Extremophiles最新文献

Extracellular proteases produced by haloarchaea, termed halolysins, possess potential applications in diverse fields including food fermentation and bio-remediation. In this study, an extracellular protease encoding gene, hly32^PRR32, from Halostella sp. PRR32 isolated from a salt mine in Anhui, China, was identified and expressed in Escherichia coli. The expressed protein MBP-Hly32 was purified and biochemically characterized. The results indicate that Hly32 belongs to the S8 family of serine proteases (halolysin). A BLAST search on NCBI reveals that Hly32 has an amino acid sequence identity of 68.87% with serine protease Hly176B from Haloarchaeobius sp. FL176. MBP-Hly32 contains a catalytic triad of Asp¹⁵⁹-His¹⁹⁸-Ser³⁵⁰ and two C-terminal extensions which are crucial for its activity. The optimal conditions for its enzyme activity are 50 °C, pH 8.0, and 4.0 M NaCl. Under these conditions, the K_m, V_max and K_cat for the MBP-Hly32 were determined to be 2.34 mM, 935.50 U·mg^-1 and 1472.40 s^-1, respectively. Metal ions and organic reagents affect its activity differently from the typical halolysins; for example, Ca²⁺, which enhances the activity of other halolysin enzymes, has no effect on MBP-Hly32. Furthermore, the activity of Hly32 was inhibited by the presence of PMSF, DTT, and EDTA. Furthermore, a three-dimensional structure prediction based on functional domains was obtained in this study which will facilitate modification and protein engineering halolysins to generate mutants with new physiological activities.

Four lakes in the same region of Aotearoa New Zealand were investigated to characterize sediment microbial communities and functions under contrasting environmental conditions. Two lakes, an acidic lake (Rototai) and a lake with elevated metals and nutrients (Killarney) were impacted by extreme stressors, while the lowland mesotrophic lake (Kaihoka East) and an alpine lake (Peel) were used as reference lakes. Using metabarcoding and metagenomics analysis, we profiled community composition, functional pathways, and resistance mechanisms in the lake sediments. Rototai contained high abundances of genes involved in sulfur cycling (assimilatory and dissimilatory sulfate reduction, sulfur oxidation) and acid tolerance (kdp potassium-transport system, ClcA antiporters). In contrast, Killarney had elevated abundances of genes involved in methanogenesis, however despite high metal concentrations, no enrichment of metal-resistance genes was detected. Kaihoka East contained the highest prokaryotic diversity and an elevated abundance of genes involved in nitrification. Although community taxonomic differences were modest across lakes, functional analyses revealed distinct metabolic adaptations. These findings highlight the utility of using metagenomic approaches to identify biogeochemical processes and stress-response strategies in lakes. Improved understanding of microbial functional diversity in surface sediments has implications for lake management, particularly in systems impacted by acidification, high nutrient loading, and metal contamination.

The identification and characterization of promoters and regulatory elements are commonly assessed using reporter genes. However, very few of these are available for haloarchaea. In this study, we describe the construction and validation of a reporter system for the haloarchaeon Haloferax volcanii based on a modified and stable version of the carotenoid biosynthesis enzyme phytoene synthase (PSY). This tool enables the analysis of a target gene expression through direct visualization of cell pigmentation and/or the extraction and quantification of carotenoid content. The modified crtB gene encoding PSY was cloned into the pTA963 vector under two regulatable promoters previously characterized in H. volcanii: PtnaA and Pxyl, inducible with tryptophan and xylose, respectively. The construct was introduced into and expressed in a non-pigmented H. volcanii strain (ΔcrtB) under varying inducer concentrations. For both promoters, a clear increase in pigmentation was visually observed in cultures and cell pellets with increasing inducer levels. These observations were corroborated by carotenoid extraction and quantification, as well as by Western blot analysis of PSY protein levels. This work presents a robust, easy-to-use, and versatile reporter system for investigating gene expression in H. volcanii, expanding the toolkit for genetic studies in haloarchaea.

Halophilic bacteria are recognized as a promising source of novel enzymes and biopolymers with various applications in biotechnology and the industry. In comparison with their mesophilic analogues, halophilic metabolites are stable under extreme conditions typically encountered in the industrial processes. In this study, the biotechnological potential of twenty strains of halophilic bacteria isolated from the Provadia salt deposit, Bulgaria was investigated for the first time. The strains were identified based on the sequencing of the 16S rRNA gene and were assigned to 13 different species falling in the Bacillota and Pseudomonadota phyla. The majority (90%) of them showed single or combined hydrolytic enzyme activity. Half of the strains (55%) were able to produce between three and eight extracellular hydrolytic enzymes-arabinase, cellulase, gelatinase, glucanase, L-glutaminase, pectinase, and xylanase. Ten strains were able to synthesise exopolysaccharides (EPS) in concentration between 32 and 227 μg/ml. The optimal EPS production kinetics (1.6 ± 0.15 g/l) by Virgibacillus halodenitrificans PSZ-34 was systematically investigated for the first time. Three strains also exhibited antimicrobial activity. The present study involved culture-dependant isolation of halophilic bacteria from the Provadia salt deposit and shed more light on their capability to synthesise hydrolytic enzymes and EPS with potential industrial exploitation.

