Biosynthetic gene clusters with biotechnological applications in novel Antarctic isolates from Actinomycetota.

Actinomycetota have been widely described as valuable sources for the acquisition of secondary metabolites. Most microbial metabolites are produced via metabolic pathways encoded by biosynthetic gene clusters (BGCs). Although many secondary metabolites are not essential for the survival of bacteria, they play an important role in their adaptation and interactions within microbial communities. This is how bacteria isolated from extreme environments such as Antarctica could facilitate the discovery of new BGCs with biotechnological potential. This study aimed to isolate rare Actinomycetota strains from Antarctic soil and sediment samples and identify their metabolic potential based on genome mining and exploration of biosynthetic gene clusters. To this end, the strains were sequenced using Illumina and Oxford Nanopore Technologies platforms. The assemblies were annotated and subjected to phylogenetic analysis. Finally, the BGCs present in each genome were identified using the antiSMASH tool, and the biosynthetic diversity of the Micrococcaceae family was evaluated. Taxonomic annotation revealed that seven strains were new and two were previously reported in the NCBI database. Additionally, BGCs encoding type III polyketide synthases (T3PKS), beta-lactones, siderophores, and non-ribosomal peptide synthetases (NRPS) have been identified, among others. In addition, the sequence similarity network showed a predominant type of BGCs in the family Micrococcaceae, and some genera were distinctly grouped. The BGCs identified in the isolated strains could be associated with applications such as antimicrobials, anticancer agents, and plant growth promoters, among others, positioning them as excellent candidates for future biotechnological applications and innovations. KEY POINTS: • Novel Antarctic rare Actinomycetota strains were isolated from soil and sediments • Genome-based taxonomic affiliation revealed seven potentially novel species • Genome mining showed metabolic potential for novel natural products.

Commonalities between the Atacama Desert and Antarctica rhizosphere microbial communities.

Plant-microbiota interactions have significant effects on plant growth, health, and productivity. Rhizosphere microorganisms are involved in processes that promote physiological responses to biotic and abiotic stresses in plants. In recent years, the interest in microorganisms to improve plant productivity has increased, mainly aiming to find promising strains to overcome the impact of climate change on crops. In this work, we hypothesize that given the desertic environment of the Antarctic and the Atacama Desert, different plant species inhabiting these areas might share microbial taxa with functions associated with desiccation and drought stress tolerance. Therefore, in this study, we described and compared the composition of the rhizobacterial community associated with Deschampsia antarctica (Da), Colobanthus quitensis (Cq) from Antarctic territories, and Croton chilensis (Cc), Eulychnia iquiquensis (Ei) and Nicotiana solanifolia (Ns) from coastal Atacama Desert environments by using 16S rRNA amplicon sequencing. In addition, we evaluated the putative functions of that rhizobacterial community that are likely involved in nutrient acquisition and stress tolerance of these plants. Even though each plant microbial rhizosphere presents a unique taxonomic pattern of 3,019 different sequences, the distribution at the genus level showed a core microbiome with a higher abundance of Haliangium, Bryobacter, Bacillus, MND1 from the Nitrosomonadaceae family, and unclassified taxa from Gemmatiamonadaceae and Chitinophagaceae families in the rhizosphere of all samples analyzed (781 unique sequences). In addition, species Gemmatirosa kalamazoonesis and Solibacter usitatus were shared by the core microbiome of both Antarctic and Desert plants. All the taxa mentioned above had been previously associated with beneficial effects in plants. Also, this microbial core composition converged with the functional prediction related to survival under harsh conditions, including chemoheterotrophy, ureolysis, phototrophy, nitrogen fixation, and chitinolysis. Therefore, this study provides relevant information for the exploration of rhizospheric microorganisms from plants in extreme conditions of the Atacama Desert and Antarctic as promising plant growth-promoting rhizobacteria.

Mammals' sperm microbiome: current knowledge, challenges, and perspectives on metagenomics of seminal samples.

Bacterial growth is highly detrimental to sperm quality and functionality. However, during the last few years, using sequencing techniques with a metagenomic approach, it has been possible to deepen the study of bacteria-sperm relationships and describe non-culturable species and synergistic and antagonistic relationships between the different species in mammalian animals. We compile the recent metagenomics studies performed on mammalian semen samples and provide updated evidence to understand the importance of the microbial communities in the results of sperm quality and sperm functionality of males, looking for future perspectives on how these technologies can collaborate in the development of andrological knowledge.

