RESUMEN

Thermophilic acetogens are gaining recognition as potent microbial cell factories, leveraging their unique metabolic capabilities to drive the development of sustainable biotechnological processes. These microorganisms, thriving at elevated temperatures, exhibit robust carbon fixation abilities via the linear Wood-Ljungdahl pathway to efficiently convert C1 substrates, including syngas (CO, CO2 and H2) from industrial waste gasses, into acetate and biomass via the central metabolite acetyl-CoA. This review summarizes recent advancements in metabolic engineering and synthetic biology efforts that have expanded the range of products derived from thermophilic acetogens after briefly discussing their autotrophic metabolic diversity. These discussions highlight their potential in the sustainable bioproduction of industrially relevant compounds. We further review the remaining challenges for implementing efficient and complex strain engineering strategies in thermophilic acetogens, significantly limiting their use in an industrial context.

RESUMEN

Plasmids are one of the key drivers of microbial adaptation and evolution. However, their diversity and role in adaptation, especially in extreme environments, remains largely unexplored. In this study, we aimed to identify, characterize and compare plasmid sequences originating from samples collected from deep-sea hydrothermal vents located in Arctic Mid-Ocean Ridges. To achieve this, we employed, and benchmarked three recently developed plasmid identification tools - PlasX, GeNomad and PLASMe - on metagenomic data from this unique ecosystem. To date, this is the first direct comparison of these computational methods in the context of data from extreme environments. Upon recovery of plasmid contigs, we performed a multi-approach analysis, focusing on identifying taxonomic and functional biases within datasets originating from each tool. Next, we implemented a majority voting system to identify high-confidence plasmid contigs, enhancing the reliability of our findings. By analyzing the consensus plasmid sequences, we gained insights into their diversity, ecological roles, and adaptive significance. Within the high-confidence sequences, we identified a high abundance of Pseudomonadota and Campylobacterota, as well as multiple toxin-antitoxin systems. Our findings ensure a deeper understanding of how plasmids contribute to shaping microbial communities living under extreme conditions of hydrothermal vents, potentially uncovering novel adaptive mechanisms.

RESUMEN

Thermophilic acetogenic bacteria have attracted attention as promising candidates for biotechnological applications such as syngas fermentation, microbial electrosynthesis, and methanol conversion. Here, we aimed to isolate and characterize novel thermophilic acetogens from diverse environments. Enrichment of heterotrophic and autotrophic acetogens was monitored by 16S rRNA gene-based bacterial community analysis. Seven novel Moorella strains were isolated and characterized by genomic and physiological analyses. Two Moorella humiferrea isolates showed considerable differences during autotrophic growth. The M. humiferrea LNE isolate (DSM 117358) fermented carbon monoxide (CO) to acetate, while the M. humiferrea OCP isolate (DSM 117359) transformed CO to hydrogen and carbon dioxide (H2 + CO2), employing the water-gas shift reaction. Another carboxydotrophic hydrogenogenic Moorella strain was isolated from the covering soil of an active charcoal burning pile and proposed as the type strain (ACPsT) of the novel species Moorella carbonis (DSM 116161T and CCOS 2103T). The remaining four novel strains were affiliated with Moorella thermoacetica and showed, together with the type strain DSM 2955T, the production of small amounts of ethanol from H2 + CO2 in addition to acetate. The physiological analyses of the novel Moorella strains revealed isolate-specific differences that considerably increase the knowledge base on thermophilic acetogens for future applications.

Asunto(s)

RESUMEN

We report the complete genomes of Geobacillus stearothermophilus strains EF60045 and SJEF4-2 from Korean hot springs, with 3,769 and 3,625 thermophilic genes, respectively. G. stearothermophilus EF60045 shows four methylation patterns. G. stearothermophilus SJEF4-2 harbors three plasmids. These findings enhance understanding of Geobacillus strains, aiding in their development as microbial platform hosts.

