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 (102-104 cells/mL), and deep (3.2 km) brine from an uranium-enriched South African gold mine. Excitation-emission matrices (EEMs), negative electrospray ionization (-ESI) 21 tesla Fourier-transform ion cyclotron resonance mass spectrometry (FT-ICR MS), and amino acid analyses suggest the brine DOC is primarily radiolytically oxidized kerogen-rich shales or reefs, methane and ethane, with trace amounts of C3-C6 hydrocarbons and organic sulfides. δ2H and δ13C of C1-C3 hydrocarbons are consistent with abiotic origins. These findings suggest water-rock processes control redox and C cycling, helping support a meagre, slow biosphere over geologic time. A radiolytic-driven, habitable brine may signal similar settings are good targets in the search for life beyond Earth.

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 (e.g., 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. To manage expectations, the scope of the SSAF was adjusted to evaluate only whether the presence of martian life can be excluded in samples returned from Mars. If the presence of martian life cannot be excluded, a Hold & Critical Review must be established to evaluate the risk management measures and decide on the next steps. The SSAF starts from a positive hypothesis (there is martian life in the samples), which is complementary to the null-hypothesis (there is no martian life in the samples) typically used for science. Testing the positive hypothesis includes four elements: (1) Bayesian statistics, (2) subsampling strategy, (3) test sequence, and (4) decision criteria. The test sequence capability covers self-replicating and non-self-replicating biology and biologically active molecules. Most of the investigations associated with the SSAF would need to be carried out within biological containment. The SSAF is described in sufficient detail to support planning activities for a Sample Receiving Facility (SRF) and for preparing science announcements, while at the same time acknowledging that further work is required before a detailed Sample Safety Assessment Protocol (SSAP) can be developed. The three major open issues to be addressed to optimize and implement the SSAF are (1) setting a value for the level of assurance to effectively exclude the presence of martian life in the samples, (2) carrying out an analogue test program, and (3) acquiring relevant contamination knowledge from all Mars Sample Return (MSR) flight and ground elements. Although the SSAF was developed specifically for assessing samples from Mars in the context of the currently planned NASA-ESA MSR Campaign, this framework and the basic safety approach are applicable to any other Mars sample return mission concept, with minor adjustments in the execution part related to the specific nature of the samples to be returned. The SSAF is also considered a sound basis for other COSPAR Planetary Protection Category V, restricted Earth return missions beyond Mars. It is anticipated that the SSAF will be subject to future review by the various MSR stakeholders.

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. Polyarginine cell-penetrating peptides are employed to deliver molecular beacons (MBs) across prokaryotic cell walls and membranes, fluorescently labeling cells when MBs hybridize to target mRNA sequences. Here, the detailed protocol of the preparation and application of FISH-TAMB is presented. To demonstrate FISH-TAMB's ability to label intracellular mRNA targets, differentiate transcriptional states, detect active and rare taxa, and keep cell viability, labeling experiments were performed that targeted the messenger RNA (mRNA) of methyl-coenzyme M reductase A (mcrA) expressed in (1) Escherichia coli containing a plasmid with a partial mcrA gene of the methanogen Methanosarcina barkeri (E. coli mcrA+); (2) M. barkeri; and (3) an anaerobic methanotrophic (ANME) enrichment from a deep continental borehole. Although FISH-TAMB was initially envisioned for mRNA of any functional gene of interest without a requirement of prior knowledge of 16S ribosomal RNA (rRNA)-based taxonomy, FISH-TAMB has the potential for multiplexing and going beyond mRNA and thus is a versatile addition to the molecular ecologist's toolkit, with potentially widespread application in the field of environmental microbiology.

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. The isolates showed minimal inhibition by the four dyes in an agar diffusions assay. Minimum inhibitory concentrations calculated from liquid culture assays of one isolate were 35 g/L for uranine, 3.5 g/L for eosin, 0.1 g/L for pyranine, and 10 mg/L for sulforhodamine B. A 14 C-glucose radiotracer experiment showed zero inhibition of overall microbial activity in a sediment sample at all dye concentrations, except at 350 g/L eosin. Thus, there are no cave-specific findings to indicate that Fort Stanton's microbes are especially sensitive to these commonly used dyes. Moreover, a literature survey of mutagenicity tests on these dyes indicates they are safe for environmental use. These results corroborate previous dye toxicity tests and suggest that these four dyes are suitable for use at Fort Stanton Cave in the concentration ranges commonly used for groundwater tracing. While broader testing of dyes with microbes from other caves is advised, the results suggest the dyes may be safe for all karst aquifers.