Metabologenomics analysis of Pseudomonas sp. So3.2b, an Antarctic strain with bioactivity against Rhizoctonia solani.

Introduction: Phytopathogenic fungi are a considerable concern for agriculture, as they can threaten the productivity of several crops worldwide. Meanwhile, natural microbial products are acknowledged to play an important role in modern agriculture as they comprehend a safer alternative to synthetic pesticides. Bacterial strains from underexplored environments are a promising source of bioactive metabolites. Methods: We applied the OSMAC (One Strain, Many Compounds) cultivation approach, in vitro bioassays, and metabolo-genomics analyses to investigate the biochemical potential of Pseudomonas sp. So3.2b, a strain isolated from Antarctica. Crude extracts from OSMAC were analyzed through HPLC-QTOF-MS/MS, molecular networking, and annotation. The antifungal potential of the extracts was confirmed against Rhizoctonia solani strains. Moreover, the whole-genome sequence was studied for biosynthetic gene clusters (BGCs) identification and phylogenetic comparison. Results and Discussion: Molecular networking revealed that metabolite synthesis has growth media specificity, and it was reflected in bioassays results against R. solani. Bananamides, rhamnolipids, and butenolides-like molecules were annotated from the metabolome, and chemical novelty was also suggested by several unidentified compounds. Additionally, genome mining confirmed a wide variety of BGCs present in this strain, with low to no similarity with known molecules. An NRPS-encoding BGC was identified as responsible for producing the banamides-like molecules, while phylogenetic analysis demonstrated a close relationship with other rhizosphere bacteria. Therefore, by combining -omics approaches and in vitro bioassays, our study demonstrates that Pseudomonas sp. So3.2b has potential application to agriculture as a source of bioactive metabolites.

Measuring the effect of climate change in Antarctic microbial communities: toward novel experimental approaches.

The Antarctic continent is undergoing a rapid warming, affecting microbial communities throughout its ecosystems. This continent is a natural laboratory for studying the effect of climate change, however, assessing the microbial communities' responses to environmental changes is challenging from a methodological point of view. We suggest novel experimental designs, including multivariable assessments that apply multiomics methods in combination with continuous environmental data recording and new warming simulation systems. Moreover, we propose that climate change studies in Antarctica should consider three main objectives, including descriptive studies, short-term temporary adaptation studies, and long-term adaptive evolution studies. This will help us to understand and manage the effects of climate change on the Earth.

Genome Sequence of Pseudomonas sp. Strain So3.2b, Isolated from a Soil Sample from Robert Island (Antarctic Specially Protected Area 112), Antarctic.

Strain So3.2b of the genus Pseudomonas was isolated from a soil sample from Robert Island (Antarctic Specially Protected Area 112), Antarctic. We report the complete genome sequence of this isolate, with a length of 6.17 Mbp and a GC content of 60.5%.

Synthesis of Antimicrobial Chitosan-Silver Nanoparticles Mediated by Reusable Chitosan Fungal Beads.

Nanoparticles, especially silver nanoparticles (Ag NPs), have gained significant attention in recent years as potential alternatives to traditional antibiotics for treating infectious diseases due to their ability to inhibit the growth of microorganisms effectively. Ag NPs can be synthesized using fungi extract, but the method is not practical for large-scale production due to time and biomass limitations. In this study, we explore the use of chitosan to encapsulate the mycelia of the white-rot fungus Stereum hirsutum and form chitosan fungal beads for use in multiple extractions and nanoparticle synthesis. The resulting nanoparticles were characterized using various techniques, including UV-vis spectrophotometry, transmission electron microscopy, dynamic light scattering, and X-ray diffraction analysis. The analysis revealed that the synthesized nanoparticles were composed of chitosan-silver nanoparticles (CS-Ag NPs) with a size of 25 nm. The chitosan fungal beads were reused in three extractions and nanoparticle synthesis before they lost their ability to produce CS-Ag NPs. The CS-Ag NPs showed potent antimicrobial activity against phytopathogenic and human pathogenic microorganisms, including Pseudomonas syringae, Escherichia coli, Staphylococcus aureus, and Candida albicans, with minimum inhibitory concentrations of 1.5, 1.6, 3.1, and 4 µg/mL, respectively. The antimicrobial activity of CS-Ag NPs was from 2- to 40-fold higher than Ag NPs synthesized using an aqueous extract of unencapsulated fungal biomass. The CS-Ag NPs were most effective at a pH of five regarding the antimicrobial activity. These results suggest that the chitosan fungal beads may be a promising alternative for the sustainable and cost-effective synthesis of CS-Ag NPs with improved antimicrobial activity.

