Structural and functional analysis of the active cow rumen's microbial community provides a catalogue of genes and microbes participating in the deconstruction of cardoon biomass.

BACKGROUND: Ruminal microbial communities enriched on lignocellulosic biomass have shown considerable promise for the discovery of microorganisms and enzymes involved in digesting cell wall compounds, a key bottleneck in the development of second-generation biofuels and bioproducts, enabling a circular bioeconomy. Cardoon (Cynara cardunculus) is a promising inedible energy crop for current and future cellulosic biorefineries and the emerging bioenergy and bioproducts industries. The rumen microbiome can be considered an anaerobic "bioreactor", where the resident microbiota carry out the depolymerization and hydrolysis of plant cell wall polysaccharides (PCWPs) through the catalytic action of fibrolytic enzymes. In this context, the rumen microbiota represents a potential source of microbes and fibrolytic enzymes suitable for biofuel production from feedstocks. In this study, metatranscriptomic and 16S rRNA sequencing were used to profile the microbiome and to investigate the genetic features within the microbial community adherent to the fiber fractions of the rumen content and to the residue of cardoon biomass incubated in the rumen of cannulated cows. RESULTS: The metatranscriptome of the cardoon and rumen fibre-adherent microbial communities were dissected in their functional and taxonomic components. From a functional point of view, transcripts involved in the methanogenesis from CO2 and H2, and from methanol were over-represented in the cardoon-adherent microbial community and were affiliated with the Methanobrevibacter and Methanosphaera of the Euryarchaeota phylum. Transcripts encoding glycoside hydrolases (GHs), carbohydrate-binding modules (CBMs), carbohydrate esterases (CEs), polysaccharide lyases (PLs), and glycoside transferases (GTs) accounted for 1.5% (6,957) of the total RNA coding transcripts and were taxonomically affiliated to major rumen fibrolytic microbes, such as Oscillospiraceae, Fibrobacteraceae, Neocallimastigaceae, Prevotellaceae, Lachnospiraceae, and Treponemataceae. The comparison of the expression profile between cardoon and rumen fiber-adherent microbial communities highlighted that specific fibrolytic enzymes were potentially responsible for the breakdown of cardoon PCWPs, which was driven by specific taxa, mainly Ruminococcus, Treponema, and Neocallimastigaceae. CONCLUSIONS: Analysis of 16S rRNA and metatranscriptomic sequencing data revealed that the cow rumen microbiome harbors a repertoire of new enzymes capable of degrading PCWPs. Our results demonstrate the feasibility of using metatranscriptomics of enriched microbial RNA as a potential approach for accelerating the discovery of novel cellulolytic enzymes that could be harnessed for biotechnology. This research contributes a relevant perspective towards degrading cellulosic biomass and providing an economical route to the production of advanced biofuels and high-value bioproducts.

Curtobacterium salicis sp. nov., isolated from willow tree stems in Washington state.

Curtobacterium sp. strain WW7 is a Gram-positive, non-motile, orange rod-shaped bacterium isolated from branches of wild willow (Salix sitchensis) trees. The WW7T strain has optimum growth in the temperature range between 25 and 30 °C, a pH range of 6-7.7, and tolerates up to 5.5% (w/v) of NaCl. The genome sequencing of strain WW7T revealed a genome size of approximately 3.8 Mbp and a G + C content of 71.3 mol%. The phylogenomic analyses support the WW7T affiliation to a novel Curtobacterium lineage, with Curtobacterium herbarum being the closest type-strain. Chemotaxonomic analysis indicates that the carbon sources assimilation profile of strain WW7T was similar to the type strains, i.e. Curtobacterium luteum, Curtobacterium albidum, and Curtobacterium flaccumfaciens, while no assimilation of the organic acids succinate, alpha-ketobutyrate, mono methyl-succinate, and lactate was observed. Finally, fatty acid methyl ester analysis identifies anteiso-C15:0 and anteiso-C17:0 as major cellular fatty acids which is a common feature for members of the Curtobacterium genus. Based on the results of phylogenomic and chemotaxonomic analyses, strain WW7T represents a novel Curtobacterium lineage, for which the name Curtobacterium salicis sp. nov. is proposed. The type strain is WW7T (DSM 34805T-NRRL B-68078T).

