Lactococcus lactis is a microbial species widely used in the dairy industry as a component of starter cultures; it is also employed as a producer of recombinant proteins and biologically active metabolites, as well as in other biotechnological processes. However, genomic editing of these bacteria aimed at conferring them the desired technological properties faces significant challenges due to the lack of convenient tools for rapid and efficient gene knockout and for insertion of large genetic cassettes (several kb long) into the chromosome. Previously, we proposed an adaptation of a system based on a CRISPR-Cas-directed transposon for use in L. lactis. In this work, we improved the previously developed tool by designing a single-plasmid variant instead of the two-plasmid one and by adapting the tetracycline resistance gene as an additional selective marker in L. lactis. The resulting system significantly simplifies and accelerates the delivery of the required genetic material into the chromosome and allows for sequential modification of three different sites using readily available resistance markers (tetracycline, chloramphenicol, and erythromycin resistance genes), which can be later deleted in a single step.
This work presents the first analysis of metagenome-assembled genomes (MAGs) of lake bottom sediments of the Barguzin Depression (Baikal Rift Zone). For soda–saline Lake Gudzhirganskoe, altogether 16 prokaryotic MAGs were distributed across six phyla: Bacillota, Bacteroidota, Deinococcota, Desulfobacterota, Gemmatimonadota, and Pseudomonadota. In soda Lake Nukhe-Nur, 21 prokaryotic MAGs represented six bacterial phyla: Bacteroidota, Bipolaricaulota, Chloroflexota, Deinococcota, Desulfobacterota, and Pseudomonadota, as well as three archaeal phyla: Aenigmatarchaeota, Halobacteriota, and Thermoplasmatota. A significant share of these MAGs (over 76
In silico analysis of the complete genome sequence of potential single-cell protein (SCP) producer, the thermotolerant methanotroph Methylococcus strain COMITA G2, was performed to clarify its taxonomic position and metabolic features. Based on phylogenetic analysis of 16S rRNA gene sequences, the closest taxonomically described phylogenetic relatives of strain COMITA G2 were Methylococcus capsulatus TexasT (98.72
The novel bacterial species Rossellomorea contaminans sp. nov. (strain FM0494T) was isolated as a contaminant from semi-finished catheters. This product originated from a medical supplies production workshop in Guangdong, China. Strain FM0494T consisted of gram-positive, aerobic, and non-motile rods with terminal oval endospores, non-flagellated rods and non-swollen sporangia whose cells formed yellow, raised, opaque and butyrous colonies. Growth was observed at a temperature range of 10–45°C, a pH range of 6.0–8.0, and 0–15
Ferromanganese nodules (FeMnNs), polymetallic ore deposits widespread in the Global Ocean, are important raw materials for metallurgy. Their largest aggregations are associated with deep-water depressions in the Pacific and Indian oceans, where the rate of nodule growth is several mm per 106 years. Contrastingly, FeMnN formation and growth at the sea shelf occurs three orders of magnitude quicker, and the estimated rate for the fastest-growing Baltic Sea nodules is several mm per 100–200 years; they may therefore be considered a renewable ore resource. The biochemogenic concept of nodule formation developed by the world scientific community implies active involvement of marine microorganisms in FeMnN formation via redox transformations of the iron and manganese minerals, which is especially important in the case of fast-growing shallow-water nodules associated with organic-rich benthic ecosystems. Isolation and characterization of the microorganisms involved in the FeMnN ore genesis is a complex task. We present a description of obtaining psychrophilic primary enrichment cultures from the Baltic FeMnN samples which have been developing for several years with oxidized and reduced manganese minerals as electron acceptors and donors, respectively. The work describes phylogenetic profiles, obtained by 16S rRNA gene amplicon sequencing, for the original FeMnNs, their changes during long-term nodule storage, and the profiles of enrichment cultures, in which we revealed taxonomic groups enriched under the conditions selective for manganese minerals transformation. Bioinformatic screening of genomes of these groups representatives for the ability to carry out extracellular electron transfer revealed the key phylotypes which may determine the processes of FeMnNs formation. Our results provide highlights for the isolation and characterization of pure cultures of the microorganisms affecting FeMnN growth. This, in turn, may open the way to directed, controlled synthesis of these ore formations.