Antarctic Sphingomonas sp. So64.6b showed evolutive divergence within its genus, including new biosynthetic gene clusters.

Introduction: The antibiotic crisis is a major human health problem. Bioprospecting screenings suggest that proteobacteria and other extremophile microorganisms have biosynthetic potential for the production novel antimicrobial compounds. An Antarctic Sphingomonas strain (So64.6b) previously showed interesting antibiotic activity and elicitation response, then a relationship between environmental adaptations and its biosynthetic potential was hypothesized. We aimed to determine the genomic characteristics in So64.6b strain related to evolutive traits for the adaptation to the Antarctic environment that could lead to its diversity of potentially novel antibiotic metabolites. Methods: The complete genome sequence of the Antarctic strain was obtained and mined for Biosynthetic Gene Clusters (BGCs) and other unique genes related to adaptation to extreme environments. Comparative genome analysis based on multi-locus phylogenomics, BGC phylogeny, and pangenomics were conducted within the closest genus, aiming to determine the taxonomic affiliation and differential characteristics of the Antarctic strain. Results and discussion: The Antarctic strain So64.6b showed a closest identity with Sphingomonas alpina, however containing a significant genomic difference of ortholog cluster related to degradation multiple pollutants. Strain So64.6b had a total of six BGC, which were predicted with low to no similarity with other reported clusters; three were associated with potential novel antibiotic compounds using ARTS tool. Phylogenetic and synteny analysis of a common BGC showed great diversity between Sphingomonas genus but grouping in clades according to similar isolation environments, suggesting an evolution of BGCs that could be linked to the specific ecosystems. Comparative genomic analysis also showed that Sphingomonas species isolated from extreme environments had the greatest number of predicted BGCs and a higher percentage of genetic content devoted to BGCs than the isolates from mesophilic environments. In addition, some extreme-exclusive clusters were found related to oxidative and thermal stress adaptations, while pangenome analysis showed unique resistance genes on the Antarctic strain included in genetic islands. Altogether, our results showed the unique genetic content on Antarctic strain Sphingomonas sp. So64.6, -a probable new species of this genetically divergent genus-, which could have potentially novel antibiotic compounds acquired to cope with Antarctic poly-extreme conditions.

Bacteria and Boar Semen Storage: Progress and Challenges.

Porcine breeding today is based on artificial insemination with chilled semen. This is stored at 5 °C with antibiotic supplementation to avoid bacteriospermia. There are many negative consequences on sperm quality and functionality as a result of bacterial contamination, as well as on the health of the sow. Nowadays, various techniques are being developed to reduce the indiscriminate use of antibiotics and thus avoid the generation of antibiotic resistance genes. This review aims to inform about the bacterial contamination consequences of storing liquid semen from boar and to provide an update on current methods and alternatives to antibiotic use in cold storage.

Metagenomic Characterization of Resistance Genes in Deception Island and Their Association with Mobile Genetic Elements.