The actinomycete Kitasatospora sp. SeTe27, subjected to adaptive laboratory evolution (ALE) in the presence of selenite, varies its cellular morphology, redox stability, and tolerance to the toxic oxyanion.

The effects of oxyanions selenite (SeO32-) in soils are of high concern in ecotoxicology and microbiology as they can react with mineral particles and microorganisms. This study investigated the evolution of the actinomycete Kitasatospora sp. SeTe27 in response to selenite. To this aim, we used the Adaptive Laboratory Evolution (ALE) technique, an experimental approach that mimics natural evolution and enhances microbial fitness for specific growth conditions. The original strain (wild type; WT) isolated from uncontaminated soil gave us a unique model system as it has never encountered the oxidative damage generated by the prooxidant nature of selenite. The WT strain exhibited a good basal level of selenite tolerance, although its growth and oxyanion removal capacity were limited compared to other environmental isolates. Based on these premises, the WT and the ALE strains, the latter isolated at the end of the laboratory evolution procedure, were compared. While both bacterial strains had similar fatty acid profiles, only WT cells exhibited hyphae aggregation and extensively produced membrane-like vesicles when grown in the presence of selenite (challenged conditions). Conversely, ALE selenite-grown cells showed morphological adaptation responses similar to the WT strain under unchallenged conditions, demonstrating the ALE strain improved resilience against selenite toxicity. Whole-genome sequencing revealed specific missense mutations in genes associated with anion transport and primary and secondary metabolisms in the ALE variant. These results were interpreted to show that some energy-demanding processes are attenuated in the ALE strain, prioritizing selenite bioprocessing to guarantee cell survival in the presence of selenite. The present study indicates some crucial points for adapting Kitasatospora sp. SeTe27 to selenite oxidative stress to best deal with selenium pollution. Moreover, the importance of exploring non-conventional bacterial genera, like Kitasatospora, for biotechnological applications is emphasized.

Insights into the Synergistic Antibacterial Activity of Silver Nitrate with Potassium Tellurite against Pseudomonas aeruginosa.

The constant, ever-increasing antibiotic resistance crisis leads to the announcement of "urgent, novel antibiotics needed" by the World Health Organization. Our previous works showed a promising synergistic antibacterial activity of silver nitrate with potassium tellurite out of thousands of other metal/metalloid-based antibacterial combinations. The silver-tellurite combined treatment not only is more effective than common antibiotics but also prevents bacterial recovery, decreases the risk of future resistance chance, and decreases the effective concentrations. We demonstrate that the silver-tellurite combination is effective against clinical isolates. Further, this study was conducted to address knowledge gaps in the available data on the antibacterial mechanism of both silver and tellurite, as well as to give insight into how the mixture provides synergism as a combination. Here, we defined the differentially expressed gene profile of Pseudomonas aeruginosa under silver, tellurite, and silver-tellurite combination stress using an RNA sequencing approach to examine the global transcriptional changes in the challenged cultures grown in simulated wound fluid. The study was complemented with metabolomics and biochemistry assays. Both metal ions mainly affected four cellular processes, including sulfur homeostasis, reactive oxygen species response, energy pathways, and the bacterial cell membrane (for silver). Using a Caenorhabditis elegans animal model we showed silver-tellurite has reduced toxicity over individual metal/metalloid salts and provides increased antioxidant properties to the host. This work demonstrates that the addition of tellurite would improve the efficacy of silver in biomedical applications. IMPORTANCE Metals and/or metalloids could represent antimicrobial alternatives for industrial and clinical applications (e.g., surface coatings, livestock, and topical infection control) because of their great properties, such as good stability and long half-life. Silver is the most common antimicrobial metal, but resistance prevalence is high, and it can be toxic to the host above a certain concentration. We found that a silver-tellurite composition has antibacterial synergistic effect and that the combination is beneficial to the host. So, the efficacy and application of silver could increase by adding tellurite in the recommended concentration(s). We used different methods to evaluate the mechanism for how this combination can be so incredibly synergistic, leading to efficacy against antibiotic- and silver-resistant isolates. Our two main findings are that (i) both silver and tellurite mostly target the same pathways and (ii) the coapplication of silver with tellurite tends not to target new pathways but targets the same pathways with an amplified change.

Effect of Arsenic on EPS Synthesis, Biofilm Formation, and Plant Growth-Promoting Abilities of the Endophytes Pseudomonas PD9R and Rahnella laticis PD12R.

