of xylose but repressed expression in glucose-supplemented media. Subsequently, we cloned the promoters of the two most stringently regulated genes, the xylosidase-like gene (XYL) and xylitol dehydrogenase (XDH), obtained from this genome-wide analysis in front of a thermostable yellow fluorescent protein (YFP) reporter. With this, we demonstrated xylose-dependent YFP expression by both Western blotting and live-cell imaging fluorescence microscopy. Prompted by these results, we expressed the C. thermophilum orthologue of a well-characterized dominant-negative ribosome assembly factor mutant, under the control of the XDH promoter, which allowed us to induce a nuclear export defect on the pre-60S subunit when C. thermophilum cells were grown in xylose- but not glucose-containing medium. Altogether, our study identified xylose-regulatable promoters in C. thermophilum, which might facilitate functional studies of genes of interest in this thermophilic eukaryotic model organism.

Optimizing Strategies for Bio-Based Ethanol Production Using Genome-Scale Metabolic Modeling of the Hyperthermophilic Archaeon, Pyrococcus furiosus.

A genome-scale metabolic model, encompassing a total of 623 genes, 727 reactions, and 865 metabolites, was developed for Pyrococcus furiosus, an archaeon that grows optimally at 100°C by carbohydrate and peptide fermentation. The model uses subsystem-based genome annotation, along with extensive manual curation of 237 gene-reaction associations including those involved in central carbon metabolism, amino acid metabolism, and energy metabolism. The redox and energy balance of P. furiosus was investigated through random sampling of flux distributions in the model during growth on disaccharides. The core energy balance of the model was shown to depend on high acetate production and the coupling of a sodium-dependent ATP synthase and membrane-bound hydrogenase, which generates a sodium gradient in a ferredoxin-dependent manner, aligning with existing understanding of P. furiosus metabolism. The model was utilized to inform genetic engineering designs that favor the production of ethanol over acetate by implementing an NADPH and CO-dependent energy economy. The P. furiosus model is a powerful tool for understanding the relationship between generation of end products and redox/energy balance at a systems-level that will aid in the design of optimal engineering strategies for production of bio-based chemicals and fuels. IMPORTANCE The bio-based production of organic chemicals provides a sustainable alternative to fossil-based production in the face of today's climate challenges. In this work, we present a genome-scale metabolic reconstruction of Pyrococcus furiosus, a well-established platform organism that has been engineered to produce a variety of chemicals and fuels. The metabolic model was used to design optimal engineering strategies to produce ethanol. The redox and energy balance of P. furiosus was examined in detail, which provided useful insights that will guide future engineering designs.

Microbe-Mineral Interactions between Asbestos and Thermophilic Chemolithoautotrophic Anaerobes.

The Fe content and the morphometry of asbestos are two major factors linked to its toxicity. This study explored the use of microbe-mineral interactions between asbestos (and asbestos-like) minerals and thermophilic chemolithoautotrophic microorganisms as possible mineral dissolution treatments targeting their toxic properties. The removal of Fe from crocidolite was tested through chemolithoautotrophic Fe(III) reduction activities at 60°C. Chrysotile and tremolite-actinolite were tested for dissolution and potential release of elements like Si and Mg through biosilicification processes at 75°C. Our results show that chemolithoautotrophic Fe(III) reduction activities by Deferrisoma palaeochoriense were supported with crocidolite as the sole source of Fe(III) used as a terminal electron acceptor during respiration. Microbial Fe(III) reduction activities resulted in higher Fe release rates from crocidolite in comparison to previous studies on Fe leaching from crocidolite through Fe assimilation activities by soil fungi. Evidence of biosilicification in Thermovibrio ammonificans did not correspond with increased Si and Mg release from chrysotile or tremolite-actinolite dissolution. However, overall Si and Mg release from chrysotile into our experimental medium outmatched previously reported capabilities for Si and Mg release from chrysotile by fungi. Differences in the profiles of elements released from chrysotile and tremolite-actinolite during microbe-mineral experiments with T. ammonificans underscored the relevance of underlying crystallochemical differences in driving mineral dissolution and elemental bioavailability. Experimental studies targeting the interactions between chemolithoautotrophs and asbestos (or asbestos-like) minerals offer new access to the mechanisms behind crystallochemical mineral alterations and their role in the development of tailored asbestos treatments. IMPORTANCE We explored the potential of chemosynthetic microorganisms growing at high temperatures to induce the release of key elements (mainly iron, silicon, and magnesium) involved in the known toxic properties (iron content and fibrous mineral shapes) of asbestos minerals. We show for the first time that the microbial respiration of iron from amphibole asbestos releases some of the iron contained in the mineral while supporting microbial growth. Another microorganism imposed on the two main types of asbestos minerals (serpentines and amphiboles) resulted in distinct elemental release profiles for each type of asbestos during mineral dissolution. Despite evidence of microbially mediated dissolution in all minerals, none of the microorganisms tested disrupted the structure of the asbestos mineral fibers. Further constraints on the relationships between elemental release rates, amount of starting asbestos, reaction volumes, and incubation times will be required to better compare asbestos dissolution treatments studied to date.

