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Summary The surge of SARS‐CoV‐2 has challenged health systems worldwide and efficient tests to detect viral particles, as well as antibodies generated against them, are needed. Specificity, sensitivity, promptness or scalability are the main parameters to estimate the final performance, but rarely all of them match in a single test. We have developed SCOVAM, a protein microarray with several viral antigens (spike, nucleocapsid, main protease Nsp5) as capturing probes in a fluorescence immunoassay for COVID‐19 serological testing. SCOVAM depicts IgG and IgM antibody responses against each of these proteins of 22 individuals in a single microscope slide. It detects specific IgM (0.094 μg ml‐1) and IgG (~0.017 μg ml‐1) and is scalable and cost‐effective. We validated SCOVAM by comparing with a widely used chemiluminescent commercial serological test (n = 742). SCOVAM showed twice the sensitivity and allowed following seroconversion in a single assay. By analysing the prevalence 4 months later in a subset of 76 positive sera, we still detected 93.42% of positives, almost doubling the detection of the commercial assay. The higher sensitivity of SCOVAM is especially relevant to screen sera for convalescent plasma‐based treatments, high‐throughput antibody response monitoring after vaccination or evaluation of vaccine efficiency. We have developed a test to discriminate antibodies against SARS‐CoV‐2 in the blood serum. It detects IgM and IgG antibodies against the Spike, Nucleocapsid and Main protease of the virus, simultaneously. We printed these viral proteins on slides forming microarrays and incubated them with human sera for fluorescent detection. We established the limit of detection and an algorithm for automated diagnostics. Our test resulted twice as sensitive as a commercial test, being able to better follow antibody response through time, what will be very useful to track the epidemiology of COVID‐19 and the effectivity of the developed vaccines.

Cyanobacteria are ecologically versatile microorganisms inhabiting most environments, ranging from marine systems to arid deserts. Although they possess several pathways for light-independent energy generation, until now their ecological range appeared to be restricted to environments with at least occasional exposure to sunlight. Here we present molecular, microscopic, and metagenomic evidence that cyanobacteria predominate in deep subsurface rock samples from the Iberian Pyrite Belt Mars analog (southwestern Spain). Metagenomics showed the potential for a hydrogen-based lithoautotrophic cyanobacterial metabolism. Collectively, our results suggest that they may play an important role as primary producers within the deep-Earth biosphere. Our description of this previously unknown ecological niche for cyanobacteria paves the way for models on their origin and evolution, as well as on their potential presence in current or primitive biospheres in other planetary bodies, and on the extant, primitive, and putative extraterrestrial biospheres.

Summary The bioleaching of metal sulfide has developed into a very important industrial process and understanding the microbial dynamic is key to advancing commercial bioleaching operations. Here we report the first quantitative description of the dynamic of active communities in an industrial bioleaching heap. Acidithiobacillus ferrooxidans was the most abundant during the first part of the leaching cycle, while the abundance of Leptospirillum ferriphilum and Ferroplasma acidiphilum increased with age of the heap. Acidithiobacillus thiooxidans kept constant throughout the leaching cycle, and Firmicutes group showed a low and a patchy distribution in the heap. The Acidiphilium‐like bacteria reached their highest abundance corresponding to the amount of autotrophs. The active microorganisms in the leaching system were determined using two RNA‐based sensitive techniques. In most cases, the 16S rRNA copy numbers of At. ferrooxidans, L. ferriphilum, At. thiooxidans and F. acidiphilum, was concomitant with the DNA copy numbers, whereas Acidiphilium‐like bacteria and some Firmicutes members did not show a clear correlation between 16S rRNA accumulation and DNA copy numbers. However, the prokaryotic acidophile microarray (PAM) analysis showed active members of Alphaproteobacteria in all samples and of Sulfobacillus genus in older ones. Also, new active groups such as Actinobacteria and Acidobacterium genus were detected by PAM. The results suggest that changes during the leaching cycle in chemical and physical conditions, such as pH and Fe3+/Fe2+ ion rate, are primary factors shaping the microbial dynamic in the heap.

