Phylogenetic perspective and the search for life on earth and elsewhere

Any search for microbial life on Mars cannot rely upon cultivation of indigenous organisms. Only a minority of even terrestrial organisms that are observed in mixed, naturally-occurring microbial populations can be cultivated in the laboratory. Consequently, methods are being developed for analyzing the phylogenetic affiliations of the constituents of natural microbial populations without the need for their cultivation. This is more than an exercise in taxonomy, for the extent of phylogenetic relatedness between unknown and known organisms is some measure of the extent of their biochemical commonalities. In one approach, total DNA is isolated from natural microbial populations and 16S rRNA genes are shotgun cloned for rapid sequence determinations and phylogenetic analyses. A second approach employs oligodeoxynucleotide hybridization probes that bind to phylogenetic group-specific sequences in 16S rRNA. Since each actively growing cell contains about 104 ribosomes, the binding of the diagnostic probes to single cells can be visualized by radioactivity or fluorescence. The application of these methods and the use of in situ cultivation techniques is illustrated using submarine hydrothermal vent communities. Recommendations are made regarding planning toward future Mars missions

Diversity of Life at the Geothermal Subsurface-Surface Interface: The Yellowstone Example Geophysical Monograph Series

Generally, studies of the terrestrial example of Yellowstone National Park indicate that the diversity of microbial life at the geothermal subsurface-surface interface is considerable. On the other hand, experiments in a subsurface well in Biscuit Basin suggest that the Yellowstone subsurface is highly reduced, with minimal in situ subsurface life at this location. The absence of life in the subsurface is likely due to low concentrations of available electron acceptors. Where subsurface thermal waters emerge to the surface, however, in the presence of oxidizing electron acceptors, microbial life blossoms on all growth surfaces at high temperatures. The geothermal subsurface-surface interface in the presence of both electron donors and acceptors, provides the key location for life to thrive and forms the cornerstone of the microbial ecosystem. Through molecular analyses, the identities of organisms present in a community can be determined by their phylogenetic types (phylotypes), their molecular signatures. Molecular sequences allow relationships to other life forms to be inferred. Comparisons of gene sequences of organisms and consideration of the geochemistry of a particular environment can help to explain how this geothermal system functions. Experimental results challenge some popular notions about the kinds of organisms that inhabit the geothermal realms and the energy sources that fuel them. In contrast to the popular notion that representatives of the phylogenetic domain Archaea dominate high-temperature ecosystems, members of the domain Bacteria are most abundant in the Yellowstone ecosystem. Moreover, while sulfur metabolism is generally proposed to be the primary energy source for life in this geothermal system, the main organisms identified by phylotype are related to organisms that utilize hydrogen, not sulfur, for energy. This implies that hydrogen is the main energy source that drives primary productivity in this and potentially other geothermal ecosystems. Primary dependence on hydrogen metabolism could be the common theme for high-temperature life in hydrothermal zones at mid-oceanic ridges, as well as for the earliest life on Earth and, potentially, for life on other planetary bodies

International cooperation for Mars exploration and sample return

The National Research Council's Space Studies Board has previously recommended that the next major phase of Mars exploration for the United States involve detailed in situ investigations of the surface of Mars and the return to earth for laboratory analysis of selected Martian surface samples. More recently, the European space science community has expressed general interest in the concept of cooperative Mars exploration and sample return. The USSR has now announced plans for a program of Mars exploration incorporating international cooperation. If the opportunity becomes available to participate in Mars exploration, interest is likely to emerge on the part of a number of other countries, such as Japan and Canada. The Space Studies Board's Committee on Cooperative Mars Exploration and Sample Return was asked by the National Aeronautics and Space Administration (NASA) to examine and report on the question of how Mars sample return missions might best be structured for effective implementation by NASA along with international partners. The committee examined alternatives ranging from scientific missions in which the United States would take a substantial lead, with international participation playing only an ancillary role, to missions in which international cooperation would be a basic part of the approach, with the international partners taking on comparably large mission responsibilities. On the basis of scientific strategies developed earlier by the Space Studies Board, the committee considered the scientific and technical basis of such collaboration and the most mutually beneficial arrangements for constructing successful cooperative missions, particularly with the USSR

Unexpected diversity and complexity of the Guerrero Negro hypersaline microbial mat

