Cave Pools in Carlsbad Caverns National Park Contain Diverse Bacteriophage Communities and Novel Viral Sequences.

Viruses are the most abundant biological entities on Earth, and they play a critical role in the environment and biosphere where they regulate microbial populations and contribute to nutrient cycling. Environmental viruses have been the most studied in the ocean, but viral investigations have now spread to other environments. Here, viral communities were characterized in four cave pools in Carlsbad Caverns National Park to test the hypotheses that (i) viral abundance is ten-fold higher than prokaryotic cell abundance in cavern pools, (ii) cavern pools contain novel viral sequences, and (iii) viral communities in pools from developed portions of the cave are distinct from those of pools in undeveloped parts of the same cave.

Radiolytic support for oxidative metabolism in an ancient subsurface brine system.

Long-isolated subsurface brine environments (Ma-Ga residence times) may be habitable if they sustainably provide substrates, e.g. through water-rock reactions, that support microbial catabolic energy yields exceeding maintenance costs.

Radiolytically reworked Archean organic matter in a habitable deep ancient high-temperature brine.

Investigations of abiotic and biotic contributions to dissolved organic carbon (DOC) are required to constrain microbial habitability in continental subsurface fluids. Here we investigate a large (101-283 mg C/L) DOC pool in an ancient (>1Ga), high temperature (45-55 °C), low biomass (10-10 cells/mL), and deep (3.2 km) brine from an uranium-enriched South African gold mine.

Kr excess and other noble gases identify a billion-year-old radiogenically-enriched groundwater system.

Deep within the Precambrian basement rocks of the Earth, groundwaters can sustain subsurface microbial communities, and are targets of investigation both for geologic storage of carbon and/or nuclear waste, and for new reservoirs of rapidly depleting resources of helium. Noble gas-derived residence times have revealed deep hydrological settings where groundwaters are preserved on millions to billion-year timescales. Here we report groundwaters enriched in the highest concentrations of radiogenic products yet discovered in fluids, with an associated Kr excess in the free fluid, and residence times >1 billion years.

COSPAR Sample Safety Assessment Framework (SSAF).

The Committee on Space Research (COSPAR) Sample Safety Assessment Framework (SSAF) has been developed by a COSPAR appointed Working Group. The objective of the sample safety assessment would be to evaluate whether samples returned from Mars could be harmful for Earth's systems ( environment, biosphere, geochemical cycles). During the Working Group's deliberations, it became clear that a comprehensive assessment to predict the effects of introducing life in new environments or ecologies is difficult and practically impossible, even for terrestrial life and certainly more so for unknown extraterrestrial life.

Toxicological Study of Fluorescent Hydrologic Tracer Dye Effects on Cave Bacteria.

Fluorescent dyes are commonly used as hydrologic tracers in a variety of surface and subsurface environments, including karst aquifers and caves, but the fragile nature of karstic groundwater ecosystems suggests a cautious approach to selecting dyes. This study tested the effects of four fluorescent dye tracers (uranine, eosin, pyranine, sulforhodamine B) on microorganisms from Fort Stanton Cave, New Mexico, United States. Toxicity of the dyes was tested on bacteria isolated from the cave and on a sediment sample collected adjacent to Snowy River in Fort Stanton Cave.

FISH-TAMB, a Fixation-Free mRNA Fluorescent Labeling Technique to Target Transcriptionally Active Members in Microbial Communities.

Keystone species or ecological engineers are vital to the health of an ecosystem; however, often, their low abundance or biomass present challenges for their discovery, identification, visualization and selection. We report the development of fluorescent in situ hybridization of transcript-annealing molecular beacons (FISH-TAMB), a fixation-free protocol that is applicable to archaea and bacteria. The FISH-TAMB method differs from existing FISH methods by the absence of fixatives or surfactants in buffers, the fast hybridization time of as short as 15 min at target cells' growth temperature, and the omission of washing steps.

Lighting Effects on the Development and Diversity of Photosynthetic Biofilm Communities in Carlsbad Cavern, New Mexico.

Photosynthetic cave communities ("lampenflora") proliferate in Carlsbad Cavern and other show caves worldwide due to artificial lighting. These biofilms mar the esthetics and can degrade underlying cave surfaces. The National Park Service recently modernized the lighting in Carlsbad Cavern to a light-emitting diode (LED) system that allows adjustment of the color temperature and intensity.