Saccharomyces boulardii (S. boulardii) is a promising probiotic microorganism with diverse health benefits. In the present study, 1 × 106 cells of this organism were inoculated in PDB and incubated at 30°C for 24 h in a rotatory shaker (120 rpm) to ensure optimal temperature and metabolite production. Secondary metabolite extraction was carried out, followed by nLC-MS analysis to identify the bioactive compounds. The files generated were analyzed using Compound Discoverer (CD) 3.3, and a ChemSpider database search was conducted to annotate the metabolites. The dataset was matched using the PubChem identifier utility. A total of 331 metabolites were identified. The metabolite spectrum analysis revealed that these bioactive metabolites possessed antimicrobial, antioxidant, anticancer, and anti-inflammatory activities. The metabolites were functionally characterized using existing literature; 25, 10, 5, and 4 of the bioactive compounds exhibited antimicrobial, anticancerous, antioxidant, and anti-inflammatory activity, respectively. This metabolomic study supports the need for further exploration of S. boulardii metabolites, as those with currently unknown activities may also possess valuable pharmacological properties.
Immobilization of members of various bacterial species, including Escherichia coli, into silanol-humate gels (SHG) results in a 10–1000 higher number of cells surviving long-term storage at room conditions. We suggested the surviving cells to possess the properties of the cells developing in biofilms (BF), the biofilm phenotype (BFP). The goal of the work was to confirm or refute the suggestion on occurrence of the cells exhibiting the BF phenotype according to their physiological, cytomorphological, and genetic characteristics in SHG-immobilized E. coli populations. The BFP cytomorphological markers were cell aggregation and extracellular specific formations (projections, cell wall protrusions termed protoprosthecae). Both in BF and SHG, some cells (15–30
Biofilms forming due to the growth of epilithic bacteria, cyanobacteria, and fungi on stone monuments (monumental rock) cause its biodeterioration and disfiguration. To investigate the possibility of controlling the biofilm growth on monumental rock surface, we studied biofilm formation by Brevundimonas faecalis strain BC1 isolated from monumental rock in microtiter plate wells in the presence of subtilisin, a popular antibiofilm enzyme, EPS solution from a fungal strain (Aspergillus niger FC1) growing on monumental rock, and a cocktail of subtilisin and the EPS solution. In this study, subtilisin at its minimum tested concentration (0.01 U/mL) showed a decrease in biofilm biomass of B. faecalis BC1 to 37.33
In ten relevant Staphylococcus aureus targets, multiple docking of 254 known antimicrobial quinazolinone derivatives and 71 new quinazolinone derivatives was performed. Using a new original architecture of a modular multi-target fully connected convolutional neural network based on the correlation convolution of multiple docking energy spectra into relevant targets, a model of anti-S. aureus activity of chemical compounds has been constructed. The threshold value of the total energy of this neural network, separating the high-affinity and low-affinity compounds, has been determined as V0 > 266.5. The limit value of anti-S. aureus activity, the minimum inhibitory concentration, which separated fairly active and low-active compounds, was determined as MIC < 112.5 μg/mL. Using the obtained model, the accuracy of the prediction on the training set for anti-S. aureus activity of quinazolinone derivatives was assessed (Acc = 78.9
The unique characteristics of mangrove sediments facilitate the ability of mangrove bacteria to produce bioactive compounds. The present study focused on the antibiotic-producing bacteria of the Bhitarkanika mangrove ecosystem, India and uncovered the nature of a bioactive compound present in the mangrove bacterium Bacillus velezensis ES8024. The impact of such external stressors as pH (pCO2), salinity, and lead (Pb) concentration on antimicrobial activities of B. velezensis ES8024 was also elucidated. The bacterium produced antimicrobial substances and was active against Aeromonas hydrophila ATCC 35654. Optimization revealed maximum production of the compound at 48 h of incubation. ATR-FTIR and 1H NMR data suggested that the extracted antibacterial compound was of a lipopeptide nature. UHPLC-TOFMS data confirmed the presence of four isoforms of the antimicrobial lipopeptide surfactin, e.g. C13 (1030.83 m/z), C14 (1044.85 m/z), and C15 (1058.87 and 1059.87 m/z) as the principle antibiotic components. The presence of two surfactin synthase genes, srfAA and srfAB, was further verified within the bacterium followed by homology modeling of SrfAA and SrfAB. Under different modifiers, lowest bacterial growth and highest intracellular reactive oxygen species (ROS) production was observed at pH 4, 4
This study provides the data on the microbial communities associated with brown trout (Salmo trutta L.) at the yolk-sac stage and within the gut of one-year-old individuals reared in a recirculating aquaculture system (RAS) in the Arkhangelsk region. Taxonomic profiling of bacterial communities in both larvae and adult fish was conducted using metabarcoding of the V4 hypervariable region of the 16S rRNA gene. The results indicate that the yolk-sac stage microbiota is predominantly composed of genera Lactobacillus, Cutibacterium, Polynucleobacter, and members of the family Comamonadaceae, whereas the gut microbiota of adult individuals is dominated by Lactobacillus, Pseudomonas, Acetobacter, and Cutibacterium. Alpha diversity indices (Shannon, Simpson, Chao1) revealed moderate abundance and homogeneity of the microbial communities at the early development stage, with a subsequent increase in microbial diversity and heterogeneity in adults. Beta diversity analysis identified overlapping yet largely distinct communities across the different ontogenetic stages. The core set of metabolically and physiologically significant bacteria appears to be primarily shaped by internal succession and dietary interactions, whereas the contribution of environmental water microbiota is limited. These findings on diversity and composition of the bacterial communities in brown trout offer a foundation for development of probiotic and management strategies in aquaculture aimed at enhancing digestive efficiency, immune competence, and post-release survival in natural water bodies.