RESUMEN

Proteases hydrolyze proteins and reduce them to smaller peptides or amino acids. Besides many biological processes, proteases play a crucial in different industrial applications. A 792 bp protease gene (nprB) from the thermophilic bacterium Streptomyces thermovulgaris was cloned and expressed in E. coli BL21 using pET 50b (+). Optimal recombinant protease expression was observed at 1 mM IPTG, 37 °C for 4 h. The resulting protease was observed in soluble form. The molecular mass estimated by SDS-PAGE and Western blot analysis of the protease (NprB) fused with His and Nus tag is ~70 KDa. The protease protein was purified by Ammonium sulfate precipitation and immobilized metal ion affinity chromatography. The optimum pH and temperature for protease activity using casein as substrate were 7.2 and 70 °C, respectively. The mature protease was active and retained 80% of its activity in a broad spectrum of pH 6-8 after 4 h of incubation. Also, the half-life of the protease at 70 °C was 4 h. EDTA (5 mM) completely inhibited the enzyme, proving the isolated protease was a metalloprotease. NprB activity was enhanced in the presence of Zn2+, Mn2+, Fe2+ and Ca2+, while Hg2+ and Ni2+ decreased its activity. Exposure to organic solvents did not affect the protease activity. The recombinant protease was stable in the presence of 10% organic solvents and surfactants. Further characterization showed that zinc-metalloprotease is promising for the detergent, laundry, leather, and pharmaceutical industries.

RESUMEN

A novel facultatively anaerobic moderately thermophilic bacteria, strains 4137-MeT and 4148-MeT, were isolated from hot springs of Karmadon and Ursdon, respectively (North Ossetia, Russian Federation). Gram-negative, motile rods were present singly, in pairs, rosettes, and aggregates, or formed biofilms. Both strains grew optimally at 50-55 °C, pH 7.0 and did not require sodium chloride or yeast extract for growth. They were chemoorganoheterotrophs, growing on mono-, di- and polysaccharides (cellulose, starch, xylan, lichenan, galactan, xyloglucan, mannan, xanthan gum, guar gum) as well as proteinaceous substrates (gelatin, peptone, beef and yeast extract). Growth under anaerobic conditions was observed in presence and absence of external electron acceptors. Sulfur, thiosulfate, arsenate, Fe-citrate, and ferrihydrite were reduced with acetate, starch, or yeast extract as electron donors. The respiratory quinone was MK-7. Major cellular fatty acids of both strains were iso-C15:0, anteiso-C17:0, C15:0, iso-C16:0 and additionally iso-C17:0 for strain 4137-MeT. The size of the genome and genomic DNA G + C content of strain 4137-MeT were 3.24 Mb. and 29.9 %, respectively; for strain 4148-MeT - 3.33 Mb and 30.7 %. According to the 16S rRNA gene sequence and conserved protein sequences phylogenies, strains 4137-MeT and 4148-MeT represented a distinct lineage of the family Melioribacteraceae within the class Ignavibacteria. Based on phylogenetic analysis and phenotypic features, the novel isolates were assigned to a novel genus, for which the name Rosettibacter gen. nov. is proposed. Strain 4148-MeT represents its type species Rosettibacter primus sp. nov., while strain 4137-MeT represents a new species Rosettibacter firmus sp. nov.

Asunto(s)

RESUMEN

BACKGROUND: The genus Geobacillus and its associated taxa have been the focal point of numerous thermophilic biotechnological investigations, both at the whole cell and enzyme level. By contrast, comparatively little research has been done on its recently delineated sister genus, Parageobacillus. Here we performed pan-genomic analyses on a subset of publicly available Parageobacillus and Saccharococcus genomes to elucidate their biotechnological potential. RESULTS: Phylogenomic analysis delineated the compared taxa into two distinct genera, Parageobacillus and Saccharococcus, with P. caldoxylosilyticus isolates clustering with S. thermophilus in the latter genus. Both genera present open pan-genomes, with the species P. toebii being characterized with the highest novel gene accrual. Diversification of the two genera is driven through the variable presence of plasmids, bacteriophages and transposable elements. Both genera present a range of potentially biotechnologically relevant features, including a source of novel antimicrobials, thermostable enzymes including DNA-active enzymes, carbohydrate active enzymes, proteases, lipases and carboxylesterases. Furthermore, they present a number of metabolic pathways pertinent to degradation of complex hydrocarbons and xenobiotics and for green energy production. CONCLUSIONS: Comparative genomic analyses of Parageobacillus and Saccharococcus suggest that taxa in both of these genera can serve as a rich source of biotechnologically and industrially relevant secondary metabolites, thermostable enzymes and metabolic pathways that warrant further investigation.

