Prokaryotic Species Research Articles

Whole Genome Sequence-Based Classification of Nonomuraea marmarensis sp. nov., Isolated from Island Soil

Actinomycetes are known to produce a vast array of bioactive secondary metabolites with potential therapeutic applications, including antimicrobials, anticancer agents, and enzyme inhibitors. Among these, members of the genus Nonomuraea have received much attention due to their broad ecological importance in nutrient cycling in soil and their ability to produce new bioactive compounds. A novel actinomycetes, designated strain M3C6T, was isolated from soil samples collected on Marmara Island, located in the Istanbul province, aiming to explore the microbial diversity of unexplored habitats, and characterized using a polyphasic approach. The isolate showed chemotaxonomic and morphological features consistent with members of the genus Nonomuraea. The 16S rRNA gene sequence analysis revealed that strain M3C6T shared the highest similarity, at 98.7% sequence identity, to Nonomuraea basaltis 160415T and Nonomuraea turkmeniaca DSM 43926T. However, the ANI and dDDH values between strain M3C6T and these reference strains were fairly low, ranging from 84.0 to 84.6% and 31.8 to 33.7%, respectively, below the generally accepted cutoffs for ANI and DDH that delineate different prokaryotic species. Genomic analysis of strain M3C6T showed that it had a genome size of 10.38 Mbp and a DNA G+C content of 69.5 mol%. Based on these chemotaxonomic, phenotypic, and genomic data, strain M3C6T is classified as a novel species within the genus Nonomuraea, for which the name Nonomuraea marmarensis sp. nov. is proposed. The type strain is M3C6T (= KCTC 49983T = CGMCC 4.8035T). Genomic analyses confirmed the high potential of M3C6T to produce specialized secondary metabolites.

Genome-Wide A → G and C → T Mutations Induced by Functional TadA Variants in Escherichia coli.

The fusion expression of deoxyribonucleic acid (DNA) replication-related proteins with nucleotide deaminase enzymes promotes random mutations in bacterial genomes, thereby increasing genetic diversity among the population. Most previous studies have focused on cytosine deaminase, which produces only C → T mutations, significantly limiting the variety of mutation types. In this study, we developed a fusion expression system by combining DnaG (RNA primase) with adenine deaminase TadA-8e (DnaG-TadA) in Escherichia coli, which is capable of rapidly introducing A → G mutations into the E. coli genome, resulting in a 664-fold increase in terms of mutation rate. Additionally, we tested a dual-functional TadA variant, TadAD, and then fused it with DnaG. This construct introduced both C → T and A → G mutations into the E. coli genome, with the mutation rate increased by 370-fold upon coexpression with a uracil glycosylase inhibitor (DnaG-TadAD-UGI). We applied DnaG-TadA and DnaG-TadAD-UGI systems to the adaptive laboratory evolution for Cd2+ and kanamycin resistance, achieving an 8.0 mM Cd2+ and 200 μg/mL kanamycin tolerance within just 17 days and 132 h, respectively. Compared to conventional evolution methods, the final tolerance levels were increased by 320 and 266%, respectively. Our work offers a novel strategy for random mutagenesis in E. coli and potentially other prokaryotic species.

Paenibacillus zeirhizosphaerae sp. nov., isolated from surface of the maize (Zea mays) roots in a horticulture field, Hungary.

A novel Gram-stain-positive, rod-shaped, endospore-forming bacterium with peritrichous flagella, designated as P96T was isolated from the surface of maize roots. Strain P96T grew optimally at 28 °C, pH 7.0. The strain contained A1γ meso-Dpm-direct in the cell-wall peptidoglycan. The dominant polar lipids were diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol. The genome size of strain P96T was 4.8 Mb, and the G+C content was 50.01%. Phylogenomic analyses based on the whole-genome sequences classified the strain into the genus Paenibacillus. The digital DNA-DNA hybridization and average nucleotide identity relatedness analysis resulted in values below the threshold for prokaryotic species delineation, with the highest values observed for Paenibacillus enshidis KCTC 33519T (29.4 and 85.2%, respectively). Genotypic data together with phenotypic properties supported the classification of strain P96T as representative of a novel species of the genus Paenibacillus, for which the name Paenibacillus zeirhizosphaerae sp. nov. is proposed. The type strain is P96T (=LMG 32802T = NCAIM B 02678T).

