I put together a brief summary (see below) of my research on salinity effects on microbial communities in hypersaline microbial mats for some people from Ingeny International (
http://www.ingeny.com/). Ingeny manufactures a system for denaturing gradient gel electrophoresis (DGGE), a tool for rapid analysis of microbial communities. I have been pretty pleased with the Ingeny system. It solves many of the problems that were endemic with the Bio-Rad DGGE system. For a discussion of DGGE systems and DGGE related problems, I recommend the yahoo group for DGGE (
http://groups.yahoo.com/group/dgge/). General molecular issues are raised on the yahoo group Molecular Diagnostics / Molecular Ecology (
http://groups.yahoo.com/group/MDME/).
Well, the rest of my day is going to be devoted to preparing for my course in molecular evolution at the Marine Biology Laboratory at Woods Hole, MA (
http://workshop.molecularevolution.org/). That, and working on a grant proposal to the NASA - Astrobiology: Exobiology and Evolutionary Biology grant request (
http://astrobiology.arc.nasa.gov). No lab work, though.
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Microbial mat communities represent, in gross morphology, some of the earliest known microbial communities on Earth. As a common form of microbial community existing on Earth during the Precambrian, and dominant in the Proterozoic era, these systems are hypothesized to have played a significant role in the development of modern oceanic and atmospheric conditions. In particular, the development of oxygen photosynthesis by cyanobacteria, which are dominant in modern mat systems, is thought to have played a particularly large role in shaping the modern environment. Fossilized lipid biomarkers from ancient mat communities, together with their stable carbon isotopic signatures, have helped establish a connection between modern mat ecosystems and their ancient counterparts. Studies of modern mat communities can help us understand ancient microbial ecosystems and the impact these and similar communities have had on the development of the modern earth. Hypersaline microbial mat communities from Guerrero Negro, Mexico have been used as model modern mat systems and are a central focus of several Ames Exobiology investigations.
Hypersaline microbial mats developing in evaporation ponds of the Guerrero Negro saltern are found growing along a salinity gradient from 6% to 16%. These systems are dependent on the primary production of phototrophic organisms, primarily cyanobacteria, but including diatoms and anoxygenic phototrophic bacteria. Prior studies have shown that salinity can affect population structure directly (e.g. salinity tolerances of individual organisms) and indirectly (e.g. secondary effects as a result of modified oxygen diffusion rates), and as a result modify communal carbon flow.
We are currently employing various molecular tools to characterize shifts in community structure of the total microbial population as a result of alterations in salinity levels. In addition to domain level analyses (Bacteria, Archae and Eukaryotes) we intend to target photosynthetic microorganisms, including oxygenic phototrophs (i.e. Diatoms and Cyanobacteria) and anoxygenic phototrophs (i.e. Green and Purple Photosynthetic Bacteria),). In most hypersaline mats, cyanobacteria are the major mat building organisms, and the largest contributors to the primary production of the system. Most primary productivity occurs via oxygenic photosynthesis by cyanobacteria or diatoms, and carbon is fixed using the Calvin cycle. Anoxygenic photosynthesis can be conducted by cyanobacteria operating photosystem I, and by anoxygenic photosynthetic bacteria (i.e. green and purple sulfur, and green non-sulfur). We hope to determine the impact of these shifts on stable carbon isotope partitioning as a means to understand carbon cycling as affected by salinity. We plan to combine nucleic-acid based molecular techniques for population analysis with lipid biomarker and carbon isotope measurements for quantitative information regarding microbial population structure and information regarding anabolic pathways utilized by mat primary producers.
Our initial molecular analyses are performed using a fairly standard DNA extraction-PCR amplification-denaturing gradient gel electrophoresis (DGGE) methodology. We are employing a wide variety of primers for PCR-DGGE analyses and while many of these primer sets amplify regions of the 16 or 18S rRNA gene, we are also exploring a variety of primer sets to target functional genes. Such functional gene analyses can provide both function and phylogeny simultaneously. We are also employing essentially the same process, but with RNA by generating copy DNA (cDNA) from RNA extraction from the same samples.
DGGE analyses are only the first stage in the characterization of microbial communities. These analyses are used to provide a broad overview of the diversity and shifts in community at different depths in the mat and accompanying changes in salinity. We show below preliminary PCR-DGGE analyses of a hypersaline microbial mat (85 ppt) removed from Guerrero Negro and transported to our greenhouse facility at NASA-Ames Research Center (
http://greencam.arc.nasa.gov/). These mats were then maintained under similar salinities or reduced salinities (20 ppt). After approximately 1 year under these conditions, the mats were sampled for lipid and DNA-based molecular analyses.
Below, two preliminary PCR-DGGE figures are presented. Figure 1 represents total bacterial communities as different depth and under two different salinities in a hypersaline microbial mat. The primers employed are the general bacterial primers pioneered by Gerard Muyzer and amplify a region of the 16S rRNA gene from most bacteria (positions 341 to 926, E. coli numbering). Figure 2 represents the cyanobacterial community from the same samples. The primers employed were published by Ulrich Nubel, Ferran Garcia-Pichel and Gerard Muyzer, and amplify a region of the 16S rRNA gene (359 to 805, E. coli numbering). Amplification products generated by PCR using both primer sets were electrophoresed in an acrylamide gel (6% acrylamide) containing a denaturing gradient (30-65% for general bacterial PCR product, 25-70% for cyanobacterial PCR product). These PCR products were electrophoresed for 17 hr at 60 C in a 1x TAE buffer with an Ingeny phorU2x2 DGGE system. The gels were stained using the GelStar Nucleic acid stain and photographed while on a UV-transilluminator.
The 18 lanes depicted below are the same for each primer set and represent a series of 8 layers of the microbial mat down to a depth of roughly 1 cm. Lanes 1 (layer 1), 4 (layer 2), 7 (layer 3), 9 (layer 4), 11 (layer 5), 13 (layer 6), 15 (layer 7), and 17 (layer 8) represent layers of the mat maintained under a native salinity of 85 ppt. Lanes 2,3 (layer 1), 5,6 (layer 2), 8 (layer 3), 10 (layer 4), 12 (layer 5), 14 (layer 6), 16 (layer 7) and 18 (layer 8) represent the same layers but from mats maintained under a lowered salinity of 20 ppt (Replicate samples for some layers were taken from different greenhouse locations). Some very general observations are noted:
* DNA amplified from replicate samples generated highly similar DGGE patterns
* Dramatic shifts in community were observed in with depth in the surface of mats from both salinities. These shifts occurred largely in the top 3 depths, and the 4th and deeper layers had many populations (but not all) in common.
* Salinity-driven shifts in general bacterial and cyanobacterial communities were observed. However, there were many populations common to both treatments. These populations have not yet been identified by sequence analysis.
*The cyanobacterial PCR-DGGE analysis revealed lower diversity than did the general bacterial analysis. At all depths, the presence or absence of some populations could be correlated to salinity.
Figure 1: DGGE Analysis of General Bacterial Populations in Microbial Mats
Figure 2: DGGE Analysis of Cyanobacterial Populations in Microbial Mats
