Search Results (43)

Manipulating the mitochondrial genome remains a significant challenge in genetic engineering, primarily due to the mitochondrial double-membrane structure. While recent advances have expanded the genetic toolkit for nuclear and cytoplasmic targets, precise editing of mitochondrial DNA (mtDNA) has remained elusive. Here we report the first successful mitochondrial import of a catalytically active RNA-guided prokaryotic Argonaute protein from the mesophilic bacterium Alteromonas macleodii (AmAgo). By guiding AmAgo to the single-stranded D- or R-loop region of mtDNA using synthetic RNA guides, we observed a nearly threefold reduction in mtDNA copy number in human cell lines. This proof of concept study demonstrates that a bacterial Argonaute can remain active within the mitochondrial environment and influence mtDNA levels. These findings establish a foundational framework for further development of programmable systems for mitochondrial genome manipulation. Full article

Argonaute (Ago) proteins are ubiquitous across all domains of life. Some prokaryotic Agos (pAgos) function as endonucleases that utilize short nucleic acid guides to recognize and cleave complementary targets. Yet, considerable diversity within pAgos leaves many of their biochemical and functional features insufficiently understood. This study characterizes CalAgo, an pAgo from the mesophilic bacterium Cohnella algarum, which demonstrates DNA-guided DNA endonuclease and RNA endonuclease activities at physiological temperatures. CalAgo’s cleavage activity depends on Mn²⁺ and Mg²⁺ ions and remains effective across a wide range of temperatures and pH levels. CalAgo utilizes only short guides ranging from 15 to 21 nucleotides (nt) in length, in contrast to other reported pAgos that target both DNA and RNA, which often exhibit broad guide selectivity. CalAgo preferentially loads 5′-phosphorylated guides and shows no significant preference among guides with different 5′-end nucleotides. CalAgo is sensitive to guide–target mismatches, and introducing a single mismatch at positions 12 or 15 of the guide strand abolished detectable activity. Structural modeling suggests that this unique guide specificity may originate from structural features in its PAZ domain involved in 3′-guide binding. In summary, this study deepens insight into mesophilic pAgos and supports their potential utility in nucleic acid-based applications. Full article

Strains of Aeromonas salmonicida subsp. salmonicida, a major pathogen of salmonids, typically do not grow at temperatures above 30 °C. The effects of thermal stress on this bacterium have been extensively studied. Recently, we demonstrated that repeated exposure to cyclical thermal stress, reaching up to 37 °C, can induce permanent thermoadaptation in certain strains of this bacterium. Many aspects of this adaptation process remain poorly understood. We generated 88 thermoadapted strains of A. salmonicida subsp. salmonicida capable of sustained growth at 33 °C or higher demonstrating that prolonged heat exposure can shift a substantial proportion of psychrophilic strains toward a more mesophilic-like behavior. Although growth at 35 °C was still weaker than in naturally mesophilic A. salmonicida strains, some thermoadapted strains were able to grow up to 37 °C. North American strains harboring the genomic island AsaGEI1a, a known biomarker, exhibited a significantly reduced capacity for thermoadaptation, suggesting a possible genetic constraint, although genomic analyses indicate that AsaGEI1a itself is not directly responsible for this limitation. Genotyping and phenotypic analyses revealed that thermoadaptation is frequently associated with the loss of Type III secretion system and the A-layer, two key virulence factors. Only 7% of the thermoadapted strains retained both features. Overall, our findings suggest that thermoadaptation may represent a potential mechanism influencing the persistence of some psychrophilic A. salmonicida subsp. salmonicida strains in warming aquatic environments under climate change. Full article

Trimethylamine (TMA) is a common malodorous pollutant known for its detrimental effects on both the natural environment and human health. In this study, strain PM-1 was successfully isolated from activated sludge in a sewage treatment plant and identified as the first Microbacterium lacticum capable of degrading TMA. Strain PM-1 is characterized as a mesophilic and mild halotolerant bacterium, thriving within a temperature range of 20–40 °C and a salinity range of 10–80 g/L NaCl. The optimal initial TMA concentrations for strain PM-1 were determined to be 0.1 wt% under aerobic conditions and 0.05 wt% under anaerobic conditions. The strain demonstrated efficient TMA degradation rates of 98.02 mg/L/h aerobically and 4.44 mg/L/h anaerobically. Additionally, beef extract and peptone significantly enhanced TMA degradation and bacterial growth by 293% and 688%, respectively, under aerobic conditions. Microbacterium lacticum strain PM-1 is the first isolated Microbacterium lacticum with the ability to convert TMA. Further research will focus on its TMA degradation pathway through the identification of key enzymes and application in TMA-containing wastewater and exhaust gas. Full article