Phytoremediation, a cost-effective, eco-friendly alternative to conventional remediation, could expand efforts to remediate arsenic-contaminated soils. As with other pollutants, the plant microbiome may improve phytoremediation outcomes for arsenic-contaminated sites. We used in vitro and in silico methods to compare the arsenic resistance mechanisms, synthesis of extracellular polymeric substances (EPS), biofilm formation, and plant growth-promoting abilities of the endophytes Pseudomonas sp. PD9R and Rahnella laticis PD12R. PD12R, which tolerates arsenate (As(V)) and arsenite (As(III)) to concentrations fivefold greater than PD9R, synthesizes high volumes of EPS in response to arsenic, and sequesters arsenic in the capsular EPS and cells. While arsenic exposure induced EPS synthesis in both strains, only PD12R continued to form biofilms at high As(III) and As(V) concentrations. The effects of endophyte inoculation on Arabidopsis growth varied by strain and As(V) concentration, and PD9R had positive effect on plants exposed to low levels of arsenic. Comparative genomic analyses exploring the EPS synthesis and arsenic resistance mechanisms against other Pseudomonas and Rahnella strains suggest that both strains possess atypical arsenic resistance mechanisms from other plant-associated strains, while the configuration of the EPS synthesis systems appeared to be more broadly distributed among plant- and non-plant-associated strains.

Insights into the microbial life in silica-rich subterranean environments: microbial communities and ecological interactions in an orthoquartzite cave (Imawarì Yeuta, Auyan Tepui, Venezuela).

Microbial communities inhabiting caves in quartz-rich rocks are still underexplored, despite their possible role in the silica cycle. The world's longest orthoquartzite cave, Imawarì Yeuta, represents a perfect arena for the investigation of the interactions between microorganisms and silica in non-thermal environments due to the presence of extraordinary amounts of amorphous silica speleothems of different kinds. In this work, the microbial diversity of Imawarì Yeuta was dissected by analyzing nineteen samples collected from different locations representative of different silica amorphization phases and types of samples. Specifically, we investigated the major ecological patterns in cave biodiversity, specific taxa enrichment, and the main ecological clusters through co-occurrence network analysis. Water content greatly contributed to the microbial communities' composition and structures in the cave leading to the sample clustering into three groups DRY, WET, and WATER. Each of these groups was enriched in members of Actinobacteriota, Acidobacteriota, and Gammaproteobacteria, respectively. Alpha diversity analysis showed the highest value of diversity and richness for the WET samples, while the DRY group had the lowest. This was accompanied by the presence of correlation patterns including either orders belonging to various phyla from WET samples or orders belonging to the Actinobacteriota and Firmicutes phyla from DRY group samples. The phylogenetic analysis of the dominant species in WET and DRY samples showed that Acidobacteriota and Actinobacteriota strains were affiliated with uncultured bacteria retrieved from various oligotrophic and silica/quartz-rich environments, not only associated with subterranean sites. Our results suggest that the water content greatly contributes to shaping the microbial diversity within a subterranean quartzite environment. Further, the phylogenetic affiliation between Imawarì Yeuta dominant microbes and reference strains retrieved from both surface and subsurface silica- and/or CO2/CO-rich environments, underlines the selective pressure applied by quartz as rock substrate. Oligotrophy probably in association with the geochemistry of silica/quartz low pH buffering activity and alternative energy sources led to the colonization of specific silica-associated microorganisms. This study provides clues for a better comprehension of the poorly known microbial life in subsurface and surface quartz-dominated environments.

Potential Biocontrol Activities of Populus Endophytes against Several Plant Pathogens Using Different Inhibitory Mechanisms.