Asunto(s)

RESUMEN

Bacillus methanolicus is a thermophilic methylotrophic bacterium that grows quickly on methanol in sea water-based media. It has been engineered for chemical bioproduction from methanol, but its efficiency needs improvement for industrialization. Synthetic biology approaches such as metabolic modeling and genome editing can reprogram B. methanolicus for low-carbon biomanufacturing.

RESUMEN

The wetsalts tiger beetle, Cicindelidia haemorrhagica (LeConte) (Coleoptera: Cicindelidae), is found in several active thermal hot spring areas in Yellowstone National Park (YNP) where substrate surface temperatures can exceed 50 °C. However, relationships between surface temperatures and the time adults spend on them remain poorly understood. Therefore, we characterized thermal profiles of Dragon Spring and Rabbit Creek, 2 thermally active research sites containing C. haemorrhagica in YNP, to quantify the time adults spend at different surface temperatures. We took 58 thermal video recordings of adults over 6 total days of observation ranging from 10 to 15 min for each adult. Thermal video analysis results indicated a positive relationship between the total time adult beetles spent on surface temperatures from Dragon Spring and Rabbit Creek as temperatures increased from 20 °C. Once surface temperatures exceeded 40 °C, the total time spent at those surface temperatures declined. Adults were recorded on substrates exceeding 50 °C at one of the 2 research locations. Rabbit Creek had substantially more instances of adults present with surface temperatures exceeding 40 °C, including one individual on a surface temperature of 61.5 °C. There were 3 instances of beetles spending more than 4 min at a particular surface temperature, all within the preferred range of 30-40 °C. Our thermal profile results and previous behavioral observations suggest that adults may be resistant to the heat produced from the thermal waters that influence the substrate temperatures but may not be subject to high surface temperatures as previously reported.

RESUMEN

Caldanaerobacter subterraneus subsp. keratinolyticus subsp. nov. strain KAk was isolated from a geothermal hot spring located in Kazakhstan. Growth occurred at temperatures ranging from 50 to 80 °C, with approximately 70 °C as optimum. It also thrived in pH conditions ranging from 4.0 to 9.0, with the best growth occurring at 6.8. Under optimal conditions in a glucose-containing medium, the cells were predominantly observed singly, in pairs, or less frequently in chains, and did not form endospores. However, under conditions involving growth with merino wool or feathers, or under suboptimal conditions, the cells of strain KAk exhibited a notably elongated and thinner morphology, with lengths ranging from 5 to 8 µm, and spores were observed. The KAk strain exhibited efficient degradation of feather keratin and merino wool at temperatures ranging from 65 to 70 °C. Analysis of the 16S rRNA gene sequence placed KAk within the genus Caldanaerobacter, family Thermoanaerobacteraceae, with the highest similarity to C. subterraneus subsp. tengcongensis MB4T (98.84% sequence identity). Furthermore, our analysis of the draft genome sequence indicated a genome size of 2.4 Mbp, accompanied by a G+C value of 37.6 mol%. This study elucidated the physiological and genomic characteristics of strain KAk, highlighting its keratinolytic capabilities and distinctiveness compared to other members of the genus Caldanaerobacter.