Thermostable cellulases and xylanases have broad acceptance in food, feed, paper and pulp, and bioconversion of lignocellulosics. Thermophilic fungi serve as an excellent source of thermostable enzymes. This study characterized four endo-β-1,4-glucanases (two glycoside hydrolase (GH) family 5 and two GH7 members) and four endo-β-1,4-xylanases (two GH10 and two GH11 members) from thermophilic fungus Thielavia terrestris, along with one GH10 endo-β-1,4-xylanase each from thermophilic fungus Chaetomium thermophilum and mesophilic fungus Chaetomium globosum. Comparative analysis was conducted against three previously reported GH10 endoxylanases: two thermostable enzymes from the thermophilic fungus Humicola insolens and thermophilic bacterium Halalkalibacterium halodurans, and one mesophilic enzyme from model fungus Neurospora crassa. The GH10 xylanase TtXyn10C (Thite_2118148; UniProt G2R8T7) from T. terrestris demonstrated high thermostability and activity, with an optimal temperature of 80–85 °C. It retained over 60% of its activity after 2 h at 70 °C, maintained approximately 30% activity after 15 min at 80 °C, and showed nearly complete stability following 1 min of exposure to 95 °C. TtXyn10C exhibited specific activity toward beechwood xylan (1130 ± 15 U/mg) that exceeded xylanases from H. insolens and H. halodurans while being comparable to N. crassa xylanase activity. Furthermore, TtXyn10C maintained stability across a pH range of 3–9 and resisted trypsin digestion, indicating its broad applicability. The study expands understanding of enzymes from thermophilic fungi. The discovery of the TtXyn10C offers a new model for investigating the high activity-stability trade-off and structure-activity relationships critical for industrial enzymes. Full article

Mesophilic microbial sources of prokaryotic Argonaute (pAgo) programmable nucleases have garnered considerable attention for their potential applications in genome editing and molecular diagnostics. In this study, we characterized a novel pAgo from the mesophilic bacterium Chroococcidiopsis sp. (ChAgo), which can cleave single-stranded DNA (ssDNA) using both 5′-phosphorylated guide DNA (5′P-gDNA) and 5′-hydroxylated guide DNA (5′OH-gDNA). Efficient cleavage occurs using 14–25 nt 5′P-gDNA and 13–20 nt 5′OH-gDNA in the presence of Mn²⁺ ions at temperatures ranging from 25 to 75 °C, with optimal activity at 55 °C. ChAgo demonstrates low tolerance for single-base mismatches, similar to other pAgo proteins. The cleavage efficiency varies based on the guide/target pair, with mismatches at specific positions significantly reducing activity. For instance, mismatches at positions 4, 5, or 12 in T-gDNA/target pairs and at positions 5 or 8–10 in g38NT-gDNA/target pairs notably decrease efficiency. ChAgo’s sensitivity to mismatches makes it a promising tool for nucleic acid manipulation and detection, requiring initial screening for high cleavage efficiency sites and subsequent identification of mismatch positions. Full article

The genome of the mildly thermophilic hot spring purple sulfur bacterium, Allochromatium (Alc.) tepidum, contains a multigene pufBA family that encodes a series of α- and β-polypeptides, collectively forming a heterogeneous light-harvesting 1 (LH1) complex. The Alc. tepidum LH1, therefore, offers a unique model for studying an intermediate phenotype between phototrophic thermophilic and mesophilic bacteria, particularly regarding their LH1 Qy transition and moderately enhanced thermal stability. Of the 16 α-polypeptides in the Alc. tepidum LH1, six α1 bind Ca²⁺ to connect with β1- or β3-polypeptides in specific Ca²⁺-binding sites. Here, we use the purple bacterium Rhodospirillum rubrum strain H2 as a host to express Ca²⁺-bound and Ca²⁺-free Alc. tepidum LH1-only complexes composed of α- and β-polypeptides that either contain or lack the calcium-binding motif WxxDxI; purified preparations of each complex were then used to test how Ca²⁺ affects their thermostability and spectral features. The cryo-EM structures of both complexes were closed circular rings consisting of 14 αβ-polypeptides. The Q_y absorption maximum of Ca²⁺-bound LH1 (α1/β1 and α1/β3) was at 894 nm, while that of Ca²⁺-free (α2/β1) was at 888 nm, indicating that Ca²⁺ imparts a Q_y transition of 6 nm. Crucially for the ecological success of Alc. tepidum, Ca²⁺-bound LH1 complexes were more thermostable than Ca²⁺-free complexes, indicating that calcium plays at least two major roles in photosynthesis by Alc. tepidum—improving photocomplex stability and modifying its spectrum. Full article