Antibiotic resistance genes (ARGs) are undergoing a remarkably rapid geographic expansion in various ecosystems, including pristine environments such as Antarctica. The study of ARGs and environmental resistance genes (ERGs) mechanisms could provide a better understanding of their origin, evolution, and dissemination in these pristine environments. Here, we describe the diversity of ARGs and ERGs and the importance of mobile genetic elements as a possible mechanism for the dissemination of resistance genes in Antarctica. We analyzed five soil metagenomes from Deception Island in Antarctica. Results showed that detected ARGs are associated with mechanisms such as antibiotic efflux, antibiotic inactivation, and target alteration. On the other hand, resistance to metals, surfactants, and aromatic hydrocarbons were the dominant ERGs. The taxonomy of ARGs showed that Pseudomonas, Psychrobacter, and Staphylococcus could be key taxa for studying antibiotic resistance and environmental resistance to stress in Deception Island. In addition, results showed that ARGs are mainly associated with phage-type mobile elements suggesting a potential role in their dissemination and prevalence. Finally, these results provide valuable information regarding the ARGs and ERGs in Deception Island including the potential contribution of mobile genetic elements to the spread of ARGs and ERGs in one of the least studied Antarctic ecosystems to date.

Association of Progranulin Gene Expression from Dyspeptic Patients with Virulent Helicobacter pylori Strains; In Vivo Model.

(1) Background: Gastric cancer, the fourth most common cause of death from tumors in the world, is closely associated with Helicobacter pylori. Timely diagnosis, therefore, is essential to achieve a higher survival rate. In Chile, deaths from gastric cancer are high, mainly due to late diagnosis. Progranulin has reflected the evolution of some cancers, but has been poorly studied in gastric lesions. Aiming to understand the role of progranulin in H. pylori infection and its evolution in development of gastric lesions, we evaluated the genic expression of progranulin in gastric tissue from infected and non-infected patients, comparing it according to the epithelial status and virulence of H. pylori strains. (2) Methods: The genic expression of progranulin by q-PCR was quantified in gastric biopsies from Chilean dyspeptic patients (n = 75) and individuals who were uninfected (n = 75) by H. pylori, after receiving prior informed consent. Bacteria were grown on a medium Columbia agar with equine-blood 7%, antibiotics (Dent 2%, OxoidTM), in a microaerophilic environment, and genetically characterized for the ureC, vacA, cagA, and iceA genes by PCR. The status of the tissue was determined by endoscopic observation. (3) Results: Minor progranulin expression was detected in atrophic tissue, with a sharp drop in the tissue colonized by H. pylori that carried greater virulence, VacAs1m1+CagA+IceA1+. (4) Conclusions: Progranulin shows a differential behavior according to the lesions and virulence of H. pylori, affecting the response of progranulin against gastric inflammation.

Mucilaginibacter sp. Strain Metal(loid) and Antibiotic Resistance Isolated from Estuarine Soil Contaminated Mine Tailing from the Fundão Dam.

In 2015 a mine dam with Mn-Fe-rich tailings collapsed releasing million tons of sediments over an estuary, in the Southwest of Brazil. The tailings have a high concentration of metals that contaminated soil until the present day. The high contaminant concentrations possibly caused a selection for microorganisms able to strive in such harsh conditions. Here, we isolated metal(loid) and anti-biotic resistance bacteria from the contaminated estuarine soil. After 16S rDNA sequencing to identify the strains, we selected the Mucilaginibacter sp. strain for a whole-genome sequence due to the bioprospective potential of the genus and the high resistance profile. We obtained a complete genome and a genome-guided characterization. Our finding suggests that the 21p strain is possibly a new species of the genus. The species presented genes for resistance for metals (i.e., As, Zn, Co, Cd, and Mn) beyond resistance and cross-resistance for antibiotics (i.e., quinolone, aminoglycoside, ß-lactamase, sulphonamide, tetracycline). The Mucilaginibacter sp. 21p description as new species should be further explored, as their extracellular polymeric substances and the potential of this strain as bioremediation and as a growth promoter in high met-al(loid) contaminated soil.

Complete Genome Sequence of a Mucilaginibacter sp. Strain Isolated from Estuarine Soil Contaminated with Mine Tailings from the Samarco Disaster at Fundão Dam.

We report the complete genome sequence of Mucilaginibacter strain 21P, which was isolated from estuarine soil contaminated with mine tailings from the Samarco disaster, which occurred in 2015 in Brazil. The genome sequence comprised 4,739,655 bp, with a G+C content of 43.2%, and harbors multiple antibiotic and metal resistance genes.

Two Archaeal Metagenome-Assembled Genomes from El Tatio Provide New Insights into the Crenarchaeota Phylum.