Microorganisms are present in snow/ice of the Antarctic Plateau, but their biogeography and metabolic state under extreme local conditions are poorly understood. Here, we show the diversity and distribution of microorganisms in air (1.5 m height) and snow/ice down to 4 m depth at three distant latitudes along a 2578 km transect on the East Antarctic Plateau on board an environmentally friendly, mobile platform. Results demonstrate the widespread distribution of microorganisms in the ice down to at least 4 m depth. Data point to geochemical and bacterial geographic distribution that correlate with wind trajectory and speed, modulated by local gathering and recirculation of microorganisms through snow drifting. Reservoir effects and community selection appear to occur over time, favoring microorganisms best adapted to hypothermal and hyperarid conditions. A new cyanobacterial species ( Gloeocapsopsis sp) was isolated from 3 to 4 m depth. Our findings suggest that some microorganisms could exhibit transient, basal metabolic activity when associated to high salt particles, contributing to set biodiversity patterns and biogeographic compartmentalization on Antarctic Plateau ice. WindSled, a zero-emission mobile science platform, is capable of traveling thousands of kilometers and doing valuable science on the Antarctic Plateau. Wind-driven aerosols condition the biogeographic distribution of bioburden from air to 4 m depth.

Background Extreme acidic environments are characterized by their high metal content and lack of nutrients (oligotrophy). Macroscopic biofilms and filaments usually grow on the water-air interface or under the stream attached to solid substrates (streamers). In the Río Tinto (Spain), brown filaments develop under the water stream where the Gram-negative iron-oxidizing bacteria Leptospirillum spp. ( L. ferrooxidans and L. ferriphilum ) and Acidithiobacillus ferrooxidans are abundant. These microorganisms play a critical role in bioleaching processes for industrial (biominery) and environmental applications (acid mine drainage, bioremediation). The aim of this study was to investigate the physiological differences between the free living (planktonic) and the sessile (biofilm associated) lifestyles of Leptospirillum spp. as part of its natural extremely acidophilic community. Results Total RNA extracted from environmental samples was used to determine the composition of the metabolically active members of the microbial community and then to compare the biofilm and planktonic environmental transcriptomes by hybridizing to a genomic microarray of L. ferrooxidans . Genes up-regulated in the filamentous biofilm are involved in cellular functions related to biofilm formation and maintenance, such as: motility and quorum sensing ( mqsR, cheAY, fliA, motAB ), synthesis of cell wall structures ( lnt, murA, murB ), specific proteases ( clpX/clpP ), stress response chaperons ( clpB, clpC, grpE-dnaKJ, groESL ), etc. Additionally, genes involved in mixed acid fermentation ( poxB , ackA ) were up-regulated in the biofilm. This result, together with the presence of small organic acids like acetate and formate (1.36 mM and 0.06 mM respectively) in the acidic (pH 1.8) water stream, suggests that either L. ferrooxidans or other member of the microbial community are producing acetate in the acidophilic biofilm under microaerophilic conditions. Conclusions Our results indicate that the acidophilic filaments are dynamic structures in which different mechanisms for biofilm formation/dispersion are operating. Specific transcriptomic fingerprints can be inferred for both planktonic and sessile cells, having the former a more active TCA cycle, while the mixed acid fermentation process dominate in the latter. The excretion of acetate may play a relevant ecological role as a source of electron donor for heterotrophic Fe 3+ reducers like some Alphaproteobacteria, Acidobacterium spp. and Sulfobacillus spp., also present in the biofilm. Additionally, acetate may have a negative effect on bioleaching by inhibiting the growth of chemolithotrophic bacteria.

Background The extracellular promastigote and the intracellular amastigote stages alternate in the digenetic life cycle of the trypanosomatid parasite Leishmania . Amastigotes develop inside parasitophorous vacuoles of mammalian phagocytes, where they tolerate extreme environmental conditions. Temperature increase and pH decrease are crucial factors in the multifactorial differentiation process of promastigotes to amastigotes. Although expression profiling approaches for axenic, cell culture- and lesion-derived amastigotes have already been reported, the specific influence of temperature increase and acidification of the environment on developmental regulation of genes has not been previously studied. For the first time, we have used custom L. infantum genomic DNA microarrays to compare the isolated and the combined effects of both factors on the transcriptome. Results Immunofluorescence analysis of promastigote-specific glycoprotein gp46 and expression modulation analysis of the amastigote-specific A2 gene have revealed that concomitant exposure to temperature increase and acidification leads to amastigote-like forms. The temperature-induced gene expression profile in the absence of pH variation resembles the profile obtained under combined exposure to both factors unlike that obtained for exposure to acidification alone. In fact, the subsequent fold change-based global iterative hierarchical clustering analysis supports these findings. Conclusions The specific influence of temperature and pH on the differential regulation of genes described in this study and the evidence provided by clustering analysis is consistent with the predominant role of temperature increase over extracellular pH decrease in the amastigote differentiation process, which provides new insights into Leishmania physiology.