Geodermatophilus maliterrae sp. nov., a member of the Geodermatophilaceae isolated from badland surfaces in the Red Desert, Wyoming, USA.

A novel Gram-stain-positive, black-pigmented bacterium, designated as WL48A T, was isolated from the surface of badland sedimentary rock in the Red Desert of Wyoming and characterized using a polyphasic taxonomic approach. Good growth occurred at 28-32 °C, pH 7-9, and NaCl less than 1% (w/v). Colonies, growing well on International Streptomyces Project media (ISP) 3 and ISP 7, were black and adhering to the agar. Phylogenetic analyses based on 16S rRNA gene and draft genome sequences showed that strain WL48AT belongs to the family Geodermatophilaceae, forming a distinct sub-branch with Geodermatophilus bullaregiensis DSM 46841T. The organism showed 16S rRNA gene sequence similarity of 98.8% with G. bullaregiensis DSM 46841T. Digital DNA-DNA hybridization value between the genome sequences of strain WL48A T and G. bullaregiensis DSM 46841T was 51.8%, below the threshold of 70% for prokaryotic species delineation. The chemotaxonomic investigation revealed the presence of galactose, glucose, mannose, xylose and ribose as well as meso-DAP in the peptidoglycan layer. The polar lipid profiles contained phosphatidylcholine (PC), phosphatidylinositol (PI), diphosphatidylglycerol (DPG), phosphatidylethanolamine (PE) phosphoglycolipid, phospholipids and an unidentified lipid. The menaquinone profile consisted of MK-9(H4) (98.2%) and MK-9(H2) (10.8%). The major fatty acid profile (>15%) comprised iso-C15 : 0 and iso-C16 : 0. Based on phenotypic, genetic and genomic data, strain WL48AT (=DSM 116197T = NCIMB 15483T=NCCB 100957T =ATCC TSD-376T) merits to be considered as a novel species for which the name Geodermatophilus maliterrae sp. nov. is proposed.

Kribbella caucasensis sp. nov. from the Soil of the North Caucasus Mountains

Mycelium-forming aerobic actinomycete, strain VKM Ас-2527T, was isolated from a soil sample collected from the North Caucasus mountains. The 16S rRNA gene sequence similarities of this strain to the type strains of the known Kribbella species ranged from 95.5 to 98.5%. The dDDH (23.1–26.3%) and ANIb (78.1–81.5%) values between VKM Ас-2527T and the type strains of the phylogenetically closest Kribbella species were below the thresholds for prokaryote species delineation. The conventional chemotaxonomic characteristics of strain VKM Ас-2527T were typical of the genus Kribbella: the LL-diaminopimelic acid (LL-A2pm) in the cell wall peptidoglycan, the major menaquinone МК-9(Н4), dominant fatty acids anteiso-C15:0, iso-C16:0, and anteiso-C17:0, and the PIII-type phospholipids (with phosphatidylcholine as the key diagnostic component). The cell wall also contained branched α-mannan characteristic of the genus and the teichulosonic acid of irregular structure. The cell wall sugars included mannose, galactose, rhamnose, 3-O-methylgalactose (madurose), and 2,3-di-O-methylgalactose. The genome size was 9.5 Mb, with 68.2% G+C content. Based on the results obtained in the present study and the previously published data, a new species, Kribbella caucasensis sp. nov., is proposed, with the type strain VKM Ас-2527T.