A phylogenomic and functional analysis of the first two Crenarchaeota MAGs belonging to El Tatio geysers fields in Chile is reported. A soil sample contiguous to a geothermal activity exposed lagoon of El Tatio was used for shotgun sequencing. Afterwards, contigs were binned into individual population-specific genomes data. A phylogenetic placement was carried out for both MAG 9-5TAT and MAG 47-5TAT. Then functional comparisons and metabolic reconstruction were carried out. Results showed that both MAG 9-5TAT and MAG 47-5TAT likely represent new species in the genus Thermoproteus and the genus Sulfolobus, respectively. These findings provide new insights into the phylogenetic and genomic diversity for archaea species that inhabit the El Tatio geysers field and expand the understanding of the Crenarchaeota phylum diversity.

Complete Genome Sequence of Bacillus safensis Strain 3A, a Heavy Metal-Resistant Bacterium Isolated from Contaminated Estuarine Sediment in Brazil.

Bacillus safensis 3A was isolated from a contaminated estuarine sediment sample with mine tailing from the Samarco dam disaster, which occurred in 2015 in Minas Gerais State, Brazil. We report here a draft genome sequence (3.6 Mb) of this bacterial strain. B. safensis exhibited strong resistance to heavy metals.

Bacterial communities in fecal samples of myotis chiloensis from Southern, Chile / Comunidades bacterianas presentes en muestras fecales de myotis chiloensis en el sur de Chile

SUMMARY: The insectivorous bat Myotis chiloensis is endemic of South America. Even though potentially pathogenic bacterial species of Mycoplasma have been reported from this species, there are no further studies regarding the bacterial communities they harbor. This may provide important insights for the better understanding of its ecology, diet and implications in cross-species pathogens transmission. Here we report a first survey on bacterial communities of M. chiloensis based on metagenomic analysis of fecal samples. We found that taxonomic profile is dominated by Proteobacteria (23.7 to 57.7 %) and Firmicutes (11.8 to 61.6 %), which main families are represented by Burkholderiaceae- Enterobacteriaceae and Veillonellaceae-Bacillaceae, respectively. Phyla Bacteroidetes, Actinobacteria, Cyanobacteria, Planctomycetes and Acidobacteria were also present with abundance above 1 % of the total reads. Variations among individuals could be observed at genus level and no significant differences were found between sex groups regarding taxonomic profiles and diversity. Potentially pathogenic species were also detected in all the samples, including Staphylococcus aureus and Clostridium perfringens. Our results highlight the significance M. chiloensis as a reservoir of pathogenic bacteria and its microbiota as an interesting ecological model due to its wide distribution. Further metagenomic studies are necessary for a better understanding of M. chiloensis diet and its host-symbiont relationships.

RESUMEN: El murciélago insectívoro Myotis chiloensis es endémico de América del Sur. A pesar de que en esta especie se han reportado bacterias potencialmente patógenas tipo Mycoplasma, no existen estudios sobre sus comunidades bacterianas, lo cual podría proporcionar información importante para una mejor comprensión de su ecología, dieta e implicaciones en la transmisión de patógenos. En el presente trabajo se realiza una descripción de las comunidades bacterianas del murciélago M. chiloensis basada en análisis metagenómico de muestras fecales. El perfil taxonómico encontradofue dominado por Proteobacterias (23,7-57,7 %) y Firmicutes (11,8-61,6 %), cuyas principales familias fueron representadas por Burkholderiaceae-Enterobacteriaceae y Veillonellaceae-Bacillaceae, respectivamente. También se encontraron los filos Bacteroidetes, Actinobacteria, Cyanobacteria, Planctomycetes y Acidobacteria con una abundancia superior al 1 %. Se observaron variaciones entre los individuos a nivel de género, sin diferencias significativas de los perfiles taxonómicos y diversidad según sexo. Se detectaron especies potencialmente patógenas en todas las muestras, entre ellos Staphylococcus aureus y Clostridium perfringens. Nuestros resultados destacan la importancia de M. chiloensis como un reservorio de bacterias patógenas y el estudio de su microbiota como un modelo ecológico debido a su amplia distribución. Más estudios metagenómicos son necesarios para comprender la dieta de M. chiloensis y sus relaciones huésped-simbionte.

