Deciphering the prokaryotic community and metabolisms in South African deep-mine biofilms through antibody microarrays and graph theory.

In the South African deep mines, a variety of biofilms growing in mine corridor walls as water seeps from intersections or from fractures represents excellent proxies for deep-subsurface environments. However, they may be greatly affected by the oxygen inputs through the galleries of mining activities. As a consequence, the interaction between the anaerobic water coming out from the walls with the oxygen inputs creates new conditions that support rich microbial communities. The inherent difficulties for sampling these delicate habitats, together with transport and storage conditions may alter the community features and composition. Therefore, the development of in situ monitoring methods would be desirable for quick evaluation of the microbial community. In this work, we report the usefulness of an antibody-microarray (EMChip66) immunoassay for a quick check of the microbial diversity of biofilms located at 1.3 km below surface within the Beatrix deep gold mine (South Africa). In addition, a deconvolution method, previously described and used for environmental monitoring, based on graph theory and applied on antibody cross-reactivity was used to interpret the immunoassay results. The results were corroborated and further expanded by 16S rRNA gene sequencing analysis. Both culture-independent techniques coincided in detecting features related to aerobic sulfur-oxidizers, aerobic chemoorganotrophic Alphaproteobacteria and metanotrophic Gammaproteobacteria. 16S rRNA gene sequencing detected phylotypes related to nitrate-reducers and anaerobic sulfur-oxidizers, whereas the EMChip66 detected immunological features from methanogens and sulfate-reducers. The results reveal a diverse microbial community with syntrophic metabolisms both anaerobic (fermentation, methanogenesis, sulphate and nitrate reduction) and aerobic (methanotrophy, sulphur oxidation). The presence of oxygen-scavenging microbes might indicate that the system is modified by the artificial oxygen inputs from the mine galleries.

Prokaryotic communities and operating metabolisms in the surface and the permafrost of Deception Island (Antarctica).

In this study we examined the microbial community composition and operating metabolisms on the surface and in the permafrost of Deception Island, (Antarctica) with an on site antibody microarray biosensor. Samples (down to a depth of 4.2 m) were analysed with LDChip300 (Life Detector Chip), an immunosensor containing more than 300 antibodies targeted to bacterial and archaeal antigens. The immunograms showed positive antigen-antibody reactions in all surface samples (lichens, pyroclasts) and the top layer of the permafrost. The results indicated the presence of exopolysaccharides, bacteria belonging to the Alpha-, Delta- and Gammaproteobacteria, Bacteroidetes, Gram-positive Actinobacteria and Firmicutes, as well as archaeal species, most probably Methanobacterium spp. Positive reactions with antibodies to proteins and peptides revealed the presence of nitrogen fixation (NifHD, GlnB, HscA), methanogenic (McrB), iron homeostasis and iron scavenging (ferritins and DPS proteins) proteins, as well as ABC transporters, which indicated that these processes were operating at the time of sampling. These results were validated with other molecular ecology techniques such as oligonucleotide microarrays, 16S bacterial rRNA gene sequence analysis, aerobic viable counts and microscopy. Molecular ecology results showed a differentiated pattern along the depth of the drill, being the top active layer the most diverse, with Acidobacteria, Actinobacteria, Proteobacteria, Bacteroidetes and the phototrophs Cyanobacteria and Chloroflexi as dominant groups. Actinobacteria and Firmicutes were dominant in depths from 0.5 to 2 m, and Betaproteobacteria from 3 to 4.2 m. The geochemical analysis revealed the presence of low molecular weight organic acids (acetate, formate) which could be used by microorganisms as energy sources for sulfate, nitrate and metal reduction under anaerobic conditions.

A microbial oasis in the hypersaline Atacama subsurface discovered by a life detector chip: implications for the search for life on Mars.