Transfer RNA (tRNA) modifications are essential for the temperature adaptation of thermophilic and psychrophilic organisms as they control the rigidity and flexibility of transcripts. To further understand how specific tRNA modifications are adjusted to maintain functionality in response to temperature fluctuations, we investigated whether tRNA modifications represent an adaptation of bacteria to different growth temperatures (minimal, optimal, and maximal), focusing on closely related psychrophilic (P. halocryophilus and E. sibiricum), mesophilic (B. subtilis), and thermophilic (G. stearothermophilus) Bacillales. Utilizing an RNA sequencing approach combined with chemical pre-treatment of tRNA samples, we systematically profiled dihydrouridine (D), 4-thiouridine (s⁴U), 7-methyl-guanosine (m⁷G), and pseudouridine (Ψ) modifications at single-nucleotide resolution. Despite their close relationship, each bacterium exhibited a unique tRNA modification profile. Our findings revealed increased tRNA modifications in the thermophilic bacterium at its optimal growth temperature, particularly showing elevated levels of s⁴U8 and Ψ55 modifications compared to non-thermophilic bacteria, indicating a temperature-dependent regulation that may contribute to thermotolerance. Furthermore, we observed higher levels of D modifications in psychrophilic and mesophilic bacteria, indicating an adaptive strategy for cold environments by enhancing local flexibility in tRNAs. Our method demonstrated high effectiveness in identifying tRNA modifications compared to an established tool, highlighting its potential for precise tRNA profiling studies. Full article

Recently, prokaryotic laccases from lactic acid bacteria (LAB), which can degrade biogenic amines, were discovered. A laccase enzyme has been cloned from Oenococcus oeni, a very important LAB in winemaking, and it has been expressed in Escherichia coli. This enzyme has similar characteristics to those previously isolated from LAB as the ability to oxidize canonical substrates such as 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 2,6-dimethoxyphenol (2,6-DMP), and potassium ferrocyanide K₄[Fe(CN₆)], and non-conventional substrates as biogenic amines. However, it presents some distinctiveness, the most characteristic being its psychrophilic behaviour, not seen before among these enzymes. Psychrophilic enzymes capable of efficient catalysis at low temperatures are of great interest due to their potential applications in various biotechnological processes. In this study, we report the discovery and characterization of a new psychrophilic laccase, a multicopper oxidase (MCO), from the bacterium Oenococcus oeni. The psychrophilic laccase gene, designated as LcOe 229, was identified through the genomic analysis of O. oeni, a Gram-positive bacterium commonly found in wine fermentation. The gene was successfully cloned and heterologously expressed in Escherichia coli, and the recombinant enzyme was purified to homogeneity. Biochemical characterization of the psychrophilic laccase revealed its optimal activity at low temperatures, with a peak at 10 °C. To our knowledge, this is the lowest optimum temperature described so far for laccases. Furthermore, the psychrophilic laccase demonstrated remarkable stability and activity at low pH (optimum pH 2.5 for ABTS), suggesting its potential for diverse biotechnological applications. The kinetic properties of LcOe 229 were determined, revealing a high catalytic efficiency (kcat/Km) for several substrates at low temperatures. This exceptional cold adaptation of LcOe 229 indicates its potential as a biocatalyst in cold environments or applications requiring low-temperature processes. The crystal structure of the psychrophilic laccase was determined using X-ray crystallography demonstrating structural features similar to other LAB laccases, such as an extended N-terminal and an extended C-terminal end, with the latter containing a disulphide bond. Also, the structure shows two Met residues at the entrance of the T1Cu site, common in LAB laccases, which we suggest could be involved in substrate binding, thus expanding the substrate-binding pocket for laccases. A structural comparison of LcOe 229 with Antarctic laccases has not revealed specific features assigned to cold-active laccases versus mesophilic. Thus, further investigation of this psychrophilic laccase and its engineering could lead to enhanced cold-active enzymes with improved properties for future biotechnological applications. Overall, the discovery of this novel psychrophilic laccase from O. oeni expands our understanding of cold-adapted enzymes and presents new opportunities for their industrial applications in cold environments. Full article