The plant microbiome can be used to bolster plant defense against abiotic and biotic stresses. Some strains of endophytes, the microorganisms within plants, can directly inhibit the growth of plant fungal pathogens. A previously isolated endophyte from wild Populus (poplar), WPB of the species Burkholderia vietnamiensis, had robust in vitro antifungal activity against pathogen strains that are highly virulent and of concern to Pacific Northwest agriculture: Rhizoctonia solani AG-8, Fusarium culmorum 70110023, and Gaemannomyces graminis var. tritici (Ggt) ARS-A1, as well as activity against the oomycete, Pythium ultimum 217. A direct screening method was developed for isolation of additional anti-fungal endophytes from wild poplar extracts. By challenging pathogens directly with dilute extracts, eleven isolates were found to be inhibitory to at least two plant pathogen strains and were therefore chosen for further characterization. Genomic analysis was conducted to determine if these endophyte strains harbored genes known to be involved in antimicrobial activities. The newly isolated Bacillus strains had gene clusters for production of bacillomycin, fengicyn, and bacillibactin, while the gene cluster for the synthesis of sessilin, viscosin and tolaasin were found in the Pseudomonas strains. The biosynthesis gene cluster for occidiofungin (ocf) was present in the Burkholderia vietnamiensis WPB genome, and an ocf deletion mutant lost inhibitory activity against 3 of the 4 pathogens. The new isolates lacked the gene cluster for occidiofungin implying they employ different modes of action. Other symbiotic traits including nitrogen fixation, phosphate solubilization, and the production of auxins and siderophores were investigated. Although it will be necessary to conduct in vivo tests of the candidates with pathogen-infected agricultural crops, the wild poplar tree microbiome may be a rich source of beneficial endophyte strains with potential for biocontrol applications against a variety of pathogens and utilizing varying modes of action.

Systems biology and metabolic engineering of Rhodococcus for bioconversion and biosynthesis processes.

Rhodococcus spp. strains are widespread in diverse natural and anthropized environments thanks to their high metabolic versatility, biodegradation activities, and unique adaptation capacities to several stress conditions such as the presence of toxic compounds and environmental fluctuations. Additionally, the capability of Rhodococcus spp. strains to produce high value-added products has received considerable attention, mostly in relation to lipid accumulation. In relation with this, several works carried out omic studies and genome comparative analyses to investigate the genetic and genomic basis of these anabolic capacities, frequently in association with the bioconversion of renewable resources and low-cost substrates into triacylglycerols. This review is focused on these omic analyses and the genetic and metabolic approaches used to improve the biosynthetic and bioconversion performance of Rhodococcus. In particular, this review summarizes the works that applied heterologous expression of specific genes and adaptive laboratory evolution approaches to manipulate anabolic performance. Furthermore, recent molecular toolkits for targeted genome editing as well as genome-based metabolic models are described here as novel and promising strategies for genome-scaled rational design of Rhodococcus cells for efficient biosynthetic processes application.

Insight into phenotypic and genotypic differences between vaginal Lactobacillus crispatus BC5 and Lactobacillus gasseri BC12 to unravel nutritional and stress factors influencing their metabolic activity.

The vaginal microbiota, normally characterized by lactobacilli presence, is crucial for vaginal health. Members belonging to L. crispatus and L. gasseri species exert crucial protective functions against pathogens, although a total comprehension of factors that influence their dominance in healthy women is still lacking. Here we investigated the complete genome sequence and comprehensive phenotypic profile of L. crispatus strain BC5 and L. gasseri strain BC12, two vaginal strains featured by anti-bacterial and anti-viral activities. Phenotype microarray (PM) results revealed an improved capacity of BC5 to utilize different carbon sources as compared to BC12, although some specific carbon sources that can be associated to the human diet were only metabolized by BC12, i.e. uridine, amygdalin, tagatose. Additionally, the two strains were mostly distinct in the capacity to utilize the nitrogen sources under analysis. On the other hand, BC12 showed tolerance/resistance towards twice the number of stressors (i.e. antibiotics, toxic metals etc.) with respect to BC5. The divergent phenotypes observed in PM were supported by the identification in either BC5 or BC12 of specific genetic determinants that were found to be part of the core genome of each species. The PM results in combination with comparative genome data provide insights into the possible environmental factors and genetic traits supporting the predominance of either L. crispatus BC5 or L. gasseri BC12 in the vaginal niche, giving also indications for metabolic predictions at the species level.

Unraveling the Metabolic Potential of Asgardarchaeota in a Sediment from the Mediterranean Hydrocarbon-Contaminated Water Basin Mar Piccolo (Taranto, Italy).