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. We hypothesized that lowering the color temperature would reduce photopigment development. We therefore assessed lampenflora responses to changes in lighting by monitoring photosynthetic communities over the course of a year. We measured photopigments using reflected-light spectrophotometric observations and analyzed microbial community composition with 16S and 18S rRNA gene amplicon sequencing. Reflected-light spectrophotometry revealed that photosynthetic biofilm development is affected by lighting intensity, color temperature, substrate type, and cleaning of the substrate. Gene sequencing showed that the most abundant phototrophs were Cyanobacteria and members of the algal phyla Chlorophyta and Ochrophyta At the end of the study, visible growth of lampenflora was seen at all sites. At sites that had no established biofilm at the start of the study period, Cyanobacteria became abundant and outpaced an increase in eukaryotic algae. Microbial diversity also increased over time at these sites, suggesting a possible pattern of early colonization and succession. Bacterial community structure showed significant effects of all variables: color temperature, light intensity, substrate type, site, and previous cleaning of the substrate. These findings provide fundamental information that can inform management practices; they suggest that altering lighting conditions alone may be insufficient to prevent lampenflora growth.IMPORTANCE Artificial lighting in caves visited by tourists ("show caves") can stimulate photosynthetic algae and cyanobacteria, called "lampenflora," which are unsightly and damage speleothems and other cave surfaces. The most common mitigation strategy employs bleach, but altering intensities and wavelengths of light might be effective and less harsh. Carlsbad Cavern in New Mexico, a U.S. National Park and UNESCO World Heritage Site, has visible lampenflora despite adjustment of LED lamps to decrease the energetic blue light. This study characterized the lampenflora communities and tested the effects of color temperature, light intensity, rock or sediment texture, and time on lampenflora development. DNA amplicon sequence data show a variety of algae and cyanobacteria and also heterotrophic bacteria. This study reveals microbial dynamics during colonization of artificially lit surfaces and indicates that while lowering the color temperature may have an effect, management of lampenflora will likely require additional chemical or UV treatment.

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.

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.

An oligotrophic deep-subsurface community dependent on syntrophy is dominated by sulfur-driven autotrophic denitrifiers.

Subsurface lithoautotrophic microbial ecosystems (SLiMEs) under oligotrophic conditions are typically supported by H2 Methanogens and sulfate reducers, and the respective energy processes, are thought to be the dominant players and have been the research foci. Recent investigations showed that, in some deep, fluid-filled fractures in the Witwatersrand Basin, South Africa, methanogens contribute <5% of the total DNA and appear to produce sufficient CH4 to support the rest of the diverse community. This paradoxical situation reflects our lack of knowledge about the in situ metabolic diversity and the overall ecological trophic structure of SLiMEs. Here, we show the active metabolic processes and interactions in one of these communities by combining metatranscriptomic assemblies, metaproteomic and stable isotopic data, and thermodynamic modeling. Dominating the active community are four autotrophic ß-proteobacterial genera that are capable of oxidizing sulfur by denitrification, a process that was previously unnoticed in the deep subsurface. They co-occur with sulfate reducers, anaerobic methane oxidizers, and methanogens, which each comprise <5% of the total community. Syntrophic interactions between these microbial groups remove thermodynamic bottlenecks and enable diverse metabolic reactions to occur under the oligotrophic conditions that dominate in the subsurface. The dominance of sulfur oxidizers is explained by the availability of electron donors and acceptors to these microorganisms and the ability of sulfur-oxidizing denitrifiers to gain energy through concomitant S and H2 oxidation. We demonstrate that SLiMEs support taxonomically and metabolically diverse microorganisms, which, through developing syntrophic partnerships, overcome thermodynamic barriers imposed by the environmental conditions in the deep subsurface.

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. Carbon monoxide dehydrogenase genes that have the potential to participate in (1) both autotrophic and heterotrophic metabolisms through the reversible oxidization of CO and subsequent transfer of electrons for sulfate reduction, (2) direct utilization of H2 and (3) methanogenesis were identified. The most abundant members of the metagenome belonged to Euryarchaeota (22%) and Firmicutes (57%)-by far, the highest relative abundance of Euryarchaeota yet reported from deep fracture fluids in South Africa and one of only five Firmicutes-dominated deep fracture fluids identified in the region. Importantly, by combining the metagenomics data and thermodynamic modeling of this study with previously published isotopic and community composition data from the South African subsurface, we are able to demonstrate that Firmicutes-dominated communities are associated with a particular hydrogeologic environment, specifically the older, more saline and more reducing waters.

