Christopher E. Carr

Life on Earth uses L-amino acids and D-sugars, whereas abiotic processes produce equal abundances of both enantiomers. Detecting enantiomeric excess among molecules of biological interest has therefore been proposed as a potential biosignature for life beyond Earth. However, robust electrical methods for determining the L/D ratios of amino acids and sugars at low concentrations, particularly in chemically complex samples, remain limited. Here we show that quantum tunneling currents electrically distinguish individual L- and D-amino acids and quantify their ratios at the single-molecule level without optical measurements or chiral recognition molecules. Individual enantiomers are distinguished with over 80% accuracy, and a blind test of 39 amino acids identifies both molecular identity and chirality with >50% accuracy, well above random chance. Mixtures containing four amino acids with different chiral compositions are quantified with >66% accuracy. Murchison meteorite and Atacama Desert extracts are analyzed using a targeted panel of 11 amino acids. Confidence-based filtering distinguishes high-confidence target assignments from low-confidence events, and the resulting compositions capture major compositional features observed by LC–MS while highlighting the effects of background and low-abundance signals. These results establish a targeted single-molecule electrical approach for chiral amino acid analysis and provide a foundation for future applications to study chemically complex samples, including those relevant to astrobiological research.

Thousands of saline to hypersaline lakes exist across the Yilgarn Craton in Western Australia, representing potential Mars analogs with diverse and varying salinity, pH, and oxygen conditions that remain poorly constrained. Here, we report geochemical trends in lake waters that vary across wet‐dry cycles as well as with regional location and geomorphic setting within the craton's paleovalley system. We identify two different geochemical regimes: (a) lakes where groundwater is more acidic than lake water, and shifts in composition reflect changing precipitation, and (b) lakes that are more acidic than local groundwater due to subsurface and surface geochemical interactions. Elevated salinity in all lakes relative to groundwater occurs due to cyclic evaporation driven by strong seasonal forcing. Most lakes occupy shallow, surficial basins that lack exposed outcrops. Lakes that are deeply incised expose truncated lateritic profiles dominated by clay minerals, quartz veins, and Fe‐oxides. These contrasting settings represent geologic and geochemical endmembers across the region, for which interactions between evaporative concentration and acidic groundwater shape the geochemical evolution of lake systems differently. These factors, in turn, influence microbial habitability, the deposition of salts and other surficial indicators of environmental change. Our observations reveal visible signatures of prolonged acidic‐groundwater interactions, including features consistent with Fe redox cycling, and recurring wet‐dry cycles. These findings suggest that paleodrainage systems within and near craters in Mars' Terra Sirenum, which host similar layered and bleached Al‐rich clay outcrops with smaller exposures of acidic weathered minerals, may record evaporitic concentration and potentially long‐duration groundwater interactions.

Anaerobic methane oxidation, typically mediated by consortia of archaea and bacteria, is a key process in the global methane cycle, but little is known about its upper salinity limits. We characterized the microbial methane cycle in the anoxic, hypersaline Orca Basin using metagenomics, metatranscriptomics, fluorescence in situ hybridization, and geochemical measurements at sub-meter resolution. In the brine, we detected transcriptional activity of the halophilic methylotrophic methanogen Methanohalophilus , consistent with a biological source for Orca Basin methane. In the particle-rich halocline (∼2 M Cl ^- ; ∼2235 meters depth), high mcrA transcription by a novel ANME-2a species was co-located with a positive shift in δ ¹³ C-CH ₄ consistent with anaerobic oxidation of methane. ANME-2a also transcribed genes for biosynthesis of the osmolyte N(ε)-acetyl-β-L-lysine, supporting adaptation to hypersaline conditions. At the same depth, consortia of sarcina-like archaea, likely ANME-2a, were observed in association with vibrioid and filamentous bacteria. Members of a halotolerant Desulfobulbales genus (family SURF-16, which includes the previously identified ANME partner Seep-DBB) were active at the same depth. At and above the oxic-anoxic interface, aerobic methane oxidation appears to be mediated by three genera of uncultivated Methylococcales bacteria. Our results extend the known upper salinity range of ANME-2a to ∼2 M Cl ^- and reveal the key microbial players in the methane bio-filter between the Orca Basin brine and overlying seawater.

