The Atacama Rover Astrobiology Drilling Studies (ARADS) Project.

With advances in commercial space launch capabilities and reduced costs to orbit, humans may arrive on Mars within a decade. Both to preserve any signs of past (and extant) martian life and to protect the health of human crews (and Earth's biosphere), it will be necessary to assess the risk of cross-contamination on the surface, in blown dust, and into the near-subsurface (where exploration and resource-harvesting can be reasonably anticipated). Thus, evaluating for the presence of life and biosignatures may become a critical-path Mars exploration precursor in the not-so-far future, circa 2030. This Special Collection of papers from the Atacama Rover Astrobiology Drilling Studies (ARADS) project describes many of the scientific, technological, and operational issues associated with searching for and identifying biosignatures in an extreme hyperarid region in Chile's Atacama Desert, a well-studied terrestrial Mars analog environment. This paper provides an overview of the ARADS project and discusses in context the five other papers in the ARADS Special Collection, as well as prior ARADS project results.

Predicting COVID-19 in very large countries: The case of Brazil.

This work presents a practical proposal for estimating health system utilization for COVID-19 cases. The novel methodology developed is based on the dynamic model known as Susceptible, Infected, Removed and Dead (SIRD). The model was modified to focus on the healthcare system dynamics, rather than modeling all cases of the disease. It was tuned using data available for each Brazilian state and updated with daily figures. A figure of merit that assesses the quality of the model fit to the data was defined and used to optimize the free parameters. The parameters of an epidemiological model for the whole of Brazil, comprising a linear combination of the models for each state, were estimated considering the data available for the 26 Brazilian states. The model was validated, and strong adherence was demonstrated in most cases.

Author Correction: Unprecedented rains decimate surface microbial communities in the hyperarid core of the Atacama Desert.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

Unprecedented rains decimate surface microbial communities in the hyperarid core of the Atacama Desert.

The hyperarid core of the Atacama Desert, the driest and oldest desert on Earth, has experienced a number of highly unusual rain events over the past three years, resulting in the formation of previously unrecorded hypersaline lagoons, which have lasted several months. We have systematically analyzed the evolution of the lagoons to provide quantitative field constraints of large-scale impacts of the rains on the local microbial communities. Here we show that the sudden and massive input of water in regions that have remained hyperarid for millions of years is harmful for most of the surface soil microbial species, which are exquisitely adapted to survive with meager amounts of liquid water, and quickly perish from osmotic shock when water becomes suddenly abundant. We found that only a handful of bacteria, remarkably a newly identified species of Halomonas, remain metabolically active and are still able to reproduce in the lagoons, while no archaea or eukaryotes were identified. Our results show that the already low microbial biodiversity of extreme arid regions greatly diminishes when water is supplied quickly and in great volumes. We conclude placing our findings in the context of the astrobiological exploration of Mars, a hyperarid planet that experienced catastrophic floodings in ancient times.

Sleep-wake detection using recurrence quantification analysis.

Actigraphy is a method for monitoring the movements of the nondominant arm, and the technology has found applications ranging from clinical devices to smart wristbands. Time series obtained from actigraphy data is used in chronobiology to define the sleep-wake cycle, as well as in sleep medicine to evaluate an individual's sleep quality. In the study described in this paper, an algorithm based on recurrence quantification analysis (RQA) was applied to a time series obtained from a commercial actigraph, which was used to collect raw data alongside polysomnography (PSG), generally considered as the gold standard for assessing sleep quality. The central hypothesis is that transitions between sleep and wakefulness are not purely random events, but are strongly influenced by two internal processes: the homeostatic pressure and the circadian cycle. On the basis of this premise, application of RQA to time series as an estimator of this system should lead to improved results and allow more reliable investigations than a purely empirical approach. To compare the results from the RQA algorithm and those from PSG, we present a detailed statistical analysis involving a bias evaluation of the two methods following an approach suggested by Bland and Altman, a comparison of data processed using the kappa coefficient, and a comparison of consolidated sleep quality data using the p -value.

Deep subsurface sulfate reduction and methanogenesis in the Iberian Pyrite Belt revealed through geochemistry and molecular biomarkers.