Bacterial thermophilic DNA polymerases: A focus on prominent biotechnological applications.

DNA polymerases are the enzymes able to replicate the genetic information in nucleic acid. As a result, they are necessary to copy the complete genome of every living creature before cell division and sustain the integrity of the genetic information throughout the life of each cell. Any organism that uses DNA as its genetic information, whether unicellular or multicellular, requires one or more thermostable DNA polymerases to thrive. Thermostable DNA polymerase is important in modern biotechnology and molecular biology because it results in methods such as DNA cloning, DNA sequencing, whole genome amplification, molecular diagnostics, polymerase chain reaction, synthetic biology, and single nucleotide polymorphism detection. There are at least 14 DNA-dependent DNA polymerases in the human genome, which is remarkable. These include the widely accepted, high-fidelity enzymes responsible for replicating the vast majority of genomic DNA and eight or more specialized DNA polymerases discovered in the last decade. The newly discovered polymerases' functions are still being elucidated. Still, one of its crucial tasks is to permit synthesis to resume despite the DNA damage that stops the progression of replication-fork. One of the primary areas of interest in the research field has been the quest for novel DNA polymerase since the unique features of each thermostable DNA polymerase may lead to the prospective creation of novel reagents. Furthermore, protein engineering strategies for generating mutant or artificial DNA polymerases have successfully generated potent DNA polymerases for various applications. In molecular biology, thermostable DNA polymerases are extremely useful for PCR-related methods. This article examines the role and importance of DNA polymerase in a variety of techniques.

Shouchella tritolerans sp. nov., a facultative anaerobic bacterium isolated from marine sediments.

An alkali, salt, and thermo-tolerant strain designated FJAT-45399T was isolated from marine sediment in Fujian Province, China. Strain FJAT-45399T was Gram-stain-positive, rod-shaped, and facultatively aerobic. It shared high 16S rRNA gene sequence similarities with the members of the genus Shouchella. Further, the phylogenetic and phylogenomic analysis also suggested strain FJAT-45399T clustered with the members of the genus Shouchella. Growth of strain FJAT-45399T was observed at 15-55 °C (optimum 45-50 °C), pH 7.0-13.0 (optimum 9.0) and 0-15% (w/v) NaCl (optimum 2%). It contained MK-7 as the menaquinone. The polar lipids were diphosphatidylglycerol (DPG), phosphatidylglycerol (PG) and an unidentified glycolipid (UGL) and lipid (UL). The major fatty acids (> 10%) were C16:0 (22.8%), iso-C15:0 (21.3%), and anteiso-C15:0 (14.0%). The genomic DNA G + C content was 44.5%. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between strain FJAT-45399T and the most closely related type strain Shouchella clausii DSM 8716T (ANI 94.1% and dDDH 55.4%) were both below the cut-off level for species delineation. Based on the above results, strain FJAT-45399T represents a novel species of the genus Shouchella, for which the name Shouchella tritolerans sp. nov., is proposed. The type strain is FJAT-45399T (= GDMCC 1.3098T = JCM 35613T).

Description of Sporanaerobium hydrogeniformans gen. nov., sp. nov., an obligately anaerobic, hydrogen-producing bacterium isolated from Aravali hot spring in India.