Increasing number of metagenome sequencing studies have proposed a central metabolic role of still understudied Archaeal members in natural and artificial ecosystems. However, their role in hydrocarbon cycling, particularly in the anaerobic biodegradation of aliphatic and aromatic hydrocarbons, is still mostly unknown in both marine and terrestrial environments. In this work, we focused our study on the metagenomic characterization of the archaeal community inhabiting the Mar Piccolo (Taranto, Italy, central Mediterranean) sediments heavily contaminated by petroleum hydrocarbons and polychlorinated biphenyls (PCB). Among metagenomic bins reconstructed from Mar Piccolo microbial community, we have identified members of the Asgardarchaeota superphylum that has been recently proposed to play a central role in hydrocarbon cycling in natural ecosystems under anoxic conditions. In particular, we found members affiliated with Thorarchaeota, Heimdallarchaeota, and Lokiarchaeota phyla and analyzed their genomic potential involved in central metabolism and hydrocarbon biodegradation. Metabolic prediction based on metagenomic analysis identified the malonyl-CoA and benzoyl-CoA routes as the pathways involved in aliphatic and aromatic biodegradation in these Asgardarchaeota members. This is the first study to give insight into the archaeal community functionality and connection to hydrocarbon degradation in marine sediment historically contaminated by hydrocarbons.

Molecular characterization of microbial communities in a peat-rich aquifer system contaminated with chlorinated aliphatic compounds.

In an aquifer-aquitard system in the subsoil of the city of Ferrara (Emilia-Romagna region, northern Italy) highly contaminated with chlorinated aliphatic toxic organics such as trichloroethylene (TCE) and tetrachloroethylene (PCE), a strong microbial-dependent dechlorination activity takes place during migration of contaminants through shallow organic-rich layers with peat intercalations. The in situ microbial degradation of chlorinated ethenes, formerly inferred by the utilization of contaminant concentration profiles and Compound-Specific Isotope Analysis (CSIA), was here assessed using Illumina sequencing of V4 hypervariable region of 16S rRNA gene and by clone library analysis of dehalogenase metabolic genes. Taxon-specific investigation of the microbial communities catalyzing the chlorination process revealed the presence of not only dehalogenating genera such as Dehalococcoides and Dehalobacter but also of numerous other groups of non-dehalogenating bacteria and archaea thriving on diverse metabolisms such as hydrolysis and fermentation of complex organic matter, acidogenesis, acetogenesis, and methanogenesis, which can indirectly support the reductive dechlorination process. Besides, the diversity of genes encoding some reductive dehalogenases was also analyzed. Geochemical and 16S rRNA and RDH gene analyses, as a whole, provided insights into the microbial community complexity and the distribution of potential dechlorinators. Based on the data obtained, a possible network of metabolic interactions has been hypothesized to obtain an effective reductive dechlorination process.

Extracellular Vesicles Generated by Gram-Positive Bacteria Protect Human Tissues Ex Vivo From HIV-1 Infection.

Vaginal microbiota dominated by lactobacilli protects women from sexually transmitted infection, in particular HIV-1. This protection is, in part, mediated by Lactobacillus-released extracellular vesicles (EVs). Here, we investigated whether EVs derived from other Gram-positive bacteria also present in healthy vaginas, in particular Staphylococcus aureus, Gardnerella vaginalis, Enterococcus faecium, and Enterococcus faecalis, can affect vaginal HIV-1 infection. We found that EVs released by these bacteria protect human cervico-vaginal tissues ex vivo and isolated cells from HIV-1 infection by inhibiting HIV-1-cell receptor interactions. This inhibition was associated with a diminished exposure of viral Env by steric hindrance of gp120 or gp120 modification evidenced by the failure of EV-treated virions to bind to nanoparticle-coupled anti-Env antibodies. Furthermore, we found that protein components associated with EV's outer surface are critical for EV-mediated protection from HIV-1 infection since treatment of bacteria-released EVs with proteinase K abolished their anti-HIV-1 effect. We identified numerous EV-associated proteins that may be involved in this protection. The identification of EVs with specific proteins that suppress HIV-1 may lead to the development of novel strategies for the prevention of HIV-1 transmission.

Robotic-Assisted Colonoscopy Platform with a Magnetically-Actuated Soft-Tethered Capsule.