The Atacama Desert has long been considered a good Mars analogue for testing instrumentation for planetary exploration, but very few data (if any) have been reported about the geomicrobiology of its salt-rich subsurface. We performed a Mars analogue drilling campaign next to the Salar Grande (Atacama, Chile) in July 2009, and several cores and powder samples from up to 5 m deep were analyzed in situ with LDChip300 (a Life Detector Chip containing 300 antibodies). Here, we show the discovery of a hypersaline subsurface microbial habitat associated with halite-, nitrate-, and perchlorate-containing salts at 2 m deep. LDChip300 detected bacteria, archaea, and other biological material (DNA, exopolysaccharides, some peptides) from the analysis of less than 0.5 g of ground core sample. The results were supported by oligonucleotide microarray hybridization in the field and finally confirmed by molecular phylogenetic analysis and direct visualization of microbial cells bound to halite crystals in the laboratory. Geochemical analyses revealed a habitat with abundant hygroscopic salts like halite (up to 260 g kg(-1)) and perchlorate (41.13 µg g(-1) maximum), which allow deliquescence events at low relative humidity. Thin liquid water films would permit microbes to proliferate by using detected organic acids like acetate (19.14 µg g(-1)) or formate (76.06 µg g(-1)) as electron donors, and sulfate (15875 µg g(-1)), nitrate (13490 µg g(-1)), or perchlorate as acceptors. Our results correlate with the discovery of similar hygroscopic salts and possible deliquescence processes on Mars, and open new search strategies for subsurface martian biota. The performance demonstrated by our LDChip300 validates this technology for planetary exploration, particularly for the search for life on Mars.

Assessing antibody microarrays for space missions: effect of long-term storage, gamma radiation, and temperature shifts on printed and fluorescently labeled antibodies.

Antibody microarrays are becoming frequently used tools for analytical purposes. A key factor for optimal performance is the stability of the immobilized (capturing) antibodies as well as those that have been fluorescently labeled to achieve the immunological test (tracers). This is especially critical for long-distance transport, field testing, or planetary exploration. A number of different environmental stresses may affect the antibody integrity, such as dryness, sudden temperature shift cycles, or, as in the case of space science, exposure to large quantities of the highly penetrating gamma radiation. Here, we report on the effect of certain stabilizing solutions for long-term storage of printed antibody microarrays under different conditions. We tested the effect of gamma radiation on printed and freeze- or vacuum-dried fluorescent antibodies at working concentrations (tracer antibodies), as well as the effect of multiple cycles of sudden and prolonged temperature shifts on the stability of fluorescently labeled tracer antibody cocktails. Our results show that (i) antibody microarrays are stable at room temperature when printed on stabilizing spotting solutions for at least 6 months, (ii) lyophilized and vacuum-dried fluorescently labeled tracer antibodies are stable for more than 9 months of sudden temperature shift cycles (-20°C to 25°C and 50°C), and (iii) both printed and freeze- or vacuum-dried fluorescent tracer antibodies are stable after several-fold excess of the dose of gamma radiation expected during a mission to Mars. Although different antibodies may exhibit different susceptibilities, we conclude that, in general, antibodies are suitable for use in planetary exploration purposes if they are properly treated and stored with the use of stabilizing substances.

SOLID3: a multiplex antibody microarray-based optical sensor instrument for in situ life detection in planetary exploration.

The search for unequivocal signs of life on other planetary bodies is one of the major challenges for astrobiology. The failure to detect organic molecules on the surface of Mars by measuring volatile compounds after sample heating, together with the new knowledge of martian soil chemistry, has prompted the astrobiological community to develop new methods and technologies. Based on protein microarray technology, we have designed and built a series of instruments called SOLID (for "Signs Of LIfe Detector") for automatic in situ detection and identification of substances or analytes from liquid and solid samples (soil, sediments, or powder). Here, we present the SOLID3 instrument, which is able to perform both sandwich and competitive immunoassays and consists of two separate functional units: a Sample Preparation Unit (SPU) for 10 different extractions by ultrasonication and a Sample Analysis Unit (SAU) for fluorescent immunoassays. The SAU consists of five different flow cells, with an antibody microarray in each one (2000 spots). It is also equipped with an exclusive optical package and a charge-coupled device (CCD) for fluorescent detection. We demonstrated the performance of SOLID3 in the detection of a broad range of molecular-sized compounds, which range from peptides and proteins to whole cells and spores, with sensitivities at 1-2 ppb (ng mL⁻¹) for biomolecules and 104 to 10³ spores per milliliter. We report its application in the detection of acidophilic microorganisms in the Río Tinto Mars analogue and report the absence of substantial negative effects on the immunoassay in the presence of 50 mM perchlorate (20 times higher than that found at the Phoenix landing site). Our SOLID instrument concept is an excellent option with which to detect biomolecules because it avoids the high-temperature treatments that may destroy organic matter in the presence of martian oxidants.