An obligately anaerobic bacterium XHS1971T, capable of degrading cellulose and xylan, was isolated from a sediment sample of Aravali hot spring, Ratnagiri, India. Cells of strain XHS1971T were Gram-stain-negative, spore-forming, motile, long-rods. Growth was observed at temperatures 30-50 °C (optimum 40-45 °C), pH 5.0-10.0 (optimum pH 8.0) and NaCl concentrations 0-0.5% (optimum 0%). Generation time of strain XHS1971T was 5 h under optimised growth conditions. Strain XHS1971T showed the ability to metabolise different complex and simple sugars constituting lignocellulosic biomass. Glucose was fermented majorly into hydrogen, formic acid, acetic acid, and ethanol, whereas carbon dioxide, butyric acid, lactic acid and succinic acid were produced in traces. 16S rRNA gene analysis of strain XHS1971T revealed < 94.5% homology with Cellulosilyticum lentocellum DSM5427T followed by Cellulosilyticum ruminicola JCM14822T, identifying strain as a distinct member of family Lachnospiraceae. The major cellular fatty acids (> 5%) were C14:0, C16:0, C18:0, and C16:1 ω7c. The genome size of the strain was 3.74 Mb with 35.3 mol% G + C content, and genes were annotated to carbohydrate metabolism, including genes involved in the degradation of cellulose and xylan and the production of hydrogen, ethanol and acetate. The uniqueness of strain was further validated by digital DNA-DNA hybridisation (dDDH), Average Nucleotide Identity (ANI), and Average Amino Acid Identity (AAI) values of 22%, 80%, and 63%, respectively, with nearest phylogenetic affiliates. Based on the detailed analyses, we propose a new genus and species, Sporanaerobium hydrogeniformans gen. nov., sp. nov., for strain XHS1971T (= MCC3498T = KCTC15729T = JCM32657T) within family Lachnospiraceae.

Post-monsoon seasonal variation of prokaryotic diversity in solfataric soil from the North Sikkim hot spring.

The solfataric soil sediments of the hot springs of Sikkim located at Yume Samdung and Lachen valley were studied for deciphering the bacterial diversity. The main aim here is to present a comparative study and generate a baseline data on the post-monsoon seasonal variation for the months of October and December, analyzed through 16S rRNA V3-V4 amplicon sequencing. The results have shown that there is not much variation at phylum level in the month of October in all the three hot springs such as New Yume Samdung (NYS), Old Yume Samdung (OYS), and Tarum (TAR) hot spring. The abundant phyla mainly present were Firmicutes, followed by Proteobacteria, Actinobacteria, and Bacteroidetes. Similarly, in the month of December, Firmicutes, Proteobacteria, Actinobacteria, and Bacteroidetes were prevalent; however, the percent relative abundance of these phyla in the month of December is relatively less. Besides this decrease in percent abundance, it was interestingly seen that relatively more phyla were found contributing towards the bacterial diversity in the month of December. Similar to phylum level, at genus level, there was not much variation seen among various prevalent genera of the three studied hot springs in both months. The major genera prevalent in both months among all the three hot springs were followed by Bacillus, Desulfotomaculum, Lactobacillus, and Paenibacillus. A similar trend was also seen at gene level that relative abundance of various genera was higher in the month of October but more genera were found to be contributing towards bacterial diversity in the month of December. Few distinct genera were found to be more abundant in the month of December such as Rhodopirellula and Blastopirellula. The results may conclude that there is not much variation in the abundance and type of bacterial communities during the post-monsoon season in the month of October and December. However, this may be assumed that there is the accumulation or increase in the bacterial communities during the winter (relatively higher temperature among hot springs) and may favor few mesophilic and more thermophilic communities as well.

A temperature dependent pilin promoter for production of thermostable enzymes in Thermus thermophilus.

BACKGROUND: Enzymes from thermophiles are of great interest for research and bioengineering due to their stability and efficiency. Thermophilic expression hosts such as Thermus thermophilus [T. thermophilus] can overcome specific challenges experienced with protein production in mesophilic expression hosts, such as leading to better folding, increased protein stability, solubility, and enzymatic activity. However, available inducible promoters for efficient protein production in T. thermophilus HB27 are limited. RESULTS: In this study, we characterized the pilA4 promoter region and evaluated its potential as a tool for production of thermostable enzymes in T. thermophilus HB27. Reporter gene analysis using a promoterless ß-glucosidase gene revealed that the pilA4 promoter is highly active under optimal growth conditions at 68 °C and downregulated during growth at 80 °C. Furthermore, growth in minimal medium led to significantly increased promoter activity in comparison to growth in complex medium. Finally, we proved the suitability of the pilA4 promoter for heterologous production of thermostable enzymes in T. thermophilus by producing a fully active soluble mannitol-1-phosphate dehydrogenase from Thermoanaerobacter kivui [T. kivui], which is used in degradation of brown algae that are rich in mannitol. CONCLUSIONS: Our results show that the pilA4 promoter is an efficient tool for gene expression in T. thermophilus with a high potential for use in biotechnology and synthetic biology applications.

Temperature - A critical abiotic paradigm that governs bacterial heterogeneity in natural ecological system.