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. Abundance of prokaryotes/individual versus animal mass scales as a nearly linear power function (exponent = 1.07, R(2) = 0.94). Combining this power function with allometry of animal abundance indicates that macrofauna have an outsized share of animal-associated microorganisms. The total number of animal-associated prokaryotes in Earth's land animals was calculated to be 1.3-1.4 × 10(25) cells and the total of marine animal-associated microbes was calculated to be 8.6-9.0 × 10(24) cells. Animal-associated microbes thus total 2.1-2.3 × 10(25) of the approximately 10(30) prokaryotes on the Earth. Microbes associated with humans comprise 3.3-3.5% of Earth's animal-associated microbes, and domestic animals harbour 14-20% of all animal-associated microbes, adding a new dimension to the scale of human impact on the biosphere. This novel allometric power function may reflect underlying mechanisms involving the transfer of energy and materials between microorganisms and their animal hosts. Microbial diversity indices of animal gut communities and gut microbial species richness for 60 mammals did not indicate significant scaling relationships with animal body mass; however, further research in this area is warranted.

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 2.9 Ga-old Witwatersrand Basin of South Africa. Between 0 and 32% of genes recovered from single cells were not present in the original, metagenomic assembly of Desulforudis, which was obtained from a neighboring subsurface fracture. We found a transposable prophage, a retron, multiple clustered regularly interspaced short palindromic repeats (CRISPRs) and restriction-modification systems, and an unusually high frequency of transposases in the analyzed single cell genomes. This indicates that recombination, HGT and viral infections are prevalent evolutionary events in the studied population of microorganisms inhabiting a highly stable deep subsurface environment.

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. Since then, a new body of highly relevant information has been generated from the Mars Reconnaissance Orbiter (launched in 2005) and Phoenix (2007) and data from Mars Express and the twin Mars Exploration Rovers (all 2003). Results have also been gleaned from the Mars Science Laboratory (launched in 2011). In addition to Mars data, there is a considerable body of new data regarding the known environmental limits to life on Earth-including the potential for terrestrial microbial life to survive and replicate under martian environmental conditions. The SR-SAG2 analysis has included an examination of new Mars models relevant to natural environmental variation in water activity and temperature; a review and reconsideration of the current parameters used to define Special Regions; and updated maps and descriptions of the martian environments recommended for treatment as "Uncertain" or "Special" as natural features or those potentially formed by the influence of future landed spacecraft. Significant changes in our knowledge of the capabilities of terrestrial organisms and the existence of possibly habitable martian environments have led to a new appreciation of where Mars Special Regions may be identified and protected. The SR-SAG also considered the impact of Special Regions on potential future human missions to Mars, both as locations of potential resources and as places that should not be inadvertently contaminated by human activity.

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. With seven metagenomic libraries generated from fracture water samples collected from five South African mines, this study was carried out to (1) screen for ubiquitous functions or pathways of biogeochemical cycling of CH4, S, and N; (2) to characterize the biodiversity represented by the common functional genes; (3) to investigate the subsurface biogeography as revealed by this subset of genes; and (4) to explore the possibility of using metagenomic data for evolutionary study. The ubiquitous functional genes are NarV, NPD, PAPS reductase, NifH, NifD, NifK, NifE, and NifN genes. Although these eight common functional genes were taxonomically and phylogenetically diverse and distinct from each other, the dissimilarity between samples did not correlate strongly with geographical or environmental parameters or residence time of the water. Por genes homologous to those of Thermodesulfovibrio yellowstonii detected in all metagenomes were deep lineages of Nitrospirae, suggesting that subsurface habitats have preserved ancestral genetic signatures that inform the study of the origin and evolution of prokaryotes.

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. The workshop aims were to identify and develop best practices for the collection, preservation, and analysis of terrestrial deep rock samples. This document summarizes the information shared during this workshop.