A novel aerotolerant anaerobic bacterium (strain M4Ah^T) was isolated from a terrestrial mud volcano (Taman Peninsula, Russia). Cells were small, cell-wall-less, non-motile cocci, 0.32–0.65 μm in diameter. The isolate was a mesophilic, neutrophilic chemoorganoheterotroph, growing on carbohydrates (D-glucose, D-trehalose, D-ribose, D-mannose, D-xylose, D-maltose, D-lactose, D-cellobiose, D-galactose, D-fructose, and D-sucrose), proteinaceous compounds (yeast extract, tryptone), and pyruvate. Strain M4Ah^T tolerated 2% oxygen in the gas phase, was catalase-positive, and showed sustainable growth under microaerobic conditions. The dominant cellular fatty acids of strain M4Ah^T were C_16:0 and C_18:0. The G+C content of the genomic DNA was 32.42%. The closest phylogenetic relative of strain M4Ah^T was Mariniplasma anaerobium from the family Acholeplasmataceae (order Acholeplasmatales, class Mollicutes). Based on the polyphasic characterization of the isolate, strain M4Ah^T is considered to represent a novel species of a new genus, for which the name Peloplasma aerotolerans gen. nov., sp. nov. is proposed. The type strain of Peloplasma aerotolerans is M4Ah^T (=DSM 112561^T = VKM B-3485^T = UQM 41475^T). This is the first representative of the order Acholeplasmatales, isolated from a mud volcano. Full article

Laccases are industrially relevant enzymes that have gained great biotechnological importance. To date, most are of fungal and mesophilic origin; however, enzymes from extremophiles possess an even greater potential to withstand industrial conditions. In this study, we evaluate the potential of a recombinant spore-coat laccase from the thermoalkaliphilic bacterium Bacillus sp. FNT (FNTL) to biodegrade antibiotics from the tetracycline, β-lactams, and fluoroquinolone families. This extremozyme was previously characterized as being thermostable and highly active in a wide range of temperatures (20–90 °C) and very versatile towards several structurally different substrates, including recalcitrant environmental pollutants such as PAHs and synthetic dyes. First, molecular docking analyses were employed for initial ligand affinity screening in the modeled active site of FNTL. Then, the in silico findings were experimentally tested with four highly consumed antibiotics, representatives of each family: tetracycline, oxytetracycline, amoxicillin, and ciprofloxacin. HPLC results indicate that FNTL with help of the natural redox mediator acetosyringone, can efficiently biodegrade 91, 90, and 82% of tetracycline (0.5 mg mL⁻¹) in 24 h at 40, 30, and 20 °C, respectively, with no apparent ecotoxicity of the products on E. coli and B. subtilis. These results complement our previous studies, highlighting the potential of this extremozyme for application in wastewater bioremediation. Full article

Some of the most extensively studied marine or estuarine bacteria belong to the genus Vibrio, with Vibrio cholerae being the most notorious species as it is the cause of cholera in humans. V. cholerae is found in tropical and temperate areas and can be classified as a mesophilic bacterium with its growth optimum at around 37 °C. One of the important factors in the activity and stability of each enzyme is its physiological environment. A previous study on the secreted mesophilic enzyme Endonuclease I from the Vibrio cholerae genus (VcEndA) showed that its activity was strongly dependent not only on temperature but also on NaCl concentration. Here, we report a structural study on the mesophilic enzyme (VcEndA) using molecular dynamics simulations at different salt concentrations (NaCl). The analysis of molecular dynamic simulation trajectories reveals that the enzyme is not tolerant and not sensitive to salt since the profile of the rmsf as a function of different concentrations does not show a large difference in the mobility of the enzyme for high values of the NaCl concentration (450 and 650 mM). However, the most flexible regions of the enzyme are recorded under the concentration of 175 mM, which coincides well with the previous experimental work. Full article