A baseline data has been presented here to prove that among the abiotic factors, temperature is the most critical factor that regulates and governs the bacterial diversity in a natural ecosystem. Present study in Yumesamdong hot springs riverine vicinity (Sikkim), parades a gamut of bacterial communities in it and hosts them from semi-frigid region (- 4-10 °C) to fervid region (50-60 °C) via an intermediate region (25-37 °C) within the same ecosystem. This is an extremely rare intriguing natural ecosystem that has no anthropogenic disturbances nor any artificial regulation of temperature. We scanned the bacterial flora through both the culture-dependent and culture-independent techniques in this naturally complex thermally graded habitat. High-throughput sequencing gave bacterial and archaeal phyla representatives of over 2000 species showcasing their biodiversity. Proteobacteria, Firmicutes, Bacteroidetes and Chloroflexi were the predominant phyla. A concave down-curve significance was found in temperature-abundance correlation as the number of microbial taxa decreased when the temperature increased from warm (35 °C) to hot (60 °C). Firmicutes showed significant linear increase from cold to hot environment whereas Proteobacteria followed the opposite trend. No significant correlation was observed for physicochemical parameters against the bacterial diversity. However, only temperature has shown significant positive correlation to the predominant phyla at their respective thermal gradients. The antibiotic resistance patterns correlated with temperature gradient where the prevalence of antibiotic resistance was higher in case of mesophiles than that of psychrophiles and there was no resistance in thermophiles. The antibiotic resistant genes obtained were solely from mesophiles as it conferred high resistance at mesophilic conditions enabling them to adapt and metabolically compete for survival. Our study concludes that the temperature is a major factor that plays a significant contribution in shaping the bacterial community structure in any thermal gradient edifice.

Inoculation with thermophiles enhanced the food waste bio-drying and complicated interdomain ecological networks between bacterial and fungal communities.

Bio-drying is a practical approach for treating food waste (FW). However, microbial ecological processes during treatment are essential for improving the dry efficiency, and have not been stressed enough. This study analyzed the microbial community succession and two critical periods of interdomain ecological networks (IDENs) during FW bio-drying inoculated with thermophiles (TB), to determine how TB affects FW bio-drying efficiency. The results showed that TB could rapidly colonize in the FW bio-drying, with the highest relative abundance of 5.13%. Inoculating TB increased the maximum temperature, temperature integrated index and moisture removal rate of FW bio-drying (55.7 °C, 219.5 °C, and 86.11% vs. 52.1 °C, 159.1 °C, and 56.02%), thereby accelerating the FW bio-drying efficiency by altering the succession of microbial communities. The structural equation model and IDEN analysis demonstrated that TB inoculation complicated the IDENs between bacterial and fungal communities by significantly and positively affecting bacterial communities (b = 0.39, p < 0.001) and fungal communities (b = 0.32, p < 0.01), thereby enhancing interdomain interactions between bacteria and fungi. Additionally, inoculation TB significantly increased the relative abundance of keystone taxa, including Clostridium sensu stricto, Ochrobactrum, Phenylobacterium, Microvirga and Candida. In conclusion, the inoculation of TB could effectively improve FW bio-drying, which is a promising technology for rapidly reducing FW with high moisture content and recovering resources from it.

Anti-Bacterial Adhesion on Abiotic and Biotic Surfaces of the Exopolysaccharide from the Marine Bacillus licheniformis B3-15.

The eradication of bacterial biofilm represents a crucial strategy to prevent a clinical problem associated with microbial persistent infection. In this study we evaluated the ability of the exopolysaccharide (EPS) B3-15, produced by the marine Bacillus licheniformis B3-15, to prevent the adhesion and biofilm formation of Pseudomonas aeruginosa ATCC 27853 and Staphylococcus aureus ATCC 29213 on polystyrene and polyvinyl chloride surfaces. The EPS was added at different times (0, 2, 4 and 8 h), corresponding to the initial, reversible and irreversible attachment, and after the biofilm development (24 or 48 h). The EPS (300 µg/mL) impaired the initial phase, preventing bacterial adhesion even when added after 2 h of incubation, but had no effects on mature biofilms. Without exerting any antibiotic activity, the antibiofilm mechanisms of the EPS were related to the modification of the (i) abiotic surface properties, (ii) cell-surface charges and hydrophobicity, and iii) cell-to-cell aggregation. The addition of EPS downregulated the expression of genes (lecA and pslA of P. aeruginosa and clfA of S. aureus) involved in the bacterial adhesion. Moreover, the EPS reduced the adhesion of P. aeruginosa (five logs-scale) and S. aureus (one log) on human nasal epithelial cells. The EPS could represent a promising tool for the prevention of biofilm-related infections.