The Iberian Pyrite Belt (IPB, southwest of Spain), the largest known massive sulfide deposit, fuels a rich chemolithotrophic microbial community in the Río Tinto area. However, the geomicrobiology of its deep subsurface is still unexplored. Herein, we report on the geochemistry and prokaryotic diversity in the subsurface (down to a depth of 166 m) of the Iberian Pyritic belt using an array of geochemical and complementary molecular ecology techniques. Using an antibody microarray, we detected polymeric biomarkers (lipoteichoic acids and peptidoglycan) from Gram-positive bacteria throughout the borehole. DNA microarray hybridization confirmed the presence of members of methane oxidizers, sulfate-reducers, metal and sulfur oxidizers, and methanogenic Euryarchaeota. DNA sequences from denitrifying and hydrogenotrophic bacteria were also identified. FISH hybridization revealed live bacterial clusters associated with microniches on mineral surfaces. These results, together with measures of the geochemical parameters in the borehole, allowed us to create a preliminary scheme of the biogeochemical processes that could be operating in the deep subsurface of the Iberian Pyrite Belt, including microbial metabolisms such as sulfate reduction, methanogenesis and anaerobic methane oxidation.

Comparative genomic analysis reveals novel facts about Leptospirillum spp. cytochromes.

Chemolithoautotrophic acidophilic bacteria, which belong to the genus Leptospirillum, can only grow with Fe(II) as electron donor and oxygen as an electron acceptor. Members of this genus play an important role in bioleaching sulfide ores. We used nearly complete genome sequences of Leptospirillum ferrooxidans (group I), Leptospirillum rubarum, Leptospirillum '5-way CG' (group II) and Leptospirillum ferrodiazotrophum (group III) to identify cytochromes that are likely involved in electron transfer chain(s). The results show the presence of genes encoding a number of c-type cytochromes (18-20 genes were identified in each species), as well as bd and cbb3 oxidases. Genes encoding cbb3 oxidase are clustered, with predicted genes involved in cbb3 maturation proteins. Duplication of cbb3 encoding genes (ccoNO) was detected in all four genomes. Interestingly, these micro-organisms also contain genes that potentially encode bc1 and b6f-like complexes organized into two putative operon structures. To date, the Leptospirillum genus includes the only organisms reported to have genes coding for two different bc complexes. This study provides detailed insights into the components of electron transfer chains of Leptospirillum spp., revealing their conservation among leptospirilla groups and suggesting that there may be a single common pathway for electron transport between Fe(II) and oxygen.

Ordered self-assembled monolayers of Peptide nucleic acids with DNA recognition capability.

We report on the formation of ordered self-assembled monolayers (SAMs) of single-stranded peptide nucleic acids (ssPNA). In spite of their remarkable length (7 nm) thiolated PNAs assemble standing up on gold surfaces similarly to the SAMs of short alkanethiols. SAMs of ssPNA recognize complementary nucleic acids, acting as specific biosensors that discriminate even a point mutation in target ssDNA. These results are obtained by surface characterization techniques that avoid labeling of the target molecule: x-ray photoemission, x-ray absorption and atomic force microscopy.

Functional analysis of the Streptomyces lividans type I signal peptidases.

Type I signal peptidases are responsible for the proteolytic cleavage of the signal peptide of secreted proteins. In the gram-positive bacterium Streptomyces lividans, four adjacent genes (sipW, sipX, sipY and sipZ) were isolated encoding putative type I signal peptidases. In this work, the different sip genes were cloned and expressed. Subsequently, the Sip proteins were purified to raise antibodies. Although the four Sip proteins share a low degree of sequence similarity and differ significantly in size and pI, anti-Sip antibodies cross-reacted intensively. Functional signal peptidase processing activity for each of these Sip proteins was shown both in vitro and in vivo. The different Sip proteins did not exhibit the same cleavage efficiency on the Bacillus subtilis pre-chitosanase.

Membrane topology of the Streptomyces lividans type I signal peptidases.