RESUMEN

The present study investigates the low temperature tolerance strategies of thermophilic bacterium Anoxybacillus rupiensis TPH1, which grows optimally at 55 °C , by subjecting it to a temperature down-shift of 10 °C (45 °C) for 4 and 6 h followed by studying its growth, morphophysiological, molecular and proteomic responses. Results suggested that although TPH1 experienced increased growth inhibition, ROS production, protein oxidation and membrane disruption after 4 h of incubation at 45 °C yet maintained its DNA integrity and cellular structure through the increased expression of DNA damage repair and cell envelop synthesizing proteins and also progressively alleviated growth inhibition by 20% within two hours i.e., 6 h, by inducing the expression of antioxidative enzymes, production of unsaturated fatty acids, capsular and released exopolysaccharides and forming biofilm along with chemotaxis proteins. Conclusively, the adaptation of Anoxybacillus rupiensis TPH1 to lower temperature is mainly mediated by the synthesis of large numbers of defense proteins and exopolysaccharide rich biofilm formation.

Asunto(s)

RESUMEN

The oxidation of sulfur compounds drives the acidification of geothermal waters. At high temperatures (>80°C) and in acidic conditions (pH <6.0), oxidation of sulfide has historically been considered an abiotic process that generates elemental sulfur (S0) that, in turn, is oxidized by thermoacidophiles of the model archaeal order Sulfolobales to generate sulfuric acid (i.e. sulfate and protons). Here, we describe five new aerobic and autotrophic strains of Sulfolobales comprising two species that were isolated from acidic hot springs in Yellowstone National Park (YNP) and that can use sulfide as an electron donor. These strains significantly accelerated the rate and extent of sulfide oxidation to sulfate relative to abiotic controls, concomitant with production of cells. Yields of sulfide-grown cultures were ∼2-fold greater than those of S0-grown cultures, consistent with thermodynamic calculations indicating more available energy in the former condition than the latter. Homologs of sulfide:quinone oxidoreductase (Sqr) were identified in nearly all Sulfolobales genomes from YNP metagenomes as well as those from other reference Sulfolobales, suggesting a widespread ability to accelerate sulfide oxidation. These observations expand the role of Sulfolobales in the oxidative sulfur cycle, the geobiological feedbacks that drive the formation of acidic hot springs, and landscape evolution.

RESUMEN

Biomass degrading thermophiles play an indispensable role in building lignocellulose-based supply chains. They operate at high temperatures to improve process efficiencies and minimize mesophilic contamination, can overcome lignocellulose recalcitrance through their native carbohydrate-active enzyme (CAZyme) inventory, and can utilize a wide range of sugar substrates. However, sugar transport in thermophiles is poorly understood and investigated, as compared to enzymatic lignocellulose deconstruction and metabolic conversion of sugars to value-added chemicals. Here, we review the general modes of sugar transport in thermophilic bacteria and archaea, covering the structural, molecular, and biophysical basis of their high-affinity sugar uptake. We also discuss recent genetic studies on sugar transporter function. With this understanding of sugar transport, we discuss strategies for how sugar transport can be engineered in thermophiles, with the potential to enhance the conversion of lignocellulosic biomass into renewable products. ONE-SENTENCE SUMMARY: Sugar transport is the understudied link between extracellular biomass deconstruction and intracellular sugar metabolism in thermophilic lignocellulose bioprocessing.

Asunto(s)

