Spatio-temporal insights into microbiology of the freshwater-to-hypersaline, oxic-hypoxic-euxinic waters of Ursu Lake

Ursu Lake is located in the Middle Miocene salt deposit of Central Romania. It is stratified, and the water column has three distinct water masses: an upper freshwater-to-moderately saline stratum (0–3 m), an intermediate stratum exhibiting a steep halocline (3–3.5 m), and a lower hypersaline stratum (4 m and below) that is euxinic (i.e. anoxic and sulphidic). Recent studies have characterized the lake's microbial taxonomy and given rise to intriguing ecological questions. Here, we explore whether the communities are dynamic or stable in relation to taxonomic composition, geochemistry, biophysics, and ecophysiological functions during the annual cycle. We found: (i) seasonally fluctuating, light-dependent communities in the upper layer (≥0.987–0.990 water-activity), a stable but phylogenetically diverse population of heterotrophs in the hypersaline stratum (water activities down to 0.762) and a persistent plate of green sulphur bacteria that connects these two (0.958–0.956 water activity) at 3–3.5 to 4 m; (ii) communities that might be involved in carbon- and sulphur-cycling between and within the lake's three main water masses; (iii) uncultured lineages including Acetothermia (OP1), Cloacimonetes (WWE1), Marinimicrobia (SAR406), Omnitrophicaeota (OP3), Parcubacteria (OD1) and other Candidate Phyla Radiation bacteria, and SR1 in the hypersaline stratum (likely involved in the anaerobic steps of carbon- and sulphur-cycling); and (iv) that species richness and habitat stability are associated with high redox-potentials. Ursu Lake has a unique and complex ecology, at the same time exhibiting dynamic fluctuations and stability, and can be used as a modern analogue for ancient euxinic water bodies and comparator system for other stratified hypersaline systems

A glimpse into the biosynthetic potential and resistome of microbial communities inhabiting sulfidic, chemoautotrophic Movile Cave ecosystem

Background: Microbial secondary metabolites (SM), especially those produced by soil microorganisms, have been a valuable source of antibiotics, antitumor agents, pigments, growth-promoting substances, etc., with tremendous market potential. These molecules are encoded by biosynthetic gene clusters (BGCs) within the bacterial genome. Their synthesis confers survival advantages by facilitating chemical defense, interspecies communication, and adaptation to well-defined ecological niches.Caves, particularly Movile Cave (Romania) - a sulfidic autotrophic-based ecosystem - can be considered extreme environments suited to investigate and discover novel bioactive microbial molecules. Here, low nutrient availability can lead to resource competition and, consequently, antimicrobial production to deter nearby microbial competitors.Aim: Our study focused on highlighting the biosynthetic gene clusters (BGCs) and biosynthetic potential of the sediment-associated microbiome in Movile Cave.Methods: Over 100 high-quality metagenome-assembled genomes (MAGs) were retrieved by whole-community shotgun-sequencing of 7 sediment samples collected in different Movile Cave’s galleries (Chiciudean et al. 2022). Detected MAGs were then analyzed for the presence of BGCs by antiSMASH (v. 6.1.1) (Blin et al. 2021) whereas antibiotic resistance genes were predicted by ResFinder (v. 4.1) (Florensa et al. 2022). The statistical analysis of BGCs data was performed by Past (v. 4.03).Results: We detected 637 BGCs across 106 high-quality MAGs that were affiliated to 22 phyla. The diversity of predicted BGCs varies across sediment samples with no apparent correlation to the number of analyzed MAGs per sample. The MAGs recovered from the sulfidic water-sediment interface (sample code PMV4) had the lowest alpha BGCs diversity among all sampled locations and it was clearly distinct in BGCs composition and abundance (β-diversity) from dry gallery samples (PMV7 and PMV8). The most abundant BGCs predicted in Movile Cave metagenomic dataset encode for terpenes, non-ribosomal peptides (NRPs) and ribosomally synthesised and post-translationally modified peptides (RIPPs). Acidobacteriota and Chloroflexota- affiliated MAGs were most enriched in BCG containing 20 and 23 BGCs per MAG, respectively, in contrast with the candidate phylum Ca. Patescibacteria-related MAGs that showed no SM biosynthetic capabilities. Two antimicrobial resistance (AR) genes (ole(C), oqxB) encoding resistance to antibiotics (i.e. oleandomycin, chloramphenicol, ciprofloxacin, trimethoprim) and disinfectants were identified in MAGs affiliated with the class Actinomycetia and Gammaproteobacteria.Based on the analyzed data, the biosynthetic potential of Movile Cave is significant compared with other microbiomes (Donia et al. 2014) and has a pronounced degree of novelty whereas the resistome (that is the genetic potential for antibiotic resistance) is reduced. Considering the uncommon futures of Movile Cave environment, future in-depth analysis of the identified BGCs might lead to the discovery of novel bioactive compounds. Additionally, the seclusion of this environment may provide an exciting opportunity for surveying the occurrence and environmental drivers of natural AR traits