Most bacterial membranes contain one or two type I signal peptidases (SPases) for the removal of signal peptides from export proteins. For Streptomyces lividans, four different type I SPases (denoted SipW, SipX, SipY, and SipZ) were previously described. In this communication, we report the experimental determination of the membrane topology of these SPases. A protease protection assay of SPase tendamistat fusions confirmed the presence of the N- as well as the C-terminal transmembrane anchor for SipY. SipX and SipZ have a predicted topology similar to that of SipY. These three S. lividans SPases are currently the only known prokaryotic-type type I SPases of gram-positive bacteria with a C-terminal transmembrane anchor, thereby establishing a new subclass of type I SPases. In contrast, S. lividans SipW contains only the N-terminal transmembrane segment, similar to most type I SPases of gram-positive bacteria. Functional analysis showed that the C-terminal transmembrane anchor of SipY is important to enhance the processing activity, both in vitro as well as in vivo. Moreover, for the S. lividans SPases, a relation seems to exist between the presence or absence of the C-terminal anchor and the relative contributions to the total SPase processing activity in the cell. SipY and SipZ, two SPases with a C-terminal anchor, were shown to be of major importance to the cell. Accordingly, for SipW, missing the C-terminal anchor, a minor role in preprotein processing was found.

Streptomyces lividans as a host for the production and secretion of Escherichia coli TEM beta-lactamase.

The regulatory region and the region coding for the signal peptide of an extracellular agarase have been used to synthesize and secrete the heterologous Escherichia coli TEM beta-lactamase in Streptomyces lividans. The transcriptional regulation of the chimeric gene, and the secretion pattern of the chimeric gene product, coincided with those of the agarase gene. The negative glucose effect on the secretion of the protein was reverted when the recombinant bacterium was grown in the chemostat under phosphate limiting conditions.

A new signal peptidase gene from Streptomyces lividans TK21.

Using synthetic oligonucleotides derived from known signal peptidase genes and a multicopy plasmid as a vector, a signal peptidase gene (sipZ) from Streptomyces lividansTK21 has been cloned. The primary structure of the gene has been determined and the amino acid composition of the SipZ protein inferred. SipZ is 258 aa long and showed homology to other type I signal peptidases, containing like them an N-terminal transmembrane anchor. Alignment of SipZ with other known SPases allowed the identification of a conserved sequence of amino acids specific for Gram-positive bacteria.

Overproduction and purification of an agarase of bacterial origin.

The agarase gene from Streptomyces coelicolor has been cloned in the non-producer bacterium Streptomyces lividans under the control of its own set of promoters and under the control of a heterologous promoter that is functional only during exponential growth. The best level of overproduction was obtained when the strain containing the natural gene was cultivated in fed batch with mannitol as carbon source. The protein, with a relative molecular mass of 32 kDa, has been purified following an affinity purification method. Contaminating activities seem to be absent from the purified enzyme preparation that can be used to purify DNA from agarose gels.

A relA/spoT homologous gene from Streptomyces coelicolor A3(2) controls antibiotic biosynthetic genes.

A 0.972-kilobase pair DNA fragment from Streptomyces lividans that induces the production of the blue-pigmented antibiotic actinorhodine in S. lividans when cloned on a multicopy plasmid has led to the isolation of a 4-kilobase pair DNA fragment from Streptomyces coelicolor containing homologous sequence. Computer-assisted analysis of the DNA sequence revealed three putative open reading frames (ORFs), ORF1, ORF2, and ORF3. ORF2 extends beyond the sequenced DNA fragment, and its deduced product shares no similarities with any other known proteins in the data bases. ORF3 is also truncated, and its 41-amino acid C-terminal product is identical to the S. coelicolor adenine phosphoribosyltransferase. The 847-amino acid ORF1 protein, with a predicted molecular mass of 94.2 kDa, strongly resembled the relA and spoT gene products from Escherichia coli and the homologs from Vibrio sp. strain S14, Haemophilus influenzae, Streptococcus equisimilis H46A, and Mycoplasma genitalium. Unlike these proteins, the ORF1 amino acid sequence analysis revealed the presence of a putative ATP/GTP-binding domain. A mutant was generated by deleting most of the ORF1 gene that showed an actinorhodine-nonproducing phenotype, while undecylprodigiosin and the calcium-dependent antibiotic were unaffected. The mutant strain grew at a much lower rate than the wild-type strain, and spore formation was delayed. When the gene was propagated on a low copy number vector, not only was actinorhodine production restored, but actinorhodine and undecylprodigiosin production was enhanced in both the mutant and wild-type and morphological differentiation returned to wild-type characteristics. (p)ppGpp synthetase activity was not detected in purified ribosomes from the ORF1-deleted mutant, while it was restored by complementation of this strain.

