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In winemaking, malolactic fermentation (MLF), which converts L-malic acid to L-lactic acid, is often applied after the alcoholic fermentation stage to improve the sensory properties of the wine and its microbiological stability. MLF is usually performed by lactic acid bacteria, which, however, are sensitive to the conditions of alcoholic fermentation. Therefore, the development of wine yeast strains capable of both alcoholic fermentation and MLF is an important task. Using genome editing, we engineered a modified variant of the triploid wine yeast strain Saccharomyces cerevisiae I-328, in which the CAR1 arginase gene was replaced by the malate permease gene from Schizosaccharomyces pombe and the malolactic enzyme gene from Oenococcus oeni. Genome-wide transcriptional profiling confirmed the expression of the introduced genes and revealed a limited effect of the modification on global gene expression. Winemaking experiments show that genome editing did not affect fermentation activity and ethanol production, while use of the modified strain resulted in a tenfold reduction in malate content with simultaneous formation of lactate. The resulting wines had a softer and more harmonious taste compared to wine obtained using the parental strain. Inactivation of arginase, which forms urea and L-ornithine through the breakdown of arginine, also resulted in a twofold decrease in the content of urea and the carcinogenic ethyl carbamate in wine. Thus, the new strain with the replacement of the arginase gene with the MLF gene cassette is promising for use in winemaking. [ABSTRACT FROM AUTHOR]

Irumamycin (Iru) is a complex polyketide with pronounced antifungal activity produced by a type I polyketide (PKS) synthase. Iru features a unique hemiketal ring and an epoxide group, making its biosynthesis and the structural diversity of related compounds particularly intriguing. In this study, we performed a detailed analysis of the iru biosynthetic gene cluster (BGC) to uncover the mechanisms underlying Iru formation. We examined the iru PKS, including the domain architecture of individual modules and the overall spatial structure of the PKS, and uncovered discrepancies in substrate specificity and iterative chain elongation. Two potential pathways for the formation of the hemiketal ring, involving either an olefin shift or electrocyclization, were proposed and assessed using 18O-labeling experiments and reaction activation energy calculations. Based on our findings, the hemiketal ring is likely formed by PKS-assisted double bond migration and TE domain-mediated cyclization. Furthermore, putative tailoring enzymes mediating epoxide formation specific to Iru were identified. The revealed Iru biosynthetic machinery provides insight into the complex enzymatic processes involved in Iru production, including macrocycle sculpting and decoration. These mechanistic details open new avenues for a targeted architecture of novel macrolide analogs through synthetic biology and biosynthetic engineering. [ABSTRACT FROM AUTHOR]

The phylum Verrucomicrobiota is one of the main groups of soil prokaryotes, which remains poorly represented by cultivated organisms. The major recognized role of Verrucomicrobiota in soils is the degradation of plant-derived organic matter. These bacteria are particularly abundant in peatlands, where xylan-type hemicelluloses represent one of the most actively decomposed peat constituents. The aim of this work was to characterize the microorganisms capable of hydrolyzing xylan under the anoxic conditions typical of peatland soils. The laboratory incubation of peat samples with xylan resulted in the pronounced enrichment of several phylotypes affiliated with the Verrucomicrobiota, Firmicutes, and Alphaproteobacteria. Sequencing of the metagenome of the enrichment culture allowed us to recover high-quality metagenome-assembled genomes (MAGs) assigned to the genera Caproiciproducens, Clostridium, Bacillus (Firmicutes), and Rhizomicrobium (Alphaproteobacteria), Cellulomonas (Actinobacteriota) and the uncultured genus-level lineage of the family Chthoniobacteraceae (Verrucomicrobiota). The latter bacterium, designated "Candidatus Chthoniomicrobium xylanophilum" SH-KS-3, dominated in the metagenome and its MAG was assembled as a complete closed chromosome. An analysis of the SH-KS-3 genome revealed potential endo-1,4-beta-xylanases, as well as xylan beta-1,4-xylosidases and other enzymes involved in xylan utilization. A genome analysis revealed the absence of aerobic respiration and predicted chemoheterotrophic metabolism with the capacity to utilize various carbohydrates, including cellulose, and to perform fermentation or nitrate reduction. An analysis of other MAGs suggested that Clostridium and Rhizomicrobium could play the role of primary xylan degraders while other community members probably took advantage of the availability of xylo-oligosaccharides and xylose or utilized low molecular weight organics. [ABSTRACT FROM AUTHOR]