Classification of modern and old Río Tinto sedimentary deposits through the biomolecular record using a life marker biochip: implications for detecting life on Mars.

The particular mineralogy formed in the acidic conditions of the Río Tinto has proven to be a first-order analogue for the acid-sulfate aqueous environments of Mars. Therefore, studies about the formation and preservation of biosignatures in the Río Tinto will provide insights into equivalent processes on Mars. We characterized the biomolecular patterns recorded in samples of modern and old fluvial sediments along a segment of the river by means of an antibody microarray containing more than 200 antibodies (LDCHIP200, for Life Detector Chip) against whole microorganisms, universal biomolecules, or environmental extracts. Samples containing 0.3-0.5 g of solid material were automatically analyzed in situ by the Signs Of LIfe Detector instrument (SOLID2), and the results were corroborated by extensive analysis in the laboratory. Positive antigen-antibody reactions indicated the presence of microbial strains or high-molecular-weight biopolymers that originated from them. The LDCHIP200 results were quantified and subjected to a multivariate analysis for immunoprofiling. We associated similar immunopatterns, and biomolecular markers, to samples with similar sedimentary age. Phyllosilicate-rich samples from modern fluvial sediments gave strong positive reactions with antibodies against bacteria of the genus Acidithiobacillus and against biochemical extracts from Río Tinto sediments and biofilms. These samples contained high amounts of sugars (mostly polysaccharides) with monosaccharides like glucose, rhamnose, fucose, and so on. By contrast, the older deposits, which are a mix of clastic sands and evaporites, showed only a few positives with LDCHIP200, consistent with lower protein and sugar content. We conclude that LDCHIP200 results can establish a correlation between microenvironments, diagenetic stages, and age with the biomarker profile associated with a sample. Our results would help in the search for putative martian biomarkers in acidic deposits with similar diagenetic maturity. Our LDCHIP200 and SOLID-like instruments may be excellent tools for the search for molecular biomarkers on Mars or other planets.

A 200-antibody microarray biochip for environmental monitoring: searching for universal microbial biomarkers through immunoprofiling.

Environmental biomonitoring approaches require the measurement of either unequivocal biomarkers or specific biological profiles. Antibody microarrays constitute new tools for fast and reliable analysis of up to hundreds of biomarkers simultaneously. Herein we report 150 new polyclonal antibodies against microbial strains and environmental extracts, as well as the construction and validation of an antibody microarray (EMCHIP200, for "Environmental Monitoring Chip") containing 200 different antibodies. Each antibody was tested against its antigen for its specificity and cross-reactivity by a sandwich microarray immunoassay. The limit of detection was 0.2 ng mL (-1) for some proteins and 10 (4)-10 (5) cells mL (-1) for bacterial cells and spores. Partial biochemical characterization allowed identification of polymeric compounds (proteins and polysaccharides) as some of the targets recognized by the antibodies. We have successfully used the EMCHIP200 for the detection of biological polymers in samples from extreme environments around the world (e.g., a deep South African mine, Antarctica's dry valleys, Yellowstone National Park, Iceland, and Rio Tinto surface and subsurface). Clustering analysis permitted us to associate similar immunoprofiles or patterns to samples from apparently very different environments, indicating that they indeed share similar universal biomarkers. Our EMCHIP200 constitutes a new generation of immunosensors for biomarker detection and profiling, for either environmental, industrial, biotechnological, or astrobiological applications.