The Genomic Enumeration of Antibiotic Resistance in Space (GEARS) flight project seeks to characterize the frequency and genomic identity of antibiotic-resistant organisms on the ISS and expand in-space sequencing-based diagnostic capabilities. This project selects for antibiotic-resistant microbes on space station surfaces using agar plates containing antibiotics, which may require storing the plates for several months. However, little published data is available on the longevity of antibiotics in agar plates. Here, we describe the process for selecting and validating antibiotic stability for GEARS. A literature review was conducted on several antibiotics for their intrinsic and acquired resistance in Enterococcus and Staphylococcus and stability in solution and in agar. Gentamicin and streptomycin were selected for a long-term study tracking changes in the bactericidal ability of the antibiotics in agar plates. After 6.5 months of storage at 4 °C, we found that both antibiotic types remained stable throughout the test period, although they both exhibited evidence of slight degradation. These findings indicated that either antibiotic would be sufficient for the GEARS study based on stability under cold stowage conditions. Furthermore, this study generated a methodology for the study of and information on the long-term stability of antibiotics in agar, which can be useful for microbiology applications beyond spaceflight studies.

Concentrated magnesium chloride brines are extreme environments that are inhospitable to life on Earth. The ionic strength of these brines significantly depresses water activity and concomitantly exerts significant chaotropic stress. Although these brines are largely considered sterile, the well-known preservative effects of magnesium chloride on certain biomolecules, such as DNA, confound life detection approaches and efforts to constrain precisely the habitable window of life on Earth. While the ability of these brines to preserve genetic material is well documented, the preservation of whole cells, which are generally thought to be preserved in magnesium chloride brines, is poorly described. This work explores the effects of long-term exposure of highly chaotropic magnesium chloride on viability, cell integrity, and DNA preservation in the model organisms Escherichia coli , Salinibacter ruber , Halobacterium salinarum , and Haloquadratum walsbyi . The selected halophiles are relevant for this study as they are abundant and globally distributed in brine environments, while E. coli was chosen to represent infall or transport of non-adapted cells. We observed unexpected resilience in E. coli , which survived in 4 M magnesium chloride for longer than the tested halophiles, and nonviable cells maintained structural whole-cell integrity for over 3 years. Whole S. ruber cells were also preserved in 4 M magnesium chloride, while the tested haloarchaea lost viability and completely degraded within hours of exposure. DNA from all tested strains was recovered from incubations after upwards of 3 years of exposure; it showed some signs of degradation but was nonetheless still amplifiable via polymerase chain reaction. Our work demonstrates that the preservation of whole cells in magnesium chloride brines is not universal. Considering the potential abundance of chaotropic brine environments within our solar system, understanding the limits of life and the preservation of biosignatures in these brines is critical to inform future life detection missions on Earth and beyond.

The Genomic Enumeration of Antibiotic Resistance in Space (GEARS) flight project seeks to characterize the frequency and genomic identity of antibiotic-resistant organisms on the ISS and expand in-space sequencing-based diagnostic capabilities. This project selects for antibiotic-resistant microbes on space station surfaces using agar plates containing antibiotics, which can require storage of these plates for several months. However, little published data is available on the longevity of antibiotics in agar plates. Here, we describe the process of antibiotic selection and validation of antibiotic stability for GEARS. A literature review was conducted on several antibiotics for their intrinsic and acquired resistance in Enterococcus and Staphylococcus and stability in solution and in agar. Gentamicin and streptomycin were selected for a long-term study tracking changes in bactericidal ability of the antibiotics in agar plates. After 6.5 months of storage at 4°C, we found that both antibiotic types remained stable throughout the test period, although they both exhibited evidence of slight degradation. These findings indicated that either antibiotic would be sufficient for the GEARS study based on stability under cold stowage conditions. Furthermore, this study generated information on long-term stability of antibiotics in agar, which can be useful for microbiology applications beyond spaceflight studies.