Xenorhabdus bharatensis sp. nov., Xenorhabdus entomophaga sp. nov., Xenorhabdus siamensis sp. nov., and Xenorhabdus thailandensis sp. nov. Isolated from Steinernema Entomopathogenic Nematodes

Four Gram-stain-negative bacterial strains, CS20T, AUT15.5T, XENO-11T, and CCN3.3T, isolated from Steinernema entomopathogenic nematodes, were found to represent novel species within the genus Xenorhabdus (Gammaproteobacteria, Morganellaceae). In this study, we described these new species using whole-genome phylogenomic reconstructions, sequence identity values from core genome sequences, and phenotypic characterization. Phylogenetic reconstructions based on 16S rRNA gene sequences showed that: (i) strain CS20T is closely related to X. stockiae DSM 17904T, (ii) strain AUT15.5T is closely related to X. budapestensis DSM 16342T, (iii) strain XENO-11T is closely related to X. khoisanae DSM 25463T, and (iv) strain CCN3.3T is closely related to X. griffiniae DSM 17911T. The 16S rRNA gene sequence similarity value between strain CS20T and X. stockiae DSM 17904T is 97.8%, between strain AUT15.5T and X. budapestensis DSM 16342T is 98.1%, between strain XENO-11T and X. khoisanae DSM 25463T is 97.8%, and between strain CCN3.3T and X. griffiniae DSM 17911T is 98.6%. Phylogenomic reconstructions using whole-genome sequences showed that: (i) strain CS20T is closely related to X. stockiae DSM 17904T and X. innexi DSM 16336T, (ii) strain AUT15.5T is closely related to X. indica DSM 17382T, (iii) strain XENO-11T is closely related to X. khoisanae DSM 25463T, and (iv) strain CCN3.3T is closely related to X. griffiniae DSM 17911T. The digital DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) values between strains CS20T, AUT15.5T, XENO-11T, and CCN3.3T and the type strains of their more closely related species are below the 70% and the 95–96% divergence thresholds, respectively, used for prokaryotic species delineation. Hence, we propose the following four new species: Xenorhabdus bharatensis sp. nov. (the type strain is CS20T=CCM 9320T=CCOS 2070T), X. entomophaga sp. nov. (the type strain is XENO-11T=CCM 9389T=CCOS 2111T), X. siamensis sp. nov. (the type strain is AUT15.5T=CCM 9405T=CCOS 2116T), and X. thailandensis sp. nov. (the type strain is CCN3.3T=CCM 9406T=CCOS 2115T). The following biochemical tests may be useful for differentiating the novel species from their more closely related taxa: acetoin production, arginine dihydrolase, citrate utilization, gelatinase, glucose oxidation, indole production, and tryptophan deaminase. Our study contributes to a better understanding of the biodiversity and phylogenetic relationships of entomopathogenic bacteria associated with insect parasitic nematodes.

Menaquinone production in genetically engineered E. coli.

Menaquinone (MK) is an important electron transporter in Escherichia coli. This isoprenoid quinone can transfer electrons to many terminal acceptors, such as fumarate and nitrate, which helps this organism survive under diverse and challenging conditions. As isoprenoid quinones with various lengths of isoprenyl tail are widely distributed in nature, the heterologous expression of polyprenyl diphosphate synthases (PDSs) has been investigated using its counterpart, ubiquinone (UQ). In this study, we investigated the MK production by the expression of various heterologous PDS genes from prokaryotic and eukaryotic species, including Saccharomyces cerevisiae COQ1 (hexa-PDS), Haemophilus influenzae hi0881 (hepta-PDS), Synechocystis sp. strain PCC6803 slr0611 (nona-PDS), and Gluconobacter suboxydans ddsA (deca-PDS) in E. coli. We detected specific isoforms of MK, including MK7, MK9, and MK10, via the expression of HI0881, Slr0611, and DdsA respectively, but barely detected MK6 via the expression of Coq1. As UQ6 was detected in E. coli harboring COQ1, the acceptance of the side chain lengths by MenA (prenyl transferase for MK) was narrower than UbiA (prenyl transferase for UQ). We also identified a mutation in menA in the E. coli AN386 strain and a transposon insertion of IS186 in menC in E. coli KO229 (∆ispB) and its parental strain FS1576. Taken together, these results elucidate the different nature of MenA from UbiA.