Background and Aims: Colorectal cancer (CRC) is a major cause of morbidity and mortality worldwide. Despite offering a prime paradigm for screening, CRC screening is often hampered by invasiveness. Endoo is a potentially painless colonoscopy method with an active locomotion tethered capsule offering diagnostic and therapeutic capabilities. Materials and Methods: The Endoo system comprises a soft-tethered capsule, which embeds a permanent magnet controlled by an external robot equipped with a second permanent magnet. Capsule navigation is achieved via closed-loop interaction between the two magnets. Ex-vivo tests were conducted by endoscopy experts and trainees to evaluate the basic key features, usability, and compliance in comparison with conventional colonoscopy (CC) in feasibility and pilot studies. Results: Endoo showed a 100% success rate in operating channel and target approach tests. Progression of the capsule was feasible and repeatable. The magnetic link was lost an average of 1.28 times per complete procedure but was restored in 100% of cases. The peak value of interaction forces was higher in the CC group than the Endoo group (4.12N vs. 1.17N). The cumulative interaction forces over time were higher in the CC group than the Endoo group between the splenic flexure and mid-transverse colon (16.53Ns vs. 1.67Ns, p < 0.001), as well as between the hepatic flexure and cecum (28.77Ns vs. 2.47Ns, p = 0.005). The polyp detection rates were comparable between groups (9.1 ± 0.9% vs. 8.7 ± 0.9%, CC and Endoo respectively, per procedure). Robotic colonoscopies were completed in 67% of the procedures performed with Endoo (53% experts and 100% trainees). Conclusions: Endoo allows smoother navigation than CC and possesses comparable features. Although further research is needed, magnetic capsule colonoscopy demonstrated promising results compared to CC.

Biotechnology of Rhodococcus for the production of valuable compounds.

Bacteria belonging to Rhodococcus genus represent ideal candidates for microbial biotechnology applications because of their metabolic versatility, ability to degrade a wide range of organic compounds, and resistance to various stress conditions, such as metal toxicity, desiccation, and high concentration of organic solvents. Rhodococcus spp. strains have also peculiar biosynthetic activities that contribute to their strong persistence in harsh and contaminated environments and provide them a competitive advantage over other microorganisms. This review is focused on the metabolic features of Rhodococcus genus and their potential use in biotechnology strategies for the production of compounds with environmental, industrial, and medical relevance such as biosurfactants, bioflocculants, carotenoids, triacylglycerols, polyhydroxyalkanoate, siderophores, antimicrobials, and metal-based nanostructures. These biosynthetic capacities can also be exploited to obtain high value-added products from low-cost substrates (industrial wastes and contaminants), offering the possibility to efficiently recover valuable resources and providing possible waste disposal solutions. Rhodococcus spp. strains have also recently been pointed out as a source of novel bioactive molecules highlighting the need to extend the knowledge on biosynthetic capacities of members of this genus and their potential utilization in the framework of bioeconomy. KEY POINTS: • Rhodococcus possesses promising biosynthetic and bioconversion capacities. • Rhodococcus bioconversion capacities can provide waste disposal solutions. • Rhodococcus bioproducts have environmental, industrial, and medical relevance. Graphical abstract.

Influences of Climate on Phyllosphere Endophytic Bacterial Communities of Wild Poplar.

Plant-associated microbial communities play a central role in the plant response to biotic and abiotic stimuli, improving plant fitness under challenging growing conditions. Many studies have focused on the characterization of changes in abundance and composition of root-associated microbial communities as a consequence of the plant response to abiotic factors such as altered soil nutrients and drought. However, changes in composition in response to abiotic factors are still poorly understood concerning the endophytic community associated to the phyllosphere, the above-ground plant tissues. In the present study, we applied high-throughput 16S rDNA gene sequencing of the phyllosphere endophytic bacterial communities colonizing wild Populus trichocarpa (black cottonwood) plants growing in native, nutrient-limited environments characterized by hot-dry (xeric) riparian zones (Yakima River, WA), riparian zones with mid hot-dry (Tieton and Teanaway Rivers, WA) and moist (mesic) climates (Snoqualmie, Skykomish and Skagit Rivers, WA). From sequencing data, 587 Amplicon Sequence Variants (ASV) were identified. Surprisingly, our data show that a core microbiome could be found in phyllosphere-associated endophytic communities in trees growing on opposite sides of the Cascades Mountain Range. Considering only taxa appearing in at least 90% of all samples within each climatic zone, the core microbiome was dominated only by two ASVs affiliated Pseudomonadaceae and two ASVs of the Enterobacteriaceae family. Alpha-diversity measures indicated that plants colonizing hot-dry environments showed a lower diversity than those from mid hot-dry and moist climates. Beta-diversity measures showed that bacterial composition was significantly different across sampling sites. Accordingly, we found that specific ASV affiliated to Pseudomonadaceae and Enterobacteriaceae were significantly more abundant in the phyllosphere endophytic community colonizing plants adapted to the xeric environment. In summary, this study highlights that sampling site is the major driver of variation and that only a few ASV showed a distribution that significantly correlated to climate variables.