RESUMEN

Deep-sea hydrothermal vents host archaeal and bacterial thermophilic communities, including taxonomically and functionally diverse Thermoproteota. Despite their prevalence in high-temperature submarine communities, Thermoproteota are chronically under-represented in genomic databases and issues have emerged regarding their nomenclature, particularly within the Aeropyrum-Thermodiscus-Caldisphaera. To resolve some of these problems, we identified 47 metagenome-assembled genomes (MAGs) within this clade, from 20 previously published deep-sea hydrothermal vent and submarine volcano metagenomes, and 24 MAGs from public databases. Using phylogenomic analysis, Genome Taxonomy Database Toolkit (GTDB-Tk) taxonomic assessment, 16S rRNA gene phylogeny, average amino acid identity (AAI) and functional gene patterns, we re-evaluated of the taxonomy of the Aeropyrum-Thermodiscus-Caldisphaera. At least nine genus-level clades were identified with two or more MAGs. In accordance with SeqCode requirements and recommendations, we propose names for three novel genera, viz. Tiamatella incendiivivens, Hestiella acidicharens and Calypsonella navitae. A fourth genus was also identified related to Thermodiscus maritimus, for which no available sequenced genome exists. We propose the novel species Thermodiscus eudorianus to describe our high-quality Thermodiscus MAG, which represents the type genome for the genus. All three novel genera and T. eudorianus are likely anaerobic heterotrophs, capable of fermenting protein-rich carbon sources, while some Tiamatella, Calypsonella and T. eudorianus may also reduce polysulfides, thiosulfate, sulfur and/or selenite, and the likely acidophile, Hestiella, may reduce nitrate and/or perchlorate. Based on phylogenomic evidence, we also propose the family Acidilobaceae be amended to include Caldisphaera, Aeropyrum, Thermodiscus and Stetteria and the novel genera described here.

Asunto(s)

RESUMEN

Parageobacillus thermoglucosidasius is emerging as a highly promising thermophilic organism for metabolic engineering. The utilization of CRISPR-Cas technologies has facilitated programmable genetic manipulation in P. thermoglucosidasius. However, the absence of thermostable NHEJ enzymes limited the capability of the endogenous type I CRISPR-Cas system to generate a variety of extensive genomic deletions. Here, two thermophilic NHEJ enzymes were identified and combined with the endogenous type I CRISPR-Cas system to develop a genetic manipulation tool that can achieve long-range genomic deletion across various lengths. By optimizing this tool-through adjusting the expression level of NHEJ enzymes and leveraging our discovery of a negative correlation between GC content of the guide RNA (gRNA) and deletion efficacy-we streamlined a comprehensive gRNA selection manual for whole-genome editing, achieving a 100 % success rate in randomly selecting gRNAs. Notably, using just one gRNA, we achieved genomic deletions spanning diverse length, exceeding 200 kilobases. This tool will facilitate the genomic manipulation of P. thermoglucosidasius for both fundamental research and applied engineering studies, further unlocking its potential as a thermophilic cell factory.

RESUMEN

Prebiotics are non-digestible compounds that promote intestinal microbiota growth and/or activity. Xylooligosaccharides (XOS) are new prebiotics derived from the hemicellulose fraction of lignocellulosic materials. Challenges in using those materials as sources for prebiotic compounds lie in the hemicellulose extraction efficiency and the safety of those ingredients. In this sense, this work aims to optimize hemicellulose extraction and XOS production through direct enzymatic hydrolysis of alkali pre-treated wheat straw without undesired byproducts. By increasing the temperature of the enzymatic step from 40 °C to 65 °C we achieved an improvement in the extraction yield from 55 % to 80 %. Products with different degrees of polymerization were also noticed: while XOS ≤ X6 where the main products at 40 °C, a mixture of long arabinoxylan derived polymers (ADPo) and XOS ≤ X6 was obtained at 65 °C, irrespective of the extraction yield. Thus, a modulatory effect of temperature on the product profile is suggested here. Among the XOS ≤ X6 produced, X2-X3 were the main products, and X4 was the minor one. At the end of the hydrolysis, 146.7 mg XOS per gram of pre-treated wheat straw were obtained.

Asunto(s)