OrthoDB and BUSCO update: annotation of orthologs with wider sampling of genomes.

OrthoDB (https://www.orthodb.org) offers evolutionary and functional annotations of orthologous genes in the widest sampling of eukaryotes, prokaryotes, and viruses, extending experimental gene function knowledge to newly sequenced genomes. We collect gene annotations, delineate hierarchical gene orthologyand annotate the orthologous groups (OGs) with functional and evolutionary traits. OrthoDB is the leading resource for species diversity, striving to sample the most diverse and well-researched organisms with the highest quality genomic data. This update expands to include 5827 eukaryotic genomes. We have also added coding DNA sequences (CDSs) and gene loci coordinates. OrthoDB can be browsed, downloaded, or accessed using REST API, SPARQL/RDFand now also via API packages for Python and R Bioconductor. OrthoLoger (https://orthologer.ezlab.org), the tool used for inferring orthologs in OrthoDB, is now available as a Conda package and through BioContainers. ODB-mapper, a component of OrthoLoger, streamlines annotation of genes from newly sequenced genomes with OrthoDB evolutionary and functional descriptors. The benchmarking sets of universal single-copy orthologs (BUSCO), derived from OrthoDB, had correspondingly a major update. The BUSCO tool (https://busco.ezlab.org) has become a standard in genomics, uniquely capable of assessing both eukaryotic and prokaryotic species. It is applicable to gene sets, transcriptomes, genome assembliesand metagenomic bins.

Parafrigoribacterium soli sp. nov. and Parafrigoribacterium humi sp. nov., two novel siderophore-synthesizing species isolated from black soil

Two siderophore-synthesizing species SYSU BS000078T and SYSU BS000231T were isolated from the black soil collected from fields located in Heilongjiang province, China. The results of phylogenetic analysis based on the 16S rRNA gene sequences indicated that these two strains showed the highest sequence similarity to Parafrigoribacterium mesophilum KCTC 19311T (98.61 % and 98.68 %, respectively). The average nucleotide identity (ANI) values between the two strains and other members of the genus Parafrigoribacterium were lower than 95 %, recommended for distinguishing novel prokaryotic species. Cells of strains SYSU BS000078T and SYSU BS000231T were aerobic, motile, Gram-stain-positive and non-spore-forming rods. The colonies of these two strains exhibited a cream pigment, with tidy edges and smooth surfaces. Growth was observed within the temperature range of 4–37 °C (optimal growth at 28 °C) and pH range of 6.0–8.0 (optimal growth at pH 7.0). The predominant polar lipids detected in these two strains included diphosphatidylglycerol and phosphatidylglycerol. The predominant respiratory quinones was MK-9. The major cellular fatty acids (>10 %) were iso-C16:0, anteiso-C15:0, and anteiso-C17:0. The genome size and G + C content of strain SYSU BS000078T were determined to be 2.89 Mbp and 66.5 %, respectively. Meanwhile, SYSU BS000231T exhibited a genome size of 2.81 Mbp, accompanied by a G + C content of 65.8 %. Based on the phenotypic, physiological, genotypic, and phylogenetic data, these two strains represent two novel species of the genus Parafrigoribacterium, which are proposed as Parafrigoribacterium soli sp. nov. SYSU BS000078T (=GDMCC 1.4599T = KCTC 59245T), and Parafrigoribacterium humi sp. nov. SYSU BS000231T (=GDMCC 1.3816T = KCTC 59001T).

Amycolatopsis melonis sp. nov., a novel protease-producing and cellulose-degrading actinobacterium isolated from soil.