Blossoms of Rot: Microbial Life in Saline Organic-Rich Sediments

The organic-rich sediments (or “sapropels”) are complex organo-mineral and microoxic/anoxic milieus underlying stagnant aquatic basins with high input of organic matter in addition to persistent salinity and density gradients. Sapropels of brackish, marine and hypersaline aquatic systems were found to harbor one of the most diverse microbiomes reported to date, and their study may contribute to elucidating the phylogenetic branching of the tree of life. In the present chapter, we explore the links between the sapropels' chemistry and innate biological diversity and postulate that highly connected systems favor phylogenetic and metabolic specialization. In these salty organic-rich sediments, the fermentative and sulfate-reducing bacteria alongside methanogenic archaea and a plethora of bacterial and archaeal candidate lineages are proposed to cooperate in the final steps of organic matter degradation. Moreover, recent findings suggest that brackish, marine and hypersaline modern sapropels-associated microbiomes include yet-uncultured Archaea that may lay close to the evolutionary roots of eukaryotes and life itself. Future investigations of both ancient and modern organic-rich sediments may have deep implications on: i) shedding light on the past events that shaped the evolutionary history of life and ii) formulating and testing hypotheses on the mechanisms that microbes may use to cope with thermodynamic (e.g., use of low Gibbs free energy metabolic conversions) and environmental (e.g., microoxic/anoxic and saline) constraints that could be considered analogous to extraterestrial conditions

Genome-wide transcriptional response to silver stress in extremely halophilic archaeon Haloferax alexandrinus DSM 27206 T

Abstract Background The extremely halophilic archaeon Haloferax (Hfx.) alexandrinus DSM 27206 T was previously documented for the ability to biosynthesize silver nanoparticles while mechanisms underlying its silver tolerance were overlooked. In the current study, we aimed to assess the transcriptional response of this haloarchaeon to varying concentrations of silver, seeking a comprehensive understanding of the molecular determinants underpinning its heavy metal tolerance. Results The growth curves confirmed the capacity of Hfx. alexandrinus to surmount silver stress, while the SEM–EDS analysis illustrated the presence of silver nanoparticles in cultures exposed to 0.5 mM silver nitrate. The RNA-Seq based transcriptomic analysis of Hfx. alexandrinus cells exposed to 0.1, 0.25, and 0.5 mM silver nitrate revealed the differential expression of multiple sets of genes potentially employed in heavy-metal stress response, genes mostly related to metal transporters, basic metabolism, oxidative stress response and cellular motility. The RT-qPCR analysis of selected transcripts was conducted to verify and validate the generated RNA-Seq data. Conclusions Our results indicated that copA, encoding the copper ATPase, is essential for the survival of Hfx. alexandrinus cells in silver-containing saline media. The silver-exposed cultures underwent several metabolic adjustments that enabled the activation of enzymes involved in the oxidative stress response and impairment of the cellular movement capacity. To our knowledge, this study represents the first comprehensive analysis of gene expression in halophillic archaea facing increased levels of heavy metals

Monitoring and risk assessment for groundwater sources in rural communities of Romania (GROUNDWATERISK)