Traditional straw return relies on microbial degradation, but cold winters delay it, risking crops. Therefore, a microbial community adapted to rice straw degradation in cold regions was constructed by restrictive consecutively sub-cultured under low-temperature limitations. The capabilities of the microbial community, such as adaptability, stability, and degradation power, were evaluated by analyzing straw degradation features, Characterization experiments and lignocellulose enzyme activities across multiple generations. 16S amplicon sequencing was used to monitor the changes in its structure over generations. Metagenomic sequencing uses CAZy and KEGG to classify gene functions. The results showed that the highest degradation efficiencies and enzyme activities were observed in the E and F generations, dominated by Proteobacteria, Bacteroidetes, and Fungi The stable microbial community was designated as LJ-7. Metagenomic analysis showed that functional genes of LJ-7 were mainly concentrated in glycoside hydrolase (GHs) and glycosyl transferase (GTs) related genes which contained many fiber and lignin-degrading enzyme genes. It is speculated that microbial enzymes degrade straw by breaking down its complex structure into monosaccharides or metabolizing quinone compounds for energy. This experiment successfully screened a microbial community capable of degrading rice straw at low temperatures, thus offering novel research insights and pathways for straw degradation in cold conditions.

Acidianus ambivalens is a metabolically flexible facultative anaerobe that can oxidize and reduce elemental sulfur with O₂ and H₂, respectively. In this work, the growth energetics of Acidianus ambivalens were determined under anaerobic conditions at 76 °C with H₂ oxidation by elemental sulfur serving as the energy-yielding catabolic reaction. The biomass yields (C-mol of biomass per mol of H₂ consumed) ranged from approximately 0.004 to 0.01. Growth rates ranged from 0.003 to 0.012 h^-1. Gibbs energies of incubation based on macrochemical equations of cell growth ranged from - 881 to - 3349 kJ/C-mol. Enthalpies of incubation determined from calorimetric measurements ranged from - 610 to - 4090 kJ/C-mol. The Gibbs energy consumed by anaerobic cultures was compared to sulfur-oxidizing cultures under aerobic and microaerobic conditions to determine the effects of environmental and substrate redox state on energetics. This comparison revealed that aerobic cultures were inefficient relative to microaerobic and anaerobic conditions. These results suggest that aerobic conditions induce a measurable oxidative stress on cultures. The similarities in growth efficiency and energy budgets under microaerobic and anaerobic conditions may allow Acidianus ambivalens to be competitive in natural environments either by oxidizing or reducing elemental sulfur.

Halophilic bacteria thrive in high-salt environments through structural modifications in their proteins. One such adaptation is seen in the DNA polymerase III beta subunit, which acts as a "sliding clamp" for the DNA polymerase III, the replication machinery's key enzyme. Like other halophilic proteins, DNA Pol III beta of Salinibacter ruber displays an increased concentration of acidic amino acids, intrinsically disordered regions, and a negatively charged surface. A detailed study using interaction and molecular dynamics simulations analyses unveils the structural intricacies of S. ruber's beta subunit dimer at the molecular level. The elliptical shape of the dimer facilitates its efficient loading onto DNA in salty environments. Modifications in the dimer enable the DNA loading-unloading mechanism by stabilizing the dimeric conformation at high salt concentrations. Additionally, the study sheds light on the molecular-level conformational changes in the interfaces of the Pol III beta subunit.

Exiguobacterium sp. S17, a polyextremophile isolated from a High-Altitude Andean Lake, exhibits a multi-resistance profile against toxic arsenic concentrations, high UV radiation, and elevated salinity. Here, we characterize the mechanisms underlying the UV resistance of Exiguobacterium sp. S17 (UV-resistome) through comparative genomics within the Exiguobacterium genus and describe morphological and ultrastructural changes using Scanning (SEM) and Transmission (TEM) Electron Microscopy.UV-resistome in Exiguobacterium species ranges from 112 to 132 genes. While we anticipated Exiguobacterium sp. S17 to lead the non-HAAL UV-resistome, it ranked eleventh with 113 genes. This larger UV-resistome in Exiguobacterium spp. aligns with their known adaptation to extreme environments. With SEM/TEM analyses we observed the formation of nanotubes (NTs), a novel finding in Exiguobacterium spp., which increased with higher UV-B doses. These NTs, confirmed to be membranous structures through sensitivity studies and imaging, suggest a role in cellular communication and environmental sensing. Genomic evidence supports the presence of essential NT biogenesis genes in Exiguobacterium sp. S17, further elucidating its adaptive capabilities.Our study highlights the complex interplay of genetic and phenotypic adaptations enabling Exiguobacterium sp. S17 to thrive in extreme UV environments. The novel discovery of NTs under UV stress presents a new avenue for understanding bacterial survival strategies in harsh conditions.

DNA methylation modification regulates gene expression during temperature stress. The adaptation mechanisms of cold-adapted microorganisms to low temperatures have been explained at the gene and metabolic levels. However, considering the important epigenetic modification in cells, the role of genomic modification in cold-adapted microorganisms remains underexplored. This study aims to discuss the regulatory role of DNA methylation in the cold response of psychrotroph Exiguobacterium undae TRM 85608. Methylome analysis shows that the methylation level of most genes in the bacterium decreases under cold stress. Combined with transcriptome results, the expression of important cold-response genes such as ABC transporter permease and ATP-binding proteins increases, but their methylation levels decrease, which is associated with a reduction of DNA adenine methyltransferase. We believe that the reduction in genomic methylation modification caused by low temperature is a major factor in stabilizing the normal growth of the cell. The bacterium counteracts cold stress by reducing the expression of methylation modification enzymes and weakening the inhibition of cold-response gene modification. These findings provide new insights into how psychrophilic organisms adapt to low temperatures.