Monascus spp. is a filamentous fungus commonly used to produce red fermented rice (RFR). Pigments extracted from Monascus spp. are currently in demand in the food industry due to many benefits to health and applications as coloring, flavoring, and preserving agents. Since citrinin (CIT) is also produced during Monascus spp. fermentation, this is raising food safety concerns. This study was designed to characterize the growth, pigments production, CIT production, and pH of M. purpureus isolates on coconut cream agar (CCA) under prolonged incubation. Two M. purpureus strains (MF1 and MS1) were isolated from RFR, inoculated onto CCA, and incubated for 30 days at 30°C. Fungal growth was determined based on colony size, fluorescence zone size, radial growth rate, and fungal biomass. During incubation, fungal growth, CIT, pigments, and pH of the extraction were determined. Two growth phases of M. purpureus were observed in this study, which were exponential (days 4–21) and stationary (days 24–30). Strong fluorescence was observed at the exponential phase together with an increase in biomass and colony diameter. CIT levels increased initially and then decreased with further incubation. Pigments production by M. purpureus increased, showing an inverse relationship with CIT decrease. The pH of the isolates on CCA increased from 7.0 to 8.6. These results provide fundamental insights into the relationship between fungal growth, CIT production, pigments, and pH during M. purpureus incubation on CCA.
Jute retting is the microbial process of decomposing the plant’s non-fibrous tissues to release and separate the usable bast fibers. The lack of understanding of the taxonomic diversity and functional profiles of microbial communities during retting stages hinders the efforts to optimize the process for enhanced retting. This study employed whole metagenome shotgun sequencing to investigate the microbial diversity, temporal community dynamics, and functional gene profiles across the retting process. Proteobacteria predominated throughout all stages, with time-specific shifts noted in Bacteroidetes and Bacillota. Aerobic genera such as Acinetobacter and Pseudomonas prevailed early on, initiating pectin degradation, whereas anaerobic Clostridium species predominated later, facilitating hemicellulose and cellulose breakdown. Fungal species, particularly Aspergillus niger and Trichoderma spp., contributed to lignocellulolytic activity, further promoting fiber release. Analysis of predicted functional genes revealed stage-specific distributions of carbohydrate-active enzyme (CAZyme) families, reflecting the potential enzymatic capacity of the microbial community during retting. Glycosyl hydrolases (GH) peaked at day 5, aiding cellulose and hemicellulose hydrolysis, while glycosyl transferases showed consistent activity for polysaccharide modification. Carbohydrate esterases and carbohydrate-binding modules demonstrated activity at days 5 and 14, enhancing ester bond hydrolysis and enzyme-substrate interactions. Polysaccharide lyases (PL) and auxiliary activity enzymes, though less abundant, peaked at day 14, facilitating lignin degradation. Pectinases (PL1, PL9, GH28), xylanases (GH8, GH10), and cellulases (GH6, GH12) exhibited sequential activity throughout the retting. This study illustrates the potential of metagenomic insights for developing microbial consortia and enzymatic formulations to enhance retting efficiency for sustainable advancements in natural fiber production.