The class Limnochordia harbors a single cultivated member, the mesophilic Limnochorda pilosa, which was isolated from a meromictic lake. Despite numerous molecular signatures reported in various ecosystems, the ecophysiological versatility of this deeply branched lineage of Firmicutes (Bacillota) remains poorly understood. The objective of this study was to use targeted cultivation, based on metagenome-assembled genomes from a deep terrestrial aquifer in Western Siberia, to isolate two new thermophilic members of the class. These isolates, described as Geochorda subterranea gen. nov. sp. nov. and Carboxydochorda subterranea gen. nov. sp. nov. within the Geochordaceae fam. nov., were capable of both anaerobic and aerobic respiration using fumarate and O2, respectively, with simple sugars as electron donors. The cultivated Geochordaceae have demonstrated fermentative growth and degradation of various polymers, including starch, maltose, maltodextrin, xylan, and chitin. The carboxydotrophic C. subterranea sp. nov. exhibited autotrophic growth via the Calvin–Benson–Bassham cycle, using CO, H2, and formate as electron donors and O2 as an electron acceptor, adding metabolic flexibility to the bacterium in the nutrient-depleted “deep biosphere” and supporting the possibility of aerobic metabolism in the deep subsurface. The broad physiological potential deciphered from physiological experiments and comparative genomic data explains the widespread distribution of uncultivated members of the class Limnochordia in various ecosystems, where they can oxidize complex organic substrates through both aerobic and anaerobic respiration, as well as pursue a chemolithotrophic lifestyle through the oxidation of H2 or CO. [ABSTRACT FROM AUTHOR]

Simple Summary: Corrosion of steel equipment during oil production, transportation, and refining is a big global issue, leading to significant economic losses. The main agents of microbially influenced corrosion (MIC) of steel equipment are sulfate-reducing bacteria (SRB) and archaea (SRA), which reduce sulfate present in the reservoir water to form sulfide. Timely detection of sulfidogens in formations is necessary for the development of measures to suppress their growth. Existing 16S rRNA gene-based molecular methods for the detection of sulfidogens make it possible to identify them in their natural habitat, and the methods based on metagenomic analysis of components of the microbial community make it possible to predict their potential functional activity. However, selection of biocides or other methods for suppressing the growth of sulfidogens requires confirmation of their effectiveness on enrichment and/or pure cultures. In order to establish a collection of sulfidogens inhabiting the oil reservoirs of Tatarstan (Russia), a number of strains of sulfate-reducing bacteria were isolated. This study describes the 5S69T strain, which, based on the physiological and biochemical characteristics and genomic analysis, has been assigned to a new species, Pseudodesulfovibrio methanolicus sp. nov. The strain is able to grow at high salinity, at reservoir temperature, and on media with alcohols or H2/CO2 in the presence of acetate, which indicates its adaptation to environmental conditions and potential in sulfide production in the oil reservoir. The search for the microorganisms responsible for sulfide formation and corrosion of steel equipment in the oil fields of Tatarstan (Russia) resulted in the isolation of a new halotolerant strictly anaerobic sulfate-reducing bacterium, strain 5S69T. The cells were motile curved Gram-negative rods. Optimal growth was observed in the presence of 2.0–4.0% (w/v) NaCl, at pH 6.5, and at 23–28 °C under sulfate-reducing conditions. The isolate was capable of chemoorganotrophic growth with sulfate and other sulfoxides as electron acceptors, resulting in sulfide formation; and of pyruvate fermentation resulting in formation of H2 and acetate. The strain utilized lactate, pyruvate, ethanol, methanol, fumarate, and fructose, as well as H2/CO2/acetate for sulfate reduction. The genome size of the type strain 5S69T was 4.16 Mb with a G + C content of 63.0 mol%. On the basis of unique physiological properties and results of the 16S rRNA gene-based phylogenetic analysis, phylogenomic analysis of the 120 conserved single copy proteins and genomic indexes (ANI, AAI, and dDDH), assigning the type strain 5S69T ((VKM B-3653T = KCTC 25499T) to a new species within the genus Pseudodesulfovibrio, is suggested, with the proposed name Pseudodesulfovibrio methanolicus sp. nov. Genome analysis of the new isolate showed several genes involved in sulfate reduction and its sulfide-producing potential in oil fields with high saline formation water. [ABSTRACT FROM AUTHOR]