MALDI-TOF MS and 16S RNA Identification of Culturable Gastric Microbiota: Variability Associated with the Presence of Helicobacter pylori.

Helicobacter pylori is the main bacteria associated with gastroduodenal diseases. Recent studies have reported that gastric microbiota might be modified by the H. pylori colonization, favoring gastric lesions' development. In Chile, the region of La Araucanía concentrates a high risk of gastric cancer associated with Helicobacter pylori colonization, rurality, poverty, and Mapuche ethnicity. Hence, we aimed to identify the culturable gastric microbiota and characterize its variability at different stages of epithelial injury, based on its H. pylori colonization in dyspeptic patients from this Chilean region. Microaerophilic bacteria strains were isolated from antrum biopsies of 155 dyspeptic patients' biopsies and identified using MALDI-TOF MS or 16sRNA gene sequencing for non-pylori species identification, and UreC gene amplification for H. pylori confirmation. We found 48 species from 18 families, mainly belonging to Neisseriaceae (21.3%), Streptococcaceae (20.0%), Actynomicetaceae (9.0%), Enterobacteriaceae, and Lactobacillaceae (4.5%); however, Streptococcaceae and Actinomycetaceae families showed a significant reduction in samples infected with H. pylori, along with a considerably lower diversity of species. Our results revealed a microbiota modification due to H. pylori colonization associated with the gastric epithelial state, suggesting a potential microbiota role for developing and progressing gastric diseases.

Genomic and Metabolomic Analysis of Antarctic Bacteria Revealed Culture and Elicitation Conditions for the Production of Antimicrobial Compounds.

Concern about finding new antibiotics against drug-resistant pathogens is increasing every year. Antarctic bacteria have been proposed as an unexplored source of bioactive metabolites; however, most biosynthetic gene clusters (BGCs) producing secondary metabolites remain silent under common culture conditions. Our work aimed to characterize elicitation conditions for the production of antibacterial secondary metabolites from 34 Antarctic bacterial strains based on MS/MS metabolomics and genome mining approaches. Bacterial strains were cultivated under different nutrient and elicitation conditions, including the addition of lipopolysaccharide (LPS), sodium nitroprusside (SNP), and coculture. Metabolomes were obtained by HPLC-QTOF-MS/MS and analyzed through molecular networking. Antibacterial activity was determined, and seven strains were selected for genome sequencing and analysis. Biosynthesis pathways were activated by all the elicitation treatments, which varies among strains and dependents of culture media. Increased antibacterial activity was observed for a few strains and addition of LPS was related with inhibition of Gram-negative pathogens. Antibiotic BGCs were found for all selected strains and the expressions of putative actinomycin, carotenoids, and bacillibactin were characterized by comparison of genomic and metabolomic data. This work established the use of promising new elicitors for bioprospection of Antarctic bacteria and highlights the importance of new "-omics" comparative approaches for drug discovery.

Computational methods for 16S metabarcoding studies using Nanopore sequencing data.

Assessment of bacterial diversity through sequencing of 16S ribosomal RNA (16S rRNA) genes has been an approach widely used in environmental microbiology, particularly since the advent of high-throughput sequencing technologies. An additional innovation introduced by these technologies was the need of developing new strategies to manage and investigate the massive amount of sequencing data generated. This situation stimulated the rapid expansion of the field of bioinformatics with the release of new tools to be applied to the downstream analysis and interpretation of sequencing data mainly generated using Illumina technology. In recent years, a third generation of sequencing technologies has been developed and have been applied in parallel and complementarily to the former sequencing strategies. In particular, Oxford Nanopore Technologies (ONT) introduced nanopore sequencing which has become very popular among molecular ecologists. Nanopore technology offers a low price, portability and fast sequencing throughput. This powerful technology has been recently tested for 16S rRNA analyses showing promising results. However, compared with previous technologies, there is a scarcity of bioinformatic tools and protocols designed specifically for the analysis of Nanopore 16S sequences. Due its notable characteristics, researchers have recently started performing assessments regarding the suitability MinION on 16S rRNA sequencing studies, and have obtained remarkable results. Here we present a review of the state-of-the-art of MinION technology applied to microbiome studies, the current possible application and main challenges for its use on 16S rRNA metabarcoding.