Life on Earth relies on mechanisms to store heritable information and translate this information into cellular machinery required for biological activity. In all known life, storage, regulation, and translation are provided by DNA, RNA, and ribosomes. Life beyond Earth, even if ancestrally or chemically distinct from life as we know it, may utilize similar structures: it has been proposed that charged linear polymers analogous to nucleic acids may be responsible for storage and regulation of genetic information in nonterran biochemical systems. We further propose that a ribosome-like structure may also exist in such a system, due to the evolutionary advantages of separating heritability from cellular machinery. In this study, we use a solid-state nanopore to detect DNA, RNA, and ribosomes, and we demonstrate that machine learning can distinguish between biomolecule samples and accurately classify new data. This work is intended to serve as a proof of principal that such biosignatures ( i.e. , informational polymers or translation apparatuses) could be detected, for example, as part of future missions targeting extant life on Ocean Worlds. A negative detection does not imply the absence of life; however, the detection of ribosome-like structures could provide a robust and sensitive method to seek extant life in combination with other methods. Key Words : RNA world—Darwinian evolution—Nucleic acids—Agnostic life detection. Astrobiology 24, 1257–1274.

The RNA World hypothesis posits that RNA was the molecule of both heredity and function during the emergence of life. This hypothesis implies that RNA templates can be copied, and ultimately replicated, without the catalytic aid of evolved enzymes. A major problem with nonenzymatic template-directed polymerization has been the very poor copying of sequences containing rA and rU. Here, we overcome that problem by using a prebiotically plausible mixture of RNA mononucleotides and random-sequence oligonucleotides, all activated by methyl isocyanide chemistry, that direct the uniform copying of arbitrary-sequence templates, including those harboring rA and rU. We further show that the use of this mixture in copying reactions suppresses copying errors while also generating a more uniform distribution of mismatches than observed for simpler systems. We find that oligonucleotide competition for template binding sites, oligonucleotide ligation and the template binding properties of reactant intermediates work together to reduce product sequence bias and errors. Finally, we show that iterative cycling of templated polymerization and activation chemistry improves the yields of random-sequence products. These results for random-sequence template copying are a significant advance in the pursuit of nonenzymatic RNA replication.

Diverse microbiota inhabit some of Earth's most extreme arid regions, enduring in environments where survival seems improbable. Despite numerous reports of microorganisms across the Atacama Desert—the oldest and driest desert on Earth—our understanding of how they adapt to extreme conditions in its hyperarid core remains limited. Prior systematic studies of the hyper-arid core have relied upon targeted gene sequencing (e.g., 16S rDNA), limiting inference of microbial community structure and function. Here we show that a diverse core group of microbes can be found in the hyperarid core, enabled by processing of large sample volumes linked to low-input metagenomic sequencing of 50 soil samples along a 450 km south-north transect. Linking gene function to soil geochemistry revealed a tipping point related to potential adaptive strategies. Our findings challenge the notion that microbial activity in the hyperarid core is predominantly transient. Among the diversity of metabolic pathways detected, we found evidence of gene circuits that encode proteins capable of using atmospheric trace gases to produce metabolic water and generate energy. Growing evidence of this previously under-recognized capability may extend the known range of habitability for soil microbial communities, with implications for the search for life beyond Earth, such as on Mars or arid exoplanets.

The RNA World hypothesis posits that RNA was the molecule of both heredity and function during the emergence of life. This hypothesis implies that RNA templates can be copied, and ultimately replicated, without the catalytic aid of evolved enzymes. A major problem with nonenzymatic templated polymerization has been the very poor copying of sequences containing rA and rU. Here we overcome that problem by using a prebiotically plausible mixture of RNA mononucleotides and random-sequence oligonucleotides, all activated by methyl isocyanide chemistry, that direct the uniform copying of arbitrary-sequence templates, including those harboring rA and rU. We further show that the use of this mixture in copying reactions suppresses copying errors while also generating a more uniform distribution of mismatches than observed for simpler systems. We find that oligonucleotide competition for template binding sites, oligonucleotide ligation, and the template binding properties of reactant intermediates work together to reduce product sequence bias and errors. Finally, we show that iterative cycling of the activation chemistry and templated polymerization improves the yield of random-sequence products. These results for random-sequence template copying are a significant advance in the pursuit of nonenzymatic RNA replication.