The composition and physicochemical characteristics of short-aged Pecorino Sardo PDO (Protected Designation of Origin) cheese makes it permissive to Listeria monocytogenes growth. The PDO product specification stipulates that this cheese is produced with whole sheep’s milk inoculated with cultures from the area of origin. Therefore, the use of bioprotective cultures for the inhibition of pathogens in PDO cheeses is allowed only if autochthonous microorganisms are used. Furthermore, bioprotective cultures are generally used on the cheese surface to prevent the outgrowth of L. monocytogenes, the application of which can be time-consuming and require specialist technical knowledge. In this study, we examine the direct addition of bioprotective cultures to the cheese vat and compare the activity of a commercial bioprotective culture (Lactiplantibacillus plantarum) and an autochthonous lactic acid bacterium with bioprotective properties (Lactobacillus delbruekii sups. sunkii), for the inhibition of L. monocytogenes in Pecorino Sardo PDO cheese. Three types of Pecorino Sardo PDO cheese were made with bioprotective cultures added directly to the cheese milk along with the starter inoculum: PSA, with the commercial bioprotective culture; PSB, with the autochthonous bioprotective culture; and a CTRL cheese with no bioprotective culture. A challenge test was performed on each of these cheeses by artificially contaminating the cheese surface with L. monocytogenes (2 Log10 CFU/g). Three batches of each cheese type were analyzed to enumerate mesophilic and thermophilic lactic acid bacteria and to investigate the growth potential of L. monocytogenes during manufacturing, at the end of ripening, at the end of shelf-life, and after 180 days from cheese production. Both bioprotective cultures tested in this study showed inhibitory action against the pathogen with 0.3–1.8 Log10 CFU/g (colony-forming unit per gram) reduction levels. The autochthonous organism, L. sunkii, was as effective as the commercially supplied culture, and the addition of the bioprotective cultures to the cheese-making procedure offered protection against L. monocytogenes. The direct addition of bioprotective cultures to the making procedure of Pecorino Sardo PDO cheese is a potentially innovative strategy to improve the safety of this product. Full article

Fine-aroma cocoa (Theobroma cacao) is one of Ecuador’s most iconic export products and comprises 63% of world production. Nevertheless, few advances have been made to improve fermentation processes that might benefit the development of chocolate’s organoleptic characteristics. The study of starter cultures, which seek to improve organoleptic properties or decrease fermentation time, has been investigated in other countries. The aim of this study was to analyze the effect of a native microbial cocktail based on two yeasts (Torulaspora delbrueckii and Hanseniaspora uvarum), a lactic acid bacterium (LAB) (Limosilactobacillus plantarum), and an acetic acid bacterium (AAB) (Acetobacter ghanensis) inoculated at the beginning of the fermentative process while tracking physical and biochemical variables, microbial population dynamics, and bean fermentation time. The starter culture caused changes in sugar and acid content and increased polyphenols, which in turn generated temperature and pH changes in the dough. The dynamics of yeast, AAB, and mesophilic microorganisms remain higher than the controls throughout the process. A decrease in filamentous fungi that affect the flavor and quality of beans was observed due to the production of acetic acid or secondary metabolites from yeasts and LAB, and resulted in 24% greater fermentation than spontaneous fermentation in only 96 h. Full article

Natural antimicrobials are effective against both food-borne pathogens and spoilage bacteria. The current study aimed to evaluate the in vitro antimicrobial activity of eight natural bioactive compounds (BACs) and one synthetic compound, butylated hydroxytoluene (BHT), and to evaluate the effect of one selected BAC (AITC) on the safety and quality of minced chicken meat. Additionally, physicochemical (pH, color, water-holding capacity (WHC), chemical forms of myoglobin (deoxymyoglobin-DeoMb, oxymyoglobin-OxyMb, and metmyoglobin-MetMb), thiobarbituric-acid-reactive substances (TBARs)), and microbiological properties (in vitro antimicrobial activity and determination of minimum inhibitory concentration (MIC)) were also evaluated through electronic-nose odor detection. Allyl-isothiocyanate (AITC), thymol, eugenol, and geraniol showed the broadest spectrum of in vitro antibacterial activity against one major meat spoilage bacterium and five pathogenic bacteria that were tested. Subsequently, AITC was selected to be applied to fresh minced chicken meat at different concentrations (at concentrations of the MIC (MIC-1), two times the MIC (MIC-2), and four times the MIC (MIC-4)). The chicken meat was then vacuum-packaged and kept for up to 14 days at 4 °C, and its quality properties were checked during storage. In this study, the addition of low concentrations of AITC (MIC-1) maintained the lightness (L*) and increased the WHC of the meat. High concentrations (MIC-2 and MIC-4) caused a significant increase in lightness (L*) and folded yellowness (b*) value, and they significantly reduced the redness (a*) and TBARS values compared to the control meat. The amounts of MetMb and DeoMb were reduced and the quantity of OxyMb was increased as a result of the addition of AITC to the chicken breast. Throughout storage, particularly at MIC-4, AITC showed the lowest numbers of aerobic mesophilic cells, as well as a reduction in Listeria monocytogenes cell numbers and a decrease in Salmonella Typhimurium counts. In addition, the meat containing MIC-4 did not exhibit growth of Pseudomonas lundensis after 10 days. During the storage period, an electronic-nose assay demonstrated a distinction in the odor buildup of AITC across the various meat groups, and meat treated with MIC-4 showed a trend that was clearly opposite to that of untreated meat. These encouraging results demonstrate the potential of AITC to improve the safety and shelf life of meat and meat products. Full article