In the past 100 years, a decreasing rainfall trend has been recorded on Romanian territory, a trend that continues today. Therefore, realistic estimation of the groundwater resources is crucial, especially for the rural communities lacking the economic power to use alternative sources of drinking water. The groundwater sources used by rural communities in Romania generally originate directly from caves, wells or springs with no proper evaluation of the water quality. Groundwater is exposed to different pollutants, as bats' guano in caves, fertilizers in agricultural areas or livestock (cattle, sheep, goats, etc.) farms on the surface. On the other hand, the water extracted directly from inside the caves is affecting groundwater ecosystems, highly vulnerable to any human impact and neglected by European legislation so far. The project aims to monitor, during two consecutive years, groundwater sources with different degrees of above- and underground pollution, from different regions of Romania. To achieve the goals of the project, a multidisciplinary monitoring strategy that will include measurements of hydrological, physico-chemical and biological (microbiology and aquatic invertebrates’ assessment) parameters alongside the quantification of radon and stable isotopes, rainfall or possible inflows of water. The specific outcomes of this project are: i) to test, develop and validate a new, more rapid and efficient method for monitoring and risk assessment of groundwater sources – and not only – by using molecular techniques, and propose this method to the water agencies in Romania; ii) to propose for Romanian authorities to implement a harmonized coherent methodology to measure radon concentration in water, as a consequence of EURATOM Directive; and iii) to educate local communities that are using groundwater as source for drinking water and raise young people’s awareness on the benefits of ecosystem services provided by the groundwater

Monitoring and risk assessment for groundwater sources in rural communities of Romania (GROUNDWATERISK)

In the past 100 years, a decreasing rainfall trend has been recorded on Romanian territory, a trend that continues today. Therefore, realistic estimation of the groundwater resources is crucial, especially for the rural communities lacking the economic power to use alternative sources of drinking water. The groundwater sources used by rural communities in Romania generally originate directly from caves, wells or springs with no proper evaluation of the water quality. Groundwater is exposed to different pollutants, as bats' guano in caves, fertilizers in agricultural areas or livestock (cattle, sheep, goats, etc.) farms on the surface. On the other hand, the water extracted directly from inside the caves is affecting groundwater ecosystems, highly vulnerable to any human impact and neglected by European legislation so far. The project aims to monitor, during two consecutive years, groundwater sources with different degrees of above- and underground pollution, from different regions of Romania. To achieve the goals of the project, a multidisciplinary monitoring strategy that will include measurements of hydrological, physico-chemical and biological (microbiology and aquatic invertebrates’ assessment) parameters alongside the quantification of radon and stable isotopes, rainfall or possible inflows of water. The specific outcomes of this project are: i) to test, develop and validate a new, more rapid and efficient method for monitoring and risk assessment of groundwater sources – and not only – by using molecular techniques, and propose this method to the water agencies in Romania; ii) to propose for Romanian authorities to implement a harmonized coherent methodology to measure radon concentration in water, as a consequence of EURATOM Directive; and iii) to educate local communities that are using groundwater as source for drinking water and raise young people’s awareness on the benefits of ecosystem services provided by the groundwater

Competition-cooperation in the chemoautotrophic ecosystem of Movile Cave: first metagenomic approach on sediments

Movile Cave (SE Romania) is a chemoautotrophically-based ecosystem fed by hydrogen sulfide-rich groundwater serving as a primary energy source analogous to the deep-sea hydrothermal ecosystems. Our current understanding of Movile Cave microbiology has been confined to the sulfidic water and its proximity, as most studies focused on the water-floating microbial mat and planktonic accumulations likely acting as the primary production powerhouse of this unique subterranean ecosystem. By employing comprehensive genomic-resolved metagenomics, we questioned the spatial variation, chemoautotrophic abilities, ecological interactions and trophic roles of Movile Cave’s microbiome thriving beyond the sulfidic-rich water

Data from: Contrasting taxonomic stratification of microbial communities in two hypersaline meromictic lakes