RESUMEN

BACKGROUND: Hot spring biofilms provide a window into the survival strategies of microbial communities in extreme environments and offer potential for biotechnological applications. This study focused on green and brown biofilms thriving on submerged plant litter within the Sungai Klah hot spring in Malaysia, characterised by temperatures of 58-74 °C. Using Illumina shotgun metagenomics and Nanopore ligation sequencing, we investigated the microbial diversity and functional potential of metagenome-assembled genomes (MAGs) with specific focus on biofilm formation, heat stress response, and carbohydrate catabolism. RESULTS: Leveraging the power of both Illumina short-reads and Nanopore long-reads, we employed an Illumina-Nanopore hybrid assembly approach to construct MAGs with enhanced quality. The dereplication process, facilitated by the dRep tool, validated the efficiency of the hybrid assembly, yielding MAGs that reflected the intricate microbial diversity of these extreme ecosystems. The comprehensive analysis of these MAGs uncovered intriguing insights into the survival strategies of thermophilic taxa in the hot spring biofilms. Moreover, we examined the plant litter degradation potential within the biofilms, shedding light on the participation of diverse microbial taxa in the breakdown of starch, cellulose, and hemicellulose. We highlight that Chloroflexota and Armatimonadota MAGs exhibited a wide array of glycosyl hydrolases targeting various carbohydrate substrates, underscoring their metabolic versatility in utilisation of carbohydrates at elevated temperatures. CONCLUSIONS: This study advances understanding of microbial ecology on plant litter under elevated temperature by revealing the functional adaptation of MAGs from hot spring biofilms. In addition, our findings highlight potential for biotechnology application through identification of thermophilic lignocellulose-degrading enzymes. By demonstrating the efficiency of hybrid assembly utilising Illumina-Nanopore reads, we highlight the value of combining multiple sequencing methods for a more thorough exploration of complex microbial communities.

RESUMEN

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.

Asunto(s)

RESUMEN

The relentless increase in atmospheric greenhouse gas concentrations as a consequence of the exploitation of fossil resources compels the adoption of sustainable routes to chemical and fuel manufacture based on biological fermentation processes. The use of thermophilic chassis in such processes is an attractive proposition; however, their effective exploitation will require improved genome editing tools. In the case of the industrially relevant chassis Parageobacillus thermoglucosidasius, CRISPR/Cas9-based gene editing has been demonstrated. The constitutive promoter used, however, accentuates the deleterious nature of Cas9, causing decreased transformation and low editing efficiencies, together with an increased likelihood of off-target effects or alternative mutations. Here, we rectify this issue by controlling the expression of Cas9 through the use of a synthetic riboswitch that is dependent on the nonmetabolized, nontoxic, and cheap inducer, theophylline. We demonstrate that the riboswitches are dose-dependent, allowing for controlled expression of the target gene. Through their use, we were then able to address the deleterious nature of Cas9 and produce an inducible system, RiboCas93. The benefits of RiboCas93 were demonstrated by increased transformation efficiency of the editing vectors, improved efficiency in mutant generation (100%), and a reduction of Cas9 toxicity, as indicated by a reduction in the number of single nucleotide polymorphisms (SNPs) observed. This new system provides a quick and efficient way to produce mutants in P. thermoglucosidasius.

Asunto(s)

RESUMEN

Over the past decade, environmental metagenomics and polymerase chain reaction-based marker gene surveys have revealed that several lineages beyond just a few well-established groups within the Euryarchaeota superphylum harbor the genetic potential for methanogenesis. One of these groups are the Archaeoglobi, a class of thermophilic Euryarchaeota that have long been considered to live non-methanogenic lifestyles. Here, we enriched Candidatus Methanoglobus hypatiae, a methanogen affiliated with the family Archaeoglobaceae, from a hot spring in Yellowstone National Park. The enrichment is sediment-free, grows at 64-70°C and a pH of 7.8, and produces methane from mono-, di-, and tri-methylamine. Ca. M. hypatiae is represented by a 1.62 Mb metagenome-assembled genome with an estimated completeness of 100% and accounts for up to 67% of cells in the culture according to fluorescence in situ hybridization. Via genome-resolved metatranscriptomics and stable isotope tracing, we demonstrate that Ca. M. hypatiae expresses methylotrophic methanogenesis and energy-conserving pathways for reducing monomethylamine to methane. The detection of Archaeoglobi populations related to Ca. M. hypatiae in 36 geochemically diverse geothermal sites within Yellowstone National Park, as revealed through the examination of previously published gene amplicon datasets, implies a previously underestimated contribution to anaerobic carbon cycling in extreme ecosystems.

Asunto(s)