The family Beggiatoaceae is currently represented by 25 genera in the Genome Taxonomy Database, of which only 6 have a definite taxonomic status. Two metagenome-assembled genomes (MAGs), WS_Bin1 and WS_Bin3, were assembled from metagenomes of the sulfur mats coating laminaria remnants in the White Sea. Using the obtained MAGs, we first applied phylogenetic analysis based on whole-genome sequences to address the systematics of Beggiatoaceae, which clarify the taxonomy of this family. According to the average nucleotide identity (ANI) and average amino acid identity (AAI) values, MAG WS_Bin3 was assigned to a new genus and a new species in the family Beggiatoaceae, namely, 'Candidatus Albibeggiatoa psychrophila' gen. nov., sp. nov., thus providing the revised taxonomic status of the candidate genus 'BB20'. Analysis of 16S rRNA gene homology allowed us to identify MAG WS_Bin1 as the only currently described species of the genus 'Candidatus Parabeggiatoa', namely, 'Candidatus Parabeggiatoa communis', and consequently assign the candidate genus 'UBA10656', including four new species, to the genus 'Ca. Parabeggiatoa'. Using comparative whole-genome analysis of the members of the genera 'Candidatus Albibeggiatoa' and 'Ca. Parabeggiatoa', we expanded information on the central pathways of carbon, sulfur and nitrogen metabolism in the family Beggiatoaceae. [ABSTRACT FROM AUTHOR]

Two metagenome-assembled genomes (MAGs), GKL-01 and GKL-02, related to the family Thiotrichaceae have been assembled from the metagenome of bacterial mat obtained from a sulfide-rich thermal spring in the North Caucasus. Based on average amino acid identity (AAI) values and genome-based phylogeny, MAG GKL-01 represented a new genus within the Thiotrichaceae family. The GC content of the GKL-01 DNA (44%) differed significantly from that of other known members of the genus Thiothrix (50.1–55.6%). We proposed to assign GKL-01 to a new species and genus 'Candidatus Thiocaldithrix dubininis' gen. nov., sp. nov. GKL-01. The phylogenetic analysis and estimated distances between MAG GKL-02 and the genomes of the previously described species of the genus Thiothrix allowed assigning GKL-02 to a new species with the proposed name 'Candidatus Thiothrix putei' sp. nov. GKL-02 within the genus Thiothrix. Genome data first revealed the presence of both Na+-ATPases and H+-ATPases in several Thiothrix species. According to genomic analysis, bacteria GKL-01 and GKL-02 are metabolically versatile facultative aerobes capable of growing either chemolithoautotrophically or chemolithoheterotrophically in the presence of hydrogen sulfide and/or thiosulfate or chemoorganoheterotrophically. [ABSTRACT FROM AUTHOR]