Hypersaline meromictic lakes are extreme environments in which water stratification is associated with powerful physicochemical gradients and high salt concentrations. Furthermore, their physical stability coupled with vertical water column partitioning makes them important research model systems in microbial niche differentiation and biogeochemical cycling. Here, we compare the prokaryotic assemblages from Ursu and Fara Fund hypersaline meromictic lakes (Transylvanian Basin, Romania) in relation to their limnological factors and infer their role in elemental cycling by matching taxa to known taxon-specific biogeochemical functions. To assess the composition and structure of prokaryotic communities and the environmental factors that structure them, deep-coverage small subunit (SSU) ribosomal RNA (rDNA) amplicon sequencing, community domain-specific quantitative PCR and physicochemical analyses were performed on samples collected along depth profiles. The analyses showed that the lakes harbored multiple and diverse prokaryotic communities whose distribution mirrored the water stratification patterns. Ursu Lake was found to be dominated by Bacteria and to have a greater prokaryotic diversity than Fara Fund Lake that harbored an increased cell density and was populated mostly by Archaea within oxic strata. In spite of their contrasting diversity, the microbial populations indigenous to each lake pointed to similar physiological functions within carbon degradation and sulfate reduction. Furthermore, the taxonomy results coupled with methane detection and its stable C isotope composition indicated the presence of a yet-undescribed methanogenic group in the lakes’ hypersaline monimolimnion. In addition, ultrasmall uncultivated archaeal lineages were detected in the chemocline of Fara Fund Lake, where the recently proposed Nanohaloarchaeota phylum was found to thrive

Spatial Distribution and Molecular Diversity of Archaeal Communities in the Extreme Hypersaline Meromictic Brâncoveanu Lake (Transylvanian Basin, Romania)

<p>Dating from the Middle Miocene, the massive halite deposits lying beneath the Transylvanian Basin (Central Romania) have been valuable mineral resources quarried for millennia. Among the numerous hypersaline pit lakes that resulted from this mining, Brâncoveanu Lake is unique by its extreme salinity. Assessment of physicochemical variables, water chemistry and trophic status indicated that Brâncoveanu Lake is a permanently stratified, pH-neutral, NaCl-rich and eutrophied system. We investigated the abundance, molecular diversity and vertical distribution of archaeal community by culture-independent approaches. Additionally, the most relevant environmental parameters shaping the archaeal community composition were evaluated by statistical methods. Archaea appeared to largely outnumber Bacteria; altogether the great prevalence of Halobacteriaceae-related sequences could imply a major contribution of this group to the biogeochemical carbon turnover. The fairly distinct composition of archaeal communities reflects the lake's physicochemical stratification. Among the limnological factors, salinity and oxygen showed a significant impact on determining the composition and structure of archaeal assemblages. Furthermore, Brâncoveanu Lake might harbor novel microorganisms such as members of the recently described phylum Nanohaloarchaea. Overall, this study reported the occurrence of halophilic Archaea in a little explored hydrogeochemical system and provided a better insight into geomicrobiology of meromictic hypersaline pit lakes.</p

Testing Different Membrane Filters for 16S rRNA Gene-Based Metabarcoding in Karstic Springs

Introduction: Karstic springs are used worldwide by rural communities as sources of fresh water for humans and livestock. In Romania, one-third of the population has no direct access to a public water supply. The present study is part of a country-wide project to develop simple, quick and cheap methods for seasonal environmental and microbiological monitoring of karstic springs used as drinking water by rural populations. Critical steps for monitoring workflow consist of evaluating water quality and selecting suitable membrane filters to efficiently capture environmental DNA for further microbial diversity estimation using 16S rRNA gene-based metabarcoding. Methods: Several commercial membrane filters of different compositions and pore sizes were tested on the water sampled from three karstic springs in Romania, followed by water chemistry and whole community 16S rRNA gene-based metabarcoding analysis. Results: We found that different types of applied membrane filters provide varying recovery in diversity and abundance of both overall and pathogenic bacteria. Conclusions: The result of the experiment with different filters shows that mixed cellulose ester, cellulose acetate, and nitrate membranes of 0.20 and 0.22 &micro;m are the best for amplicon-based metabarcoding monitoring of karst springs