Effect of glucose on agarase overproduction by Streptomyces.

The Streptomyces coelicolor dagA gene, coding for an extracellular agarase, has been propagated on a multicopy plasmid in S. coelicolor A3(2), the natural agarase producer strain and in S. lividans TK21, a closely related, nonproducer strain. The effect of the carbon source on the production of agarase by both strains, upon cultivation in liquid medium, revealed that the glucose repression affected the synthesis of agarase at the level of secretion, rather than at the level of transcription. In the presence of glucose, the pre-agarase was degraded intracellularly and the overall secretion of proteases decreased considerably in both strains, suggesting a negative regulatory role for glucose in the overall secretion in Streptomyces.

Expression of an heterologous gene activating actinorhodin biosynthesis in Streptomyces lividans and Streptomyces coelicolor.

Production of the blue-pigmented antibiotic actinorhodin resulted in activation in the non-producer strain Streptomyces lividans, but not in the natural producer strain Streptomyces coelicolor, when transformed with an heterologous activator gene from Streptomyces fradiae. The gene encodes a 132 nucleotide-long transcript, responsible for the actinorhodin production phenotype, and thought to act as a putative antisense RNA, which has been detected in the transformed S. lividans cultures by reverse transcription followed by cyclic amplification.

Heterologous recognition in vivo of promoter sequences from the Streptomyces coelicolor dagA gene.

The Streptomyces coelicolor dagA gene that codes for an extracellular agarase was cloned in the closely related bacterium S. lividans and transferred to the distantly related low G+C Gram-positive bacterium Bacillus subtilis and to the far more distantly related Gram-negative bacterium Escherichia coli. S1 nuclease mapping experiments identified a putative fifth promoter from which transcription of the dagA gene can take place, and accurately mapped the transcription termination site. The transcription terminator was specific for the Streptomyces strains and could terminate transcription initiated by promoters other than those of dagA. The agarase gene is efficiently transcribed in B. subtilis and E. coli, although pulse-chase experiments failed to detect the synthesis of agarase in these two bacteria.

Streptomyces lividans possesses a GroEL-like chaperonin.

Streptomyces lividans grown at 45 degrees C produces a GroEL-like chaperonin. This protein is specifically synthesized in bacterial cell cultures upon heat shock induction. It has a similar size (62 kDa) to the GroEL-like proteins from Escherichia coli and Bacillus subtilus and shows immunological cross-reaction with serum raised against GroEL from E. coli. The S. lividans 62-kDa protein assembles into oligomers around 20S that show a morphology consistent with a barrel showing six-fold and seven-fold symmetries as previously described in E. coli and B. subtilis.

Heterologous activation of the actinorhodin biosynthetic pathway in Streptomyces lividans.

A DNA fragment of Streptomyces fradiae is able to activate the antibiotic actinorhodin biosynthetic pathway when cloned in Streptomyces lividans. The activator DNA region has been sequenced and its transcription initiation and termination sites accurately mapped in vivo. This DNA encodes a 132 nucleotides long transcript which is apparently responsible for the actinorhodin production phenotype, possibly acting as an antisense RNA. The sequence of the activator gene revealed no homology with any other known Streptomyces coelicolor genes concerned with actinorhodin biosynthesis or its pleiotropic regulation.

Transcription of genes involved in the earliest steps of actinorhodin biosynthesis in Streptomyces coelicolor.

A 170bp long BamHI-Sau3A DNA fragment from the actIII-actI intergenic region of the actinorhodin (Act) biosynthetic gene cluster of Streptomyces coelicolor A3(2) contains two promoters directing transcription in a divergent manner. One of them, the actIII promoter, is responsible for the transcription of the actIII gene and the other controls transcription of the adjacent actI region in the opposite direction. Weak activity of the actIII promoter can be detected in Streptomyces lividans and Bacillus subtilis in the absence but not in the presence of glucose. Neither promoter seems to function in Escherichia coli.