Author Posting. © The Author(s), 2006. This is the author's version of the work. It is posted here by permission of American Society for Microbiology for personal use, not for redistribution. The definitive version was published in Applied and Environmental Microbiology 72 (2006): 3685-3695, doi:10.1128/AEM.72.5.3685-3695.2006.We applied nucleic acids-based molecular methods, combined with estimates of biomass (ATP), pigments and microelectrode measurements of chemical gradients, to map microbial diversity vertically on the mm-scale in a hypersaline microbial mat from Guerrero Negro, Baja California Sur, Mexico. To identify the constituents of the mat, small-subunit ribosomal RNA genes were amplified by PCR from community genomic DNA extracted from layers, cloned and sequenced. Bacteria dominated the mat and displayed unexpected and unprecedented diversity. The majority (1336) of 1586 bacterial 16S rRNA sequences generated were unique, representing 752 species (≥97% rRNA sequence identity) in 42 of the main bacterial phyla, including 15 novel candidate phyla. The diversity of the mat samples differentiated according to the chemical milieu defined by concentrations of O2 and H2S. Chloroflexi formed the majority of the biomass by percentage of bulk rRNA and of clones in rRNA gene libraries. This result contradicts the general belief that Cyanobacteria dominate these communities. Although Cyanobacteria constituted a large fraction of the biomass in the upper few mm (>80% of total rRNA and photosynthetic pigments), Chloroflexi sequences were conspicuous throughout the mat. Filamentous Chloroflexi were identified by fluorescent in-situ hybridization within the polysaccharide sheaths of the prominent cyanobacterium Microcoleus chthonoplastes in addition to free-living in the mat. The biological complexity of the mat far exceeds that observed in other polysaccharide-rich microbial ecosystems, such as human and mouse distal guts, and suggests that positive feedbacks exist between chemical complexity and biological diversity.R. Ley was supported in part by an NRC- NASA Astrobiology Institutes Post Doctoral Associateship, J. Spear by an Agouron Institute postdoctoral fellowship. This work was supported by the NASA Cooperative Agreement with the University of Colorado Center for Astrobiology to N. R. Pace

Inflammatory Bowel Diseases Phenotype, C. difficile and NOD2 Genotype Are Associated with Shifts in Human Ileum Associated Microbial Composition

We tested the hypothesis that Crohn’s disease (CD)-related genetic polymorphisms involved in host innate immunity are associated with shifts in human ileum–associated microbial composition in a cross-sectional analysis of human ileal samples. Sanger sequencing of the bacterial 16S ribosomal RNA (rRNA) gene and 454 sequencing of 16S rRNA gene hypervariable regions (V1–V3 and V3–V5), were conducted on macroscopically disease-unaffected ileal biopsies collected from 52 ileal CD, 58 ulcerative colitis and 60 control patients without inflammatory bowel diseases (IBD) undergoing initial surgical resection. These subjects also were genotyped for the three major NOD2 risk alleles (Leu1007fs, R708W, G908R) and the ATG16L1 risk allele (T300A). The samples were linked to clinical metadata, including body mass index, smoking status and Clostridia difficile infection. The sequences were classified into seven phyla/subphyla categories using the Naïve Bayesian Classifier of the Ribosome Database Project. Centered log ratio transformation of six predominant categories was included as the dependent variable in the permutation based MANCOVA for the overall composition with stepwise variable selection. Polymerase chain reaction (PCR) assays were conducted to measure the relative frequencies of the Clostridium coccoides – Eubacterium rectales group and the Faecalibacterium prausnitzii spp. Empiric logit transformations of the relative frequencies of these two microbial groups were included in permutation-based ANCOVA. Regardless of sequencing method, IBD phenotype, Clostridia difficile and NOD2 genotype were selected as associated (FDR ≤0.05) with shifts in overall microbial composition. IBD phenotype and NOD2 genotype were also selected as associated with shifts in the relative frequency of the C. coccoides – E. rectales group. IBD phenotype, smoking and IBD medications were selected as associated with shifts in the relative frequency of F. prausnitzii spp. These results indicate that the effects of genetic and environmental factors on IBD are mediated at least in part by the enteric microbiota

Millimeter-scale genetic gradients and community-level molecular convergence in a hypersaline microbial mat

To investigate the extent of genetic stratification in structured microbial communities, we compared the metagenomes of 10 successive layers of a phylogenetically complex hypersaline mat from Guerrero Negro, Mexico. We found pronounced millimeter-scale genetic gradients that were consistent with the physicochemical profile of the mat. Despite these gradients, all layers displayed near-identical and acid-shifted isoelectric point profiles due to a molecular convergence of amino-acid usage, indicating that hypersalinity enforces an overriding selective pressure on the mat community

Disease phenotype and genotype are associated with shifts in intestinal-associated microbiota in inflammatory bowel diseases:

Abnormal host-microbe interactions are implicated in the pathogenesis of inflammatory bowel diseases. Previous 16S rRNA sequence analysis of intestinal tissues demonstrated that a subset of Crohn’s disease (CD) and ulcerative colitis (UC) samples exhibited altered intestinal associated microbial compositions characterized by depletion of Bacteroidetes and Firmicutes (particularly Clostridium taxa). We hypothesize that NOD2 and ATG16L1 risk alleles may be associated with these alterations

Culture-Independent Microbiological Analysis of Foley Urinary Catheter Biofilms

Background: Prevention of catheter-associated urinary tract infection (CAUTI), a leading cause of nosocomial disease, is complicated by the propensity of bacteria to form biofilms on indwelling medical devices [1,2,3,4,5]. Methodology/Principal Findings: To better understand the microbial diversity of these communities, we report the results of a culture-independent bacterial survey of Foley urinary catheters obtained from patients following total prostatectomy. Two patient subsets were analyzed, based on treatment or no treatment with systemic fluoroquinolone antibiotics during convalescence. Results indicate the presence of diverse polymicrobial assemblages that were most commonly observed in patients who did not receive systemic antibiotics. The communities typically contained both Gram-positive and Gramnegative microorganisms that included multiple potential pathogens. Conclusion/Significance: Prevention and treatment of CAUTI must take into consideration the possible polymicrobial nature of any particular infection