Geomicrobiology of a seawater-influenced active sulfuric acid cave.

Fetida Cave is an active sulfuric acid cave influenced by seawater, showing abundant microbial communities that organize themselves under three main different morphologies: water filaments, vermiculations and moonmilk deposits. These biofilms/deposits have different cave distribution, pH, macro- and microelement and mineralogical composition, carbon and nitrogen content. In particular, water filaments and vermiculations had circumneutral and slightly acidic pH, respectively, both had abundant organic carbon and high microbial diversity. They were rich in macro- and microelements, deriving from mineral dissolution, and, in the case of water filaments, from seawater composition. Vermiculations had different color, partly associated with their mineralogy, and unusual minerals probably due to trapping capacities. Moonmilk was composed of gypsum, poor in organic matter, had an extremely low pH (0-1) and low microbial diversity. Based on 16S rRNA gene analysis, the microbial composition of the biofilms/deposits included autotrophic taxa associated with sulfur and nitrogen cycles and biomineralization processes. In particular, water filaments communities were characterized by bacterial taxa involved in sulfur oxidation and reduction in aquatic, aphotic, microaerophilic/anoxic environments (Campylobacterales, Thiotrichales, Arenicellales, Desulfobacterales, Desulforomonadales) and in chemolithotrophy in marine habitats (Oceanospirillales, Chromatiales). Their biodiversity was linked to the morphology of the water filaments and their collection site. Microbial communities within vermiculations were partly related to their color and showed high abundance of unclassified Betaproteobacteria and sulfur-oxidizing Hydrogenophilales (including Sulfuriferula), and Acidiferrobacterales (including Sulfurifustis), sulfur-reducing Desulfurellales, and ammonia-oxidizing Planctomycetes and Nitrospirae. The microbial community associated with gypsum moonmilk showed the strong dominance (>60%) of the archaeal genus Thermoplasma and lower abundance of chemolithotrophic Acidithiobacillus, metal-oxidizing Metallibacterium, Sulfobacillus, and Acidibacillus. This study describes the geomicrobiology of water filaments, vermiculations and gypsum moonmilk from Fetida Cave, providing insights into the microbial taxa that characterize each morphology and contribute to biogeochemical cycles and speleogenesis of this peculiar seawater-influenced sulfuric acid cave.

Time-Dependent Changes in Morphostructural Properties and Relative Abundances of Contributors in Pleurotus ostreatus/Pseudomonas alcaliphila Mixed Biofilms.

Pleurotus ostreatus dual biofilms with bacteria are known to be involved in rock phosphate solubilization, endophytic colonization, and even in nitrogen fixation. Despite these relevant implications, no information is currently available on the architecture of P. ostreatus-based dual biofilms. In addition to this, there is a limited amount of information regarding the estimation of the temporal changes in the relative abundances of the partners in such binary systems. To address these issues, a dual biofilm model system with this fungus was prepared by using Pseudomonas alcaliphila 34 as the bacterial partner due to its very fast biofilm-forming ability. The application of the bacterial inoculum to already settled fungal biofilm on a polystyrene surface coated with hydroxyapatite was the most efficient approach to the production of the mixed system the ultrastructure of which was investigated by a multi-microscopy approach. Transmission electron microscopy analysis showed that the adhesion of bacterial cells onto the mycelial cell wall appeared to be mediated by the presence of an abundant layer of extracellular matrix (ECM). Scanning electron microscopy analysis showed that ECM filaments of bacterial origin formed initially a reticular structure that assumed a tabular semblance after 72 h, thus overshadowing the underlying mycelial network. Across the thickness of the mixed biofilms, the presence of an extensive network of channels with large aggregates of viable bacteria located on the edges of their lumina was found by confocal laser scanning microscopy; on the outermost biofilm layer, a significant fraction of dead bacterial cells was evident. Albeit with tangible differences, similar results regarding the estimation of the temporal shifts in the relative abundances of the two partners were obtained by two independent methods, the former relying on qPCR targeting of 16S and 18S rRNA genes and the latter on ester-linked fatty acid methyl esters analysis.