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 3.1 km. The major cation and anion composition of these thermal springs are very similar to that of the deep fracture water with the exception of the dissolved inorganic carbon and dissolved O2, both of which are typically higher in the springs than in the deep fracture water. The in situ biological relatedness of such thermal springs and the subsurface fracture fluids that feed them has not previously been evaluated. In this study, we evaluated the microbial diversity of six thermal spring and six subsurface sites in South Africa using high-throughput sequencing of 16S rRNA gene hypervariable regions. Proteobacteria were identified as the dominant phylum within both subsurface and thermal spring environments, but only one genera, Rheinheimera, was identified among all samples. Using Morisita similarity indices as a metric for pairwise comparisons between sites, we found that the communities of thermal springs are highly distinct from subsurface datasets. Although the Limpopo thermal springs do not appear to provide a new window for viewing subsurface bacterial communities, we report that the taxonomic compositions of the subsurface sites studied are more similar than previous results would indicate and provide evidence that the microbial communities sampled at depth are more correlated to subsurface conditions than geographical distance.

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. Two different arginine-functionalized chitosans (6% arginine-substituted and 30% arginine-substituted) each strongly inhibited P. fluorescens and E. coli growth. Time-dependent killing efficacy experiments showed that 5000 mg l(-1) of 6%- and 30%-substituted chitosan-arginine killed 2.7 logs and 4.5 logs of P. fluorescens, and 4.8 logs and 4.6 logs of E. coli in 4h, respectively. At low concentrations, the 6%-substituted chitosan-arginine was more effective in inhibiting cell growth even though the 30%-substituted chitosan-arginine appeared to be more effective in permeabilizing the cell membranes of both P. fluorescens and E. coli. Studies using fluorescent probes, 1-N-phenyl-naphthylamine (NPN), nile red (NR) and propidium iodide (PI), and field emission scanning electron microscopy (FESEM) suggest that chitosan-arginine's antibacterial activity is, at least in part, due to its interaction with the cell membrane, in which it increases membrane permeability.

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.) and was compared to the community from the same borehole after 2 weeks of being plugged with an expandable packer. Statistical analyses with UniFrac revealed a significant shift in community structure following the addition of the packer. Phospholipid fatty acid (PLFA) analysis suggested that Nitrosomonadales dominated the oxic borehole, while PLFAs indicative of anaerobic bacteria were most abundant in the samples from the plugged borehole. Microbial sequences were represented primarily by Firmicutes, Proteobacteria, and a lineage of sequences which did not group with any identified bacterial division; phylogenetic analyses confirmed the presence of a novel candidate division. This "Henderson candidate division" dominated the clone libraries from the dilute anoxic fluids. Sequences obtained from the granitic rock core (1,740 m below the surface) were represented by the divisions Proteobacteria (primarily the family Ralstoniaceae) and Firmicutes. Sequences grouping within Ralstoniaceae were also found in the clone libraries from metal-rich fluids yet were absent in more dilute fluids. Lineage-specific comparisons, combined with phylogenetic statistical analyses, show that geochemical variance has an important effect on microbial community structure in deep, subsurface systems.

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. pneumophila genomic DNA. However, cycle threshold (C(T)) were yielded and DNA product detected in our no-template negative controls and the phenomenon persisted when two separate batches of PCR reagents and water from two different biochemical companies were tested. Since L. pneumophila can be widespread in municipal water supplies, the commercial reagents, especially the reagent water (80% of the reaction volume), could be the source of contamination. To test this hypothesis, we treated Millipore Milli-Q water by filtering through a 0.2 microm-pore-size polycarbonate filter to remove bacteria prior to autoclaving. Real-time PCR using this water had no contamination. Our finding is indirect evidence that commercially available purified water can harbor low level contamination by L. pneumophila DNA that has escaped purification processes. This presents a challenge when developing a sensitive DNA-based bacterial detection method if the target organism or its DNA is a common contaminant of necessary reagents.

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. A specific and sensitive method for detecting the Cry1Ac toxin would facilitate monitoring for this protein in crop and non-crop plants and also in foods. The purpose of this study was to develop an immuno-PCR technique for detecting this toxin. Immuno-PCR combines the specificity of an ELISA reaction with the sensitivity of assays that use a PCR-amplification step. In our assay, anti-Cry1Ac antibodies were covalently bound to reporter DNA via a linker molecule, succinimidyl-4-[N-maleimidomethyl]-cyclohexane-1-carboxylate (SMCC). Antigen was coated onto the surfaces of polyvinyl chloride microtiter plates or onto streptavidin-coated beads. Each of these solid-surface platforms was tested in immuno-PCR reactions. Both the microtiter plate- and bead-based assays showed a high degree of specificity and sensitivity, with minimum detection limits of 21.6 and 432 ng of toxin, respectively. This sensitive immuno-PCR method could be modified for detecting a variety of other protein toxins.