Microbial ecology and biogeochemistry of hypersaline sediments in Orca Basin.

In deep ocean hypersaline basins, the combination of high salinity, unusual ionic composition and anoxic conditions represents significant challenges for microbial life. We used geochemical porewater characterization and DNA sequencing based taxonomic surveys to enable environmental and microbial characterization of anoxic hypersaline sediments and brines in the Orca Basin, the largest brine basin in the Gulf of Mexico. Full-length bacterial 16S rRNA gene clone libraries from hypersaline sediments and the overlying brine were dominated by the uncultured halophilic KB1 lineage, Deltaproteobacteria related to cultured sulfate-reducing halophilic genera, and specific lineages of heterotrophic Bacteroidetes. Archaeal clones were dominated by members of the halophilic methanogen genus Methanohalophilus, and the ammonia-oxidizing Marine Group I (MG-I) within the Thaumarchaeota. Illumina sequencing revealed higher phylum- and subphylum-level complexity, especially in lower-salinity sediments from the Orca Basin slope. Illumina and clone library surveys consistently detected MG-I Thaumarchaeota and halotolerant Deltaproteobacteria in the hypersaline anoxic sediments, but relative abundances of the KB1 lineage differed between the two sequencing methods. The stable isotopic composition of dissolved inorganic carbon and methane in porewater, and sulfate concentrations decreasing downcore indicated methanogenesis and sulfate reduction in the anoxic sediments. While anaerobic microbial processes likely occur at low rates near their maximal salinity thresholds in Orca Basin, long-term accumulation of reaction products leads to high methane concentrations and reducing conditions within the Orca Basin brine and sediments.

Mobile elements in archaeal genomes.

The recent availability of several archaeal genome sequences has provided a basis for detailed analyses of the frequency, location and phylogeny of archaeal mobile elements. All the known elements fall into two main types, autonomous insertion sequence (IS) elements and the non-autonomous miniature inverted repeat element (MITE)-like elements. Both classes are considered to be mobilized via transposases that are encoded by the IS elements, although mobility has only been demonstrated experimentally for a few elements. The number, and diversity, of the elements differs greatly between the genomes. At one extreme Sulfolobus solfataricus P2 and Halobacterium NRC-1 are very rich in elements while Methanobacterium thermoautotrophicum contains none. The former also show examples of complex clusters of interwoven elements. An analysis of the genomic distribution in S. solfataricus suggests that the putative oriC and terC regions act as barriers for the mobility of both IS and MITE-like elements. Moreover, the very high level of truncated IS elements in the genomes of S. solfataricus, Sulfolobus tokodaii and Thermoplasma volcanium suggests that there may be a cellular mechanism for selectively inactivating IS elements at a point when they become too numerous and disadvantageous for the cell. Phylogenetically, archaeal IS elements are confined to 11 of the 17 known families of bacterial and eukaryal IS elements where some generate distinct subgroups. Finally, DNA viruses, plasmids and DNA fragments can also be inserted into, and excised from, archaeal genomes by means of an integrase-mediated mechanism that has special archaeal characteristics.

Whole cell immunomagnetic enrichment of environmental microbial consortia using rRNA-targeted Magneto-FISH.

Magneto-FISH, in combination with metagenomic techniques, explores the middle ground between single-cell analysis and complex community characterization in bulk samples to better understand microbial partnerships and their roles in ecosystems. The Magneto-FISH method combines the selectivity of catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH) with immunomagnetic capture to provide targeted molecular and metagenomic analysis of co-associated microorganisms in the environment. This method was originally developed by Pernthaler et al. (Pernthaler et al., 2008; Pernthaler & Orphan, 2010). It led to the discovery of new bacterial groups associated with anaerobic methane-oxidizing (ANME-2) archaea in methane seeps, as well as provided insight into their physiological potential using metagenomics. Here, we demonstrate the utility of this method for capturing aggregated consortia using a series of nested oligonucleotide probes of differing specificity designed to target either the ANME archaea or their Deltaproteobacteria partner, combined with 16S rRNA and mcrA analysis. This chapter outlines a modified Magneto-FISH protocol for large- and small-volume samples and evaluates the strengths and limitations of this method predominantly focusing on (1) the relationship between FISH probe specificity and sample selectivity, (2) means of improving DNA yield from paraformaldehyde-fixed samples, and (3) suggestions for adapting the Magneto-FISH method for other microbial systems, including potential for single-cell recovery.

Growth temperatures of archaeal communities can be estimated from the guanine-plus-cytosine contents of 16S rRNA gene fragments.

Prokaryote growth temperatures in environmental samples are difficult to measure because it is hard to culture viable prokaryotes in natural environments. We comprehensively surveyed growth temperatures and 16S rRNA sequences of prokaryotes to estimate their growth temperatures based on guanine-plus-cytosine contents (P(GC)) of their 16S rRNA sequences. We focused on archaea because of the wide range of growth temperatures within this group. Their minimum (Tmin), optimum (Topt) and maximum (Tmax) growth temperatures correlated strongly with PGC of their 16S rRNA genes. Linear regression equations were established to approximate Tmin, Topt and Tmax from P(GC). We also established a linear regression equation for calculating P(GC) of 16S rRNA genes based on the melting temperatures (Tm) of PCR fragments, without using a clone library or sequencing. Environmental samples were obtained from a wide variety of microbial natural habitats. Tm of archaeal 16S rRNA genes amplified by real-time PCR were determined by melting curve analysis. Based on those values, P(GC) of 16S rRNA genes and mean Tmin, Topt and Tmax were calculated using the linear regression equations. These temperatures correlated strongly with the in situ temperatures. Tmax agreed particularly well with these temperatures, suggesting many archaea live at their maximum growth temperatures.

Metagenomic insights into anaerobic metabolism along an Arctic peat soil profile.

A metagenomic analysis was performed on a soil profile from a wet tundra site in northern Alaska. The goal was to link existing biogeochemical knowledge of the system with the organisms and genes responsible for the relevant metabolic pathways. We specifically investigated how the importance of iron (Fe) oxides and humic substances (HS) as terminal electron acceptors in this ecosystem is expressed genetically, and how respiratory and fermentative processes varied with soil depth into the active layer and into the upper permafrost. Overall, the metagenomes reflected a microbial community enriched in a diverse range of anaerobic pathways, with a preponderance of known Fe reducing species at all depths in the profile. The abundance of sequences associated with anaerobic metabolic processes generally increased with depth, while aerobic cytochrome c oxidases decreased. Methanogenesis genes and methanogen genomes followed the pattern of CH4 fluxes: they increased steeply with depth into the active layer, but declined somewhat over the transition zone between the lower active layer and the upper permafrost. The latter was relatively enriched in fermentative and anaerobic respiratory pathways. A survey of decaheme cytochromes (MtrA, MtrC and their homologs) revealed that this is a promising approach to identifying potential reducers of Fe(III) or HS, and indicated a possible role for Acidobacteria as Fe reducers in these soils. Methanogens appear to coexist in the same layers, though in lower abundance, with Fe reducing bacteria and other potential competitors, including acetogens. These observations provide a rich set of hypotheses for further targeted study.