High-Density Electrical Recording and Impedance Imaging With a Multi-Modal CMOS Multi-Electrode Array Chip.

Multi-electrode arrays, both active or passive, emerged as ideal technologies to unveil intricated electrophysiological dynamics of cells and tissues. Active MEAs, designed using complementary metal oxide semiconductor technology (CMOS), stand over passive devices thanks to the possibility of achieving single-cell resolution, the reduced electrode size, the reduced crosstalk and the higher functionality and portability. Nevertheless, most of the reported CMOS MEA systems mainly rely on a single operational modality, which strongly hampers the applicability range of a single device. This can be a limiting factor considering that most biological and electrophysiological dynamics are often based on the synergy of multiple and complex mechanisms acting from different angles on the same phenomena. Here, we designed a CMOS MEA chip with 16,384 titanium nitride electrodes, 6 independent operational modalities and 1,024 parallel recording channels for neuro-electrophysiological studies. Sixteen independent active areas are patterned on the chip surface forming a 4 × 4 matrix, each one including 1,024 electrodes. Electrodes of four different sizes are present on the chip surface, ranging from 2.5 × 3.5 µm2 up to 11 × 11.0 µm2, with 15 µm pitch. In this paper, we exploited the impedance monitoring and voltage recording modalities not only to monitor the growth and development of primary rat hippocampal neurons, but also to assess their electrophysiological activity over time showing a mean spike amplitude of 144.8 ± 84.6 µV. Fixed frequency (1 kHz) and high sampling rate (30 kHz) impedance measurements were used to evaluate the cellular adhesion and growth on the chip surface. Thanks to the high-density configuration of the electrodes, as well as their dimension and pitch, the chip can appreciate the evolutions of the cell culture morphology starting from the moment of the seeding up to mature culture conditions. The measurements were confirmed by fluorescent staining. The effect of the different electrode sizes on the spike amplitudes and noise were also discussed. The multi-modality of the presented CMOS MEA allows for the simultaneous assessment of different physiological properties of the cultured neurons. Therefore, it can pave the way both to answer complex fundamental neuroscience questions as well as to aid the current drug-development paradigm.

Identification of Resistance Genes and Response to Arsenic in Rhodococcus aetherivorans BCP1.

Arsenic (As) ranks among the priority metal(loid)s that are of public health concern. In the environment, arsenic is present in different forms, organic or inorganic, featured by various toxicity levels. Bacteria have developed different strategies to deal with this toxicity involving different resistance genetic determinants. Bacterial strains of Rhodococcus genus, and more in general Actinobacteria phylum, have the ability to cope with high concentrations of toxic metalloids, although little is known on the molecular and genetic bases of these metabolic features. Here we show that Rhodococcus aetherivorans BCP1, an extremophilic actinobacterial strain able to tolerate high concentrations of organic solvents and toxic metalloids, can grow in the presence of high concentrations of As(V) (up to 240 mM) under aerobic growth conditions using glucose as sole carbon and energy source. Notably, BCP1 cells improved their growth performance as well as their capacity of reducing As(V) into As(III) when the concentration of As(V) is within 30-100 mM As(V). Genomic analysis of BCP1 compared to other actinobacterial strains revealed the presence of three gene clusters responsible for organic and inorganic arsenic resistance. In particular, two adjacent and divergently oriented ars gene clusters include three arsenate reductase genes (arsC1/2/3) involved in resistance mechanisms against As(V). A sequence similarity network (SSN) and phylogenetic analysis of these arsenate reductase genes indicated that two of them (ArsC2/3) are functionally related to thioredoxin (Trx)/thioredoxin reductase (TrxR)-dependent class and one of them (ArsC1) to the mycothiol (MSH)/mycoredoxin (Mrx)-dependent class. A targeted transcriptomic analysis performed by RT-qPCR indicated that the arsenate reductase genes as well as other genes included in the ars gene cluster (possible regulator gene, arsR, and arsenite extrusion genes, arsA, acr3, and arsD) are transcriptionally induced when BCP1 cells were exposed to As(V) supplied at two different sub-lethal concentrations. This work provides for the first time insights into the arsenic resistance mechanisms of a Rhodococcus strain, revealing some of the unique metabolic requirements for the environmental persistence of this bacterial genus and its possible use in bioremediation procedures of toxic metal contaminated sites.