A metagenomic analysis of microbial communities in the water and sediments of the West Aral Sea, an extreme ecosystem with a salinity of 22
The ability of nodule bacteria to form a nitrogen-fixing legume–rhizobium symbiosis is ensured by a cluster of symbiotic genes that determine both the processes of interaction with the legume, leading to the formation of specialized structures called nodules on the roots, and the fixation of atmospheric nitrogen. The organization and localization of these genomic clusters facilitate their active participation in horizontal gene transfer. The frequency of transfer correlates with the degree of relationship between the bacteria. Recombination events lead to formation of numerous rhizobial strains with varying symbiotic efficiency. In this study, we investigated the possible mechanisms underlying the formation of genetic diversity in nodule bacteria and the involvement of the legume in this diversity. It was demonstrated that the pangenome of a rhizobial population in the rhizosphere of wild legumes may contain several allelic variants of symbiotic genes, which, upon recombination, can form various combinations. This suggests that the evolution of the symbiotic system of nodule bacteria may occur not through the formation of an optimal cluster of symbiotic genes, but through individual evolution of each gene component, which, when assembled into different mosaics, produce diverse symbiotic phenotypes of rhizobia. This allows the host plant to select the most effective microorganisms based on the changing environmental conditions.
Bacteriophage Mimir124, a virulent N4-like phage isolated against a multidrug-resistant uropathogenic E. coli (UPEC) strain N124 (O101 serotype), exhibits unusual phenotypic heterogeneity during plaque formation. Under standard conditions (37°C), Mimir124 produces two stable plaque morphotypes: small (1–2 mm, transparent, sharp-edged) and large (4–10 mm, surrounded by an expanding enzymatic halo). Whole-genome sequencing revealed that this dichotomy presumably stems from mutations in the tail fiber protein gene: a six-amino-acid in-frame deletion in the “large” variant and a premature stop codon in the “small” variant. The halo formation is believed to be due to the phage-encoded depolymerase activity, enabling diffusion-mediated enzymatic cell lysis beyond the primary plaque and maintaining infectivity even at low temperatures (4°C). However, viable phage particles were found within the halo zone up to 25 mm from the plaque center, suggesting enhanced ecological adaptability, particularly the ability to infect metabolically inactive or stationary-phase bacteria—a feature of high relevance for personalized phage therapy against persistent or relapsing urological infections. The sequencing of the phage sub-strains with large- and small-plaque phenotypes revealed the deletion.
In the RC-LH1 complex of Rhodopseudomonas (Rps.) palustris KM 286 (le5), a γ-protein, which was recently identified in the light-harvesting LH2 complex of this bacterium, was identified by proteomic analysis. For the first time, the method of adsorptive chromatography on hydroxyapatite was developed and applied to isolate RC-LH1. The bioinformatics analysis of Rps. palustris CGA009 (ATCC BAA-98) genome revealed a close location of the genes encoding α-, β-, and γ-proteins and similar functional groups in the γ-proteins of both complexes interacting with bacteriochlorophyll and the α- and β-proteins, which may indicate that γ-proteins have similar functions. The differences in the structure of the LH2 and LH1 complexes leave open the question of the localization of the γ-protein in RC-LH1, which requires further structural studies of this complex.
An Erratum to this paper has been published: https://doi.org/10.1134/S0026261726601338
Taxonogenomic analysis was conducted on an alphaproteobacterium, designated as KMU-71T, that was isolated from seawater taken at Dadaepo Harbor in South Korea. Strain KMU-71T was gram-stain-negative, light apricot-colored, rod-shaped, strictly aerobic, and showed no motility. The novel strain could grow at 0–3.0
Deferribacter autotrophicus SL50T is a thermophilic anaerobic bacterium capable of autotrophic growth, but its carbon fixation mechanism has remained unclear. Here, radioisotopic assays and comparative proteomics were used to identify the pathway supporting CO2 assimilation. During autotrophic growth on hydrogen and ferrihydrite, the rate of 14C incorporation was sixfold higher than under heterotrophic conditions, coinciding with the logarithmic growth phase. Proteomic analysis detected over 1400 proteins in each growth mode and showed high abundance of all major tricarboxylic acid (TCA) cycle enzymes, with citrate synthase and malate dehydrogenase among the most strongly expressed. Genomic and proteomic data indicate that D. autotrophicus SL50T lacks phosphoenolpyruvate carboxylase and instead converts pyruvate directly to oxaloacetate via pyruvate:ferredoxin oxidoreductase. Unexpectedly, acetate kinase and phosphate acetyltransferase were expressed threefold higher during autotrophic growth than during growth on acetate. These results suggest a previously unrecognized feature of the reverse oxidative TCA (roTCA) cycle, in which excess acetyl-CoA is recycled through acetyl-CoA:succinate CoA-transferase and subsequently reintegrated into the cycle. Together, these findings provide strong evidence that D. autotrophicus SL50T fixes CO2 via the roTCA cycle and broadens the understanding of the metabolic diversity of anaerobic microorganisms.