A novel protease-producing and cellulose-degrading actinobacterium, designated strain NEAU-NG30T, was isolated from a melon rhizosphere soil sample collected in Harbin, Heilongjiang Province, China, and established its status using a polyphasic taxonomic study. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain NEAU-NG30T was closely related to Amycolatopsis bullii DSM 45802T (98.7%) and Amycolatopsis vancoresmycina DSM 44592T (98.3%). The phospholipid profile contained diphosphatidylglycerol, phosphatidyl methylethanolamine, phosphatidylethanolamine and phosphatidylinositol. The diagnostic sugars in cell hydrolysates were determined to be galactose and arabinose. Cell walls contained meso-diaminopimelic acid as the diagnostic diamino acid. The predominant menaquinone was MK-9(H4). The major fatty acids were iso-C15:0 and iso-C16:0. Meanwhile, genome analysis of sequences revealed a genome size of 9 338 250 bp and a DNA G+C content of 71.6%. In addition, the average nucleotide identity values and the level of digital DNA-DNA hybridization between strain NEAU-NG30T and its reference strains fall below the thresholds typically used for delineating prokaryote species. According to phenotypic, chemotaxonomic and genotypic studies, it is indicated that strain NEAU-NG30T is considered to be a novel species of the genus Amycolatopsis, for which the name Amycolatopsis melonis sp. nov. is proposed, with NEAU-NG30T (=MCCC 1K08677T=JCM 35654T) as the type strain.

Flavobacterium capsici sp. nov., isolated from the rhizospheric soils of bell pepper (Capsicum annuum L.).

Two separate bacterial strains, PMTSA4T and PMR2A8, were isolated from the rhizospheric soils of bell pepper plants grown in a plant nursery. These strains are Gram-negative, non-motile and rod-shaped and grow in aerobic conditions. They exhibit a positive reaction for catalase activity but negative results for oxidase activity. Phylogenetic analysis of the 16S rRNA gene sequences revealed that the strains PMTSA4T and PMR2A8 are closely related to Flavobacterium piscinae ICH-30T (95.6%, respectively), Flavobacterium ahnfeltiae 10Alg 130T (95.5%) and Flavobacterium maris KMM 9535T (95.3%), aligning them within the genus Flavobacterium. Digital DNA-DNA hybridization (dDDH) values and average nucleotide identities (ANIs) of the whole-genome sequences for the two strains and related Flavobacterium species were significantly below the established thresholds for prokaryotic species delineation (<70% for dDDH and <95% for ANI). The observed values were as follows: Flavobacterium aquatile LMG 4008T (dDDH: 19.8% and ANI: 75.5%), F. piscinae ICH-30T (dDDH: 18.6% and ANI: 73.3%) and F. stagni WWJ 16T (dDDH: 18.5% and ANI: 72.0%). The strains have genome sizes of 3 068 185 bp and 3 068 330 bp, with a G+C content of 32.5 mol%. In phenotypic characterization, the new strains grew at 10-35 °C and tolerated up to 4% NaCl at pH 5-9 (optimum pH 8). The predominant cellular fatty acids were observed to be iso-C15:0, iso-C17:0 3-OH and iso-C15:0 3-OH. Menaquinone-6 was the predominant quinone. Considering the results from phenotypic, chemotaxonomic, phylogenetic and genomic analyses, it is proposed that the strains PMTSA4T and PMR2A8 represent a novel species within the genus Flavobacterium.

Structural Engineering of Cationic Block Copolymer Architectures for Selective Breaching of Prokaryotic and Eukaryotic Biological Species.