Can an Off-Nominal Landing by an MMRTG-Powered Spacecraft Induce a Special Region on Mars When No Ice Is Present?

This work aims at addressing whether a catastrophic failure of an entry, descent, and landing event of a Multimission Radioisotope Thermoelectric Generator-based lander could embed the heat sources into the martian subsurface and create a local environment that (1) would temporarily satisfy the conditions for a martian Special Region and (2) could establish a transport mechanism through which introduced terrestrial organisms could be mobilized to naturally occurring Special Regions elsewhere on Mars. Two models were run, a primary model by researchers at the Lawrence Berkeley National Laboratory and a secondary model by researchers at the Jet Propulsion Laboratory, both of which were based on selected starting conditions for various surface composition cases that establish the worst-case scenario, including geological data collected by the Mars Science Laboratory at Gale Crater. The summary outputs of both modeling efforts showed similar results: that the introduction of the modeled heat source could temporarily create the conditions established for a Special Region, but that there would be no transport mechanism by which an introduced terrestrial microbe, even if it was active during the temporarily induced Special Region conditions, could be transported to a naturally occurring Special Region of Mars.

Fluctuations in populations of subsurface methane oxidizers in coordination with changes in electron acceptor availability.

The concentrations of electron donors and acceptors in the terrestrial subsurface biosphere fluctuate due to migration and mixing of subsurface fluids, but the mechanisms and rates at which microbial communities respond to these changes are largely unknown. Subsurface microbial communities exhibit long cellular turnover times and are often considered relatively static-generating just enough ATP for cellular maintenance. Here, we investigated how subsurface populations of CH4 oxidizers respond to changes in electron acceptor availability by monitoring the biological and geochemical composition in a 1339 m-below-land-surface (mbls) fluid-filled fracture over the course of both longer (2.

New Allometric Scaling Laws Revealed for Microorganisms.

Allometric scaling relationships based on microbial data sets are revealing novel biological principles; for example, the abundance and diversity of animal-associated microbes scale with individual animal mass. The global abundance of animal-associated microbes and biosphere species richness have also been estimated. The potential for further microbe-inclusive macroecological insights is high.

A metagenomic window into carbon metabolism at 3 km depth in Precambrian continental crust.

Subsurface microbial communities comprise a significant fraction of the global prokaryotic biomass; however, the carbon metabolisms that support the deep biosphere have been relatively unexplored. In order to determine the predominant carbon metabolisms within a 3-km deep fracture fluid system accessed via the Tau Tona gold mine (Witwatersrand Basin, South Africa), metagenomic and thermodynamic analyses were combined. Within our system of study, the energy-conserving reductive acetyl-CoA (Wood-Ljungdahl) pathway was found to be the most abundant carbon fixation pathway identified in the metagenome.

Allometry of animal-microbe interactions and global census of animal-associated microbes.

Animals live in close association with microorganisms, mostly prokaryotes, living in or on them as commensals, mutualists or parasites, and profoundly affecting host fitness. Most animal-microbe studies focus on microbial community structure; for this project, allometry (scaling of animal attributes with animal size) was applied to animal-microbe relationships across a range of species spanning 12 orders of magnitude in animal mass, from nematodes to whales. Microbial abundances per individual animal were gleaned from published literature and also microscopically counted in three species.

Single cell genomics indicates horizontal gene transfer and viral infections in a deep subsurface Firmicutes population.

A major fraction of Earth's prokaryotic biomass dwells in the deep subsurface, where cellular abundances per volume of sample are lower, metabolism is slower, and generation times are longer than those in surface terrestrial and marine environments. How these conditions impact biotic interactions and evolutionary processes is largely unknown. Here we employed single cell genomics to analyze cell-to-cell genome content variability and signatures of horizontal gene transfer (HGT) and viral infections in five cells of Candidatus Desulforudis audaxviator, which were collected from a 3 km-deep fracture water in the 2.

Comparisons of the composition and biogeographic distribution of the bacterial communities occupying South African thermal springs with those inhabiting deep subsurface fracture water.

South Africa has numerous thermal springs that represent topographically driven meteoric water migrating along major fracture zones. The temperature (40-70°C) and pH (8-9) of the thermal springs in the Limpopo Province are very similar to those of the low salinity fracture water encountered in the South African mines at depths ranging from 1.0 to 3.