Sulphate-reducing bacteria, primarily Desulfovibrio, are responsible for the active generation of H2S in swine production waste. The model species for sulphate reduction studies, Desulfovibrio vulgaris strain L2, was previously isolated from swine manure characterized by high rates of dissimilatory sulphate reduction. The source of electron acceptors in low-sulphate swine waste for the high rate of H2S formation remains uncertain. Here, we demonstrate the ability of the L2 strain to use common animal farming supplements including L-lysine-sulphate, gypsum and gypsum plasterboards as electron acceptors for H2S production. Genome sequencing of strain L2 revealed the presence of two megaplasmids and predicted resistance to various antimicrobials and mercury, which was confirmed in physiological experiments. Most of antibiotic resistance genes (ARG) are carried by two class 1 integrons located on the chromosome and on the plasmid pDsulf-L2-2. These ARGs, predicted to confer resistance to beta-lactams, aminoglycosides, lincosamides, sulphonamides, chloramphenicol and tetracycline, were probably laterally acquired from various Gammaproteobacteria and Firmicutes. Resistance to mercury is likely enabled by two mer operons also located on the chromosome and on pDsulf-L2-2 and acquired via horizontal gene transfer. The second megaplasmid, pDsulf-L2-1, encoded nitrogenase, catalase and type III secretion system suggesting close contact of the strain with intestinal cells in the swine gut. The location of ARGs on mobile elements allows us to consider D. vulgaris strain L2 as a possible vector transferring antimicrobials resistance determinants between the gut microbiote and microbial communities in environmental biotopes. [ABSTRACT FROM AUTHOR]

Desulfurococcus kamchatkensis is an anaerobic organotrophic hyperthermophilic crenarchaeon isolated from a terrestrial hot spring. Its genome consists of a single circular chromosome of 1,365,223 bp with no extra-chromosomal elements. A total of 1,474 protein-encoding genes were annotated, among which 205 are exclusive for D. kamchatkensis. The search for a replication origin site revealed a single region coinciding with a global extreme of the nucleotide composition disparity curve and containing a set of crenarchaeon-type origin recognition boxes. Unlike in most archaea, two genes encoding homologs of the eukaryotic initiator proteins Orc1 and Cdc6 are located distantly from this site. A number of mobile elements are present in the genome, including seven transposons representing IS607 and IS200/IS605 families and multiple copies of miniature inverted repeat transposable elements. Two large clusters of regularly interspaced repeats are present; none of the spacer sequences matches known archaeal extrachromosomal elements, except one spacer matches the sequence of a resident gene of D. kamchatkensis. Many of the predicted metabolic enzymes are associated with the fermentation of peptides and sugars, including more than 30 peptidases with diverse specificities, a number of polysaccharide degradation enzymes, and many transporters. Consistently, the genome encodes both enzymes of the modified Embden-Meyerhof pathway of glucose oxidation and a set of enzymes needed for gluconeogenesis. The genome structure and content reflect the organism's nutritionally diverse, competitive natural environment, which is periodically invaded by viruses and other mobile elements.

The metagenome of foulings from sulfidic spring "Serovodorodny" (Tatarstan, Russia), where members of the genus Thiothrix was observed, was sequenced. Representatives of the phyla Gammaproteobacteria, Cyanobacteria and Campilobacteriota dominated in the microbial community. The complete genome of Thiothrix sp. KT was assembled from the metagenome. It displayed 93.93–99.72% 16S rRNA gene sequence identity to other Thiothrix species. The average nucleotide identity (ANI) и digital DNA-DNA hybridization (dDDH) showed that the genome designated KT represents a new species within the genus Thiothrix, 'Candidatus Thiothrix sulfatifontis' sp. nov. KT. The taxonomic status has been determined of the strain Thiothrix sp. CT3, isolated about 30 years ago and not assigned to any of Thiothrix species due to high 16S rRNA gene sequence identity with related species (i.e., 98.8–99.4%). The complete genome sequence of strain CT3 was determined. The ANI between CT3 and other Thiothrix species was below 82%, and the dDDH values were less than 40%, indicating that strain CT3 belongs to a novel species, Thiothrix winogradskyi sp. nov. A genome analysis showed that both strains are chemo-organoheterotrophs, chemolithotrophs (in the presence of hydrogen sulfide and thiosulfate) and chemoautotrophs. For the first time, representatives of Thiothrix showed anaerobic growth in the presence of thiosulfate. [ABSTRACT FROM AUTHOR]