Positively charged antimicrobial polymers are known to cause severe damage to biological systems, and thus synthetic strategies are urgently required to design next-generation nontoxic cationic macromolecular architectures for healthcare applications. Here, we report a structural-engineering strategy to build cationic linear and star-block copolymer nanoarchitectures having identical chemical composition, molar mass, nanoparticle size, and positive surface charge, yet they differ distinctly in their biological action in breaching prokaryotic species such as E. coli (Gram-negative bacteria) without affecting eukaryotic species like red-blood and mammalian cells. For this purpose, linear and star-block structures are built on a polycaprolactone biodegradable platform having an imidazolium positive handle. Under physiological conditions, the linear architecture exhibits toxicity indiscriminately to all biological species, whereas its star counterpart is remarkably selective in membrane breaching action toward bacteria while maintaining inertness toward eukaryotic species. Confocal microscopy analysis of HPTS fluorescent dye-loaded star-polymer nanoparticles substantiated their antimicrobial action in E. coli. Tissue-penetrable near-infrared fluorescent dye (IR-780) loaded NP aided the in vivo biodistribution analysis and ex vivo quantification of cationic species' accumulations in vital organs in mice. Azithromycin, a clinical water-insoluble macrolide, is delivered from the star platform to accomplish synergistic antimicrobial activity by the combination of bactericidal-bacteriostatic action of the polymer carrier and drug together in a single system.

Pangenomes of human gut microbiota uncover links between genetic diversity and stress response

The genetic diversity of the gut microbiota has a central role in host health. Here, we created pangenomes for 728 human gut prokaryotic species, quadrupling the genes of strain-specific genomes. Each of these species has a core set of a thousand genes, differing even between closely related species, and an accessory set of genes unique to the different strains. Functional analysis shows high strain variability associates with sporulation, whereas low variability is linked with antibiotic resistance. We further map the antibiotic resistome across the human gut population and find 237 cases of extreme resistance even to last-resort antibiotics, with a predominance among Enterobacteriaceae. Lastly, the presence of specific genes in the microbiota relates to host age and sex. Our study underscores the genetic complexity of the human gut microbiota, emphasizing its significant implications for host health. The pangenomes and antibiotic resistance map constitute a valuable resource for further research.

Options and considerations for validation of prokaryotic names under the SeqCode

Stable taxon names for Bacteria and Archaea are essential for capturing and documenting prokaryotic diversity. They are also crucial for scientific communication, effective accumulation of biological data related to the taxon names and for developing a comprehensive understanding of prokaryotic evolution. However, after more than a hundred years, taxonomists have succeeded in valid publication of only around 30 000 species names, based mostly on pure cultures under the International Code of Nomenclature of Prokaryotes (ICNP), out of the millions estimated to reside in the biosphere. The vast majority of prokaryotic species have not been cultured and are becoming increasingly known to us via culture-independent sequence-based approaches. Until recently, such taxa could only be addressed nomenclaturally via provisional names such as Candidatus or alphanumeric identifiers. Here, we present options and considerations to facilitate validation of names for these taxa using the recently established Code of Nomenclature of Prokaryotes Described from Sequence Data (SeqCode). Community engagement and participation of relevant taxon specialists are critical and encouraged for the success of endeavours to formally name the uncultured majority.

Time-series sewage metagenomics distinguishes seasonal, human-derived and environmental microbial communities potentially allowing source-attributed surveillance

Sewage metagenomics has risen to prominence in urban population surveillance of pathogens and antimicrobial resistance (AMR). Unknown species with similarity to known genomes cause database bias in reference-based metagenomics. To improve surveillance, we seek to recover sewage genomes and develop a quantification and correlation workflow for these genomes and AMR over time. We use longitudinal sewage sampling in seven treatment plants from five major European cities to explore the utility of catch-all sequencing of these population-level samples. Using metagenomic assembly methods, we recover 2332 metagenome-assembled genomes (MAGs) from prokaryotic species, 1334 of which were previously undescribed. These genomes account for ~69% of sequenced DNA and provide insight into sewage microbial dynamics. Rotterdam (Netherlands) and Copenhagen (Denmark) show strong seasonal microbial community shifts, while Bologna, Rome, (Italy) and Budapest (Hungary) have occasional blooms of Pseudomonas-dominated communities, accounting for up to ~95% of sample DNA. Seasonal shifts and blooms present challenges for effective sewage surveillance. We find that bacteria of known shared origin, like human gut microbiota, form communities, suggesting the potential for source-attributing novel species and their ARGs through network community analysis. This could significantly improve AMR tracking in urban environments.