A new analysis of Mars "Special Regions": findings of the second MEPAG Special Regions Science Analysis Group (SR-SAG2).

A committee of the Mars Exploration Program Analysis Group (MEPAG) has reviewed and updated the description of Special Regions on Mars as places where terrestrial organisms might replicate (per the COSPAR Planetary Protection Policy). This review and update was conducted by an international team (SR-SAG2) drawn from both the biological science and Mars exploration communities, focused on understanding when and where Special Regions could occur. The study applied recently available data about martian environments and about terrestrial organisms, building on a previous analysis of Mars Special Regions (2006) undertaken by a similar team.

Phylogeny and phylogeography of functional genes shared among seven terrestrial subsurface metagenomes reveal N-cycling and microbial evolutionary relationships.

Comparative studies on community phylogenetics and phylogeography of microorganisms living in extreme environments are rare. Terrestrial subsurface habitats are valuable for studying microbial biogeographical patterns due to their isolation and the restricted dispersal mechanisms. Since the taxonomic identity of a microorganism does not always correspond well with its functional role in a particular community, the use of taxonomic assignments or patterns may give limited inference on how microbial functions are affected by historical, geographical and environmental factors.

Trends and future challenges in sampling the deep terrestrial biosphere.

Research in the deep terrestrial biosphere is driven by interest in novel biodiversity and metabolisms, biogeochemical cycling, and the impact of human activities on this ecosystem. As this interest continues to grow, it is important to ensure that when subsurface investigations are proposed, materials recovered from the subsurface are sampled and preserved in an appropriate manner to limit contamination and ensure preservation of accurate microbial, geochemical, and mineralogical signatures. On February 20th, 2014, a workshop on "Trends and Future Challenges in Sampling The Deep Subsurface" was coordinated in Columbus, Ohio by The Ohio State University and West Virginia University faculty, and sponsored by The Ohio State University and the Sloan Foundation's Deep Carbon Observatory.

Does aspartic acid racemization constrain the depth limit of the subsurface biosphere?

Previous studies of the subsurface biosphere have deduced average cellular doubling times of hundreds to thousands of years based upon geochemical models. We have directly constrained the in situ average cellular protein turnover or doubling times for metabolically active micro-organisms based on cellular amino acid abundances, D/L values of cellular aspartic acid, and the in vivo aspartic acid racemization rate. Application of this method to planktonic microbial communities collected from deep fractures in South Africa yielded maximum cellular amino acid turnover times of ~89 years for 1 km depth and 27 °C and 1-2 years for 3 km depth and 54 °C.

Antibacterial action of a novel functionalized chitosan-arginine against Gram-negative bacteria.

The antimicrobial activity of chitosan and chitosan derivatives has been well established. However, although several mechanisms have been proposed, the exact mode of action is still unclear. Here we report on the investigation of antibacterial activity and the antibacterial mode of action of a novel water-soluble chitosan derivative, arginine-functionalized chitosan, on the Gram-negative bacteria Pseudomonas fluorescens and Escherichia coli.

Subsurface microbial diversity in deep-granitic-fracture water in Colorado.

A microbial community analysis using 16S rRNA gene sequencing was performed on borehole water and a granite rock core from Henderson Mine, a >1,000-meter-deep molybdenum mine near Empire, CO. Chemical analysis of borehole water at two separate depths (1,044 m and 1,004 m below the mine entrance) suggests that a sharp chemical gradient exists, likely from the mixing of two distinct subsurface fluids, one metal rich and one relatively dilute; this has created unique niches for microorganisms. The microbial community analyzed from filtered, oxic borehole water indicated an abundance of sequences from iron-oxidizing bacteria (Gallionella spp.

Sensitive, real-time PCR detects low-levels of contamination by Legionella pneumophila in commercial reagents.

In a real-time PCR assay of Legionella pneumophila (targeting the L. pneumophila-specific mip gene and using SYBR Green dye for DNA detection in conjunction with the iCycler system) we detected as few as 1.3 copies of a mip gene in a 50-microl reaction from serially diluted L.

An immuno-PCR method for detecting Bacillus thuringiensis Cry1Ac toxin.

The Cry1Ac toxin is an insecticidal protein produced by Bacillus thuringiensis var. kurstaki. Recently, the gene encoding the toxin was genetically transformed into crop plants.