A random genomic library from an environmental isolate of the Gram-negative bacterium Leptospirillum ferrooxidans has been printed on a microarray. Gene expression analysis was carried out with total RNA extracted from L. ferrooxidans cultures in the presence or absence of ammonium as nitrogen source under aerobic conditions. Although practically nothing is known about the genome sequence of this bacterium, this approach allowed us the selection and sequencing of only those clones bearing genes that showed an altered expression pattern. By sequence comparison, we have identified most of the genes of nitrogen fixation regulon in L. ferrooxidans, like the nifHDKENX operon, encoding the structural components of Mo-Fe nitrogenase; nifSU-hesB-hscBA-fdx operon, for Fe-S cluster assembly; the amtB gene (ammonium transporter); modA (molybdenum ABC type transporter); some regulatory genes like ntrC, nifA (the specific activator of nif genes); or two glnB-like genes (encoding the PII regulatory protein). Our results show that shotgun DNA microarrays are very powerful tools to accomplish gene expression studies with environmental bacteria whose genome sequence is still unknown, avoiding the time and effort necessary for whole genome sequencing projects.

Abstract Chaperonins are essential protein-folding machines, classified into three structural and phylogenetic groups: Group I (bacterial GroEL), Group II (archaeal thermosome and eukaryotic CCT), and Group III (bacterial thermosome-like). Using ancestral sequence reconstruction (ASR) and protein resurrection, we inferred and experimentally characterized the last common ancestors of these groups (ancestral chaperonins ACI, ACII, and ACIII). The resurrected proteins exhibited ATPase activity (except ACII) and protected client proteins from heat-induced inactivation. Structural analyses by electron microscopy and Cryo-EM revealed that ACI forms single 7-mer rings, whereas ACII adopts a mixed population of single/double 8-mer rings, representing the first experimental observation of intermediate oligomeric states. ACII also features a unique cochaperonin-independent closure mechanism, distinct from modern Group I and II chaperonins. Together, these findings provide the experimental structural reconstruction of the most ancient and complex multimeric proteins so far, uncover novel intermediate states in chaperonin evolution, and offer a direct empirical framework for studying the emergence of multimeric complexity in early cellular life.

Solar salterns represent unique systems with low diversity microbial communities that serve as an excellent model for studying the evolution and ecology of archaeal viruses and the interactions with their hosts. This is particularly relevant for the extremely abundant “square” archaeon Haloquadratum walsbyi, for which isolated viruses have remained elusive despite the fact that this microbe governs the salt-saturated ponds of most solar salterns worldwide. In this work, we have used cutting-edge imaging techniques, based on virusFISH, and a combination of -omic techniques, at both population and single-cell levels, to provide an in-depth characterization of the Hqr. walsbyi virosphere. Our analyses have led to the identification of a new subfamily of tailed low-GC dsDNA viruses, which we propose to name “Haloquadravirinae”, with host assignment confirmed by virusFISH in natural samples. Haloquadraviruses can represent more than 50% of the viral community in solar saltern viromes and infect nearly 40% of square cells in natural environments. The genetic imprint of these viruses, which are globally distributed in hypersaline environments, has provided insights into the structure of their virions and their potential life strategy. Along with the identification of other virus-like elements associated with Hqr. walsbyi through single-cell genomics, this work expands our current understanding of the archaeal virosphere.

Hypersaline environments harbour the highest number of viruses reported for aquatic environments. In crystallizer ponds from solar salterns, haloviruses coexist with extremely halophilic Archaea and Bacteria and present a high diversity although little is known about their activity. In this work, we analyzed the viral expression in one crystallizer using a metatranscriptomic approach in which clones from a metaviromic library were immobilized in a microarray and used as probes against total mRNA extracted from the hypersaline community. This approach has two advantages: (i) it overcomes the fact that there is no straightforward, unambiguous way to extract viral mRNA from bulk mRNAs and (ii) it makes the sequencing of all mRNAs unnecessary. Transcriptomic data indicated that the halovirus assemblage was highly active at the time of sampling and the viral groups with the highest expression levels were those related to high GC content haloarchaea and Salinibacter representatives, which are minor components in the environment. Moreover, the changes in the viral expression pattern and in the numbers of free viral particles were analyzed after submitting the samples to two stress conditions: ultraviolet-radiation and dilution. Results showed that Archaea were more sensitive than Bacteria to these stress conditions. The overexpression in the predicted archaeal virus fraction raised and the total numbers of free viruses increased. Furthermore, we identified some very closely related viral clones, displaying single-nucleotide polymorphisms, which were expressed only under certain conditions. These clones could be part of very closely related virus genomes for which we propose the term ‘ecoviriotypes’.