Rufibacter psychrotolerans sp. nov., a Cold-Tolerating Novel Species Isolated from Desert Soil.

Strain SYSU D00308T, a short-rod-shaped bacterium, was isolated from a sandy soil collected from the Gurbantunggut Desert, Xinjiang, PR China. Strain SYSU D00308T was Gram-stain-negative, aerobic, pink-pigmented, non-motile, catalase- and oxidase-positive. The strain grew at 4-37 ℃, pH 5.0-8.0 and 0-1.5% (w/v) NaCl. 16S rRNA gene sequencing analyses demonstrated that strain SYSU D00308T belonged to the genus Rufibacter and exhibited the highest sequence similarity (97.4%) to Rufibacter glacialis MDT1-10-3T. Summed features 3, 4, and iso-C15:0 were the major fatty acids, and menaquinone 7 (MK-7) was the sole respiratory menaquinone. The polar lipid profiles comprised phosphatidylethanolamine, an unidentified glycolipid, an unidentified phospholipid, two unidentified aminophospholipids, and two unidentified lipids. The genome size and DNA G + C content of strain SYSU D00308T were 5,176,683 bp and 54.8%, respectively. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between SYSU D00308T and members of the genus Rufibacter were 77.7-81.8% and 20.4-23.4% respectively, which were less than the corresponding thresholds (ANI: 95-96%; dDDH: 70%) for prokaryotic species definition. According to the genotypic, phenotypic and phylogenetic characteristics, strain SYSU D00308T represents a novel species of the genus Rufibacter. We propose the name, Rufibacter psychrotolerans sp. nov., with SYSU D00308T (= CGMCC 1.18621T = KCTC 82275T = MCCC 1K04970T) as the type strain.

Advancing Source Tracking: Systematic Review and Source-Specific Genome Database Curation of Fecally Shed Prokaryotes.

Advancements within fecal source tracking (FST) studies are complicated by a lack of knowledge regarding the genetic content and distribution of fecally shed microbial populations. To address this gap, we performed a systematic literature review and curated a large collection of genomes (n = 26,018) representing fecally shed prokaryotic species across broad and narrow source categories commonly implicated in FST studies of recreational waters (i.e., cats, dogs, cows, seagulls, chickens, pigs, birds, ruminants, human feces, and wastewater). We find that across these sources the total number of prokaryotic genomes recovered from materials meeting our initial inclusion criteria varied substantially across fecal sources: from none in seagulls to 9,085 in pigs. We examined genome sequences recovered from these metagenomic and isolation-based studies extensively via comparative genomic approaches to characterize trends across source categories and produce a finalized genome database for each source category which is available online (n = 12,730). On average, 81% of the genomes representing species-level populations occur only within a single source. Using fecal slurries to test the performance of each source database, we report read capture rates that vary with fecal source alpha diversity and database size. We expect this resource to be useful to FST-related objectives, One Health research, and sanitation efforts globally.

Database size positively correlates with the loss of species-level taxonomic resolution for the 16S rRNA and other prokaryotic marker genes.

For decades, the 16S rRNA gene has been used to taxonomically classify prokaryotic species and to taxonomically profile microbial communities. However, the 16S rRNA gene has been criticized for being too conserved to differentiate between distinct species. We argue that the inability to differentiate between species is not a unique feature of the 16S rRNA gene. Rather, we observe the gradual loss of species-level resolution for other nearly-universal prokaryotic marker genes as the number of gene sequences increases in reference databases. This trend was strongly correlated with how represented a taxonomic group was in the database and indicates that, at the gene-level, the boundaries between many species might be fuzzy. Through our study, we argue that any approach that relies on a single marker to distinguish bacterial taxa is fraught even if some markers appear to be discriminative in current databases.

C(P)XCG Proteins of Haloferax volcanii with Predicted Zinc Finger Domains: The Majority Bind Zinc, but Several Do Not.

In recent years, interest in very small proteins (µ-proteins) has increased significantly, and they were found to fulfill important functions in all prokaryotic and eukaryotic species. The halophilic archaeon Haloferax volcanii encodes about 400 µ-proteins of less than 70 amino acids, 49 of which contain at least two C(P)XCG motifs and are, thus, predicted zinc finger proteins. The determination of the NMR solution structure of HVO_2753 revealed that only one of two predicted zinc fingers actually bound zinc, while a second one was metal-free. Therefore, the aim of the current study was the homologous production of additional C(P)XCG proteins and the quantification of their zinc content. Attempts to produce 31 proteins failed, underscoring the particular difficulties of working with µ-proteins. In total, 14 proteins could be produced and purified, and the zinc content was determined. Only nine proteins complexed zinc, while five proteins were zinc-free. Three of the latter could be analyzed using ESI-MS and were found to contain another metal, most likely cobalt or nickel. Therefore, at least in haloarchaea, the variability of predicted C(P)XCG zinc finger motifs is higher than anticipated, and they can be metal-free, bind zinc, or bind another metal. Notably, AlphaFold2 cannot correctly predict whether or not the four cysteines have the tetrahedral configuration that is a prerequisite for metal binding.

Technique of PCR analysis of MT2A genes in different animal species

Metallothioneins are a family of low-molecular-weight cysteine-rich proteins that participate in the metabolism of essential metals and protect against heavy metal toxicity. Metallothioneins are represented by different isoforms in many eukaryotic and prokaryotic species. The promoter regions of metallothionein genes contain sequences of elements that regulate expression in response to metals (MRE), which makes it an urgent task to investigate polymorphisms in the promoter regions of metallothionein genes. Objective. To develop and optimize the technique of PCR analysis of orthologous metallothionein genes in various biological species using the example of the MT2A isoform of rats and pigs. Methods. Analysis of the primary structure of MT2A isoform genes was performed using the NCBI and Ensembl databases. The design ofprimers for PCR analysis was carried out using the Primer3 program. Biomaterial samples were used for the study: livers of Wistar rats and blood of large white pigs of intrabreed type ULW-1. DNA was isolatedfrom rat liver using the NeoPrep DNA Magnet plant DNA kit, and from pig blood using Chelex 100 reagent. Amplification of DNA fragments was carried out using PCR followed by electrophoretic separation of the amplicons in a 2 % agarose gel. Results. In the course of the study, the primary structure of the MT2A genes of rats and pigs was analyzed. Primers were designed to cover the promoter regions of the MT2A genes of these two animal species. The expected sizes of the amplicons are 583 bp and 512 bp for rats andpigs, respectively. The conditions for the synthesis of PCR amplicons were selected, the optimality of which is confirmed by the presence of the corresponding PCR amplicons on the electrophoregram. Conclusions. The developed PCR analysis system for the MT2A genes of rats and pigs allows to study polymorphisms in the promoter regions that contain MRE sites. Considering the importance of MRE sequences for the regulation of metallothioneins’ expression under the influence of metals, as well as the participation of metallothioneins in various physiological and pathological processes, it can be expected that polymorphisms in the studied areas may have associations with adaptation potential, resistance to heavy metals, economic qualities of organisms, etc. In the future, research involving the MT2A gene as a candidate for the development of molecular genetic markers and their use in the practice of marker-associated selection is promising. Keywords: metallothioneins, polymorphism, Rattus norvegicus, Sus scrofa domesticus, polymerase chain reaction, promoter.