Michigan State University's Center for Genomics and Evolutionary Studies on Microbial Life at Low TemperaturesMember of NASA's Astrobiology Institute Research Findings for First Year of Project Our first year as members of the NASA Astrobiology
Institute has been rewarding and exciting.
In this “getting up to speed”
year, we have successfully attracted graduate
students and postdoctoral researchers
to the research projects being conducted
and have begun to generate results that
provide important foundations for our
overall research goals. In the Genomic
and Proteomic Analysis project, we completed
a phenotypic survey of 20 permafrost bacterial
isolates and chose two, Exiguobacterium
255-15 and Psychrobacter
273-4,
for complete genome sequencing that is
being accomplished in collaboration with
the DOE Joint Genome Institute. The strains,
which were isolated from 2-3 million and
20-40 thousand year old permafrost soils,
respectively, were determined to have
genomes of about 2.5 Mb (determined by
pulse field gel electrophoresis) and to
be capable of growth at low temperature
(-2.5°C) and low water activity (5 osmolar
salt), distinctive characteristics that
one would anticipate for microbes inhabiting
the permafrost. Within the past month,
DNA sequence representing a 10-fold coverage
of the Exiguobacterium isolate
has been obtained and similar sequencing
of the Psychrobacter strain should
be accomplished within the next couple
of months. We should, therefore, soon
be in a position to determine the complete
“informational content” of both
of these permafrost bacteria. Not only
will this be an important milestone for
our particular project, but in addition,
will be significant for microbial genome
sequencing in general, as no genome sequence
has yet been published for a psychroactive
bacterium. In the Bacterial Adaptation to Low Temperature project, E. coli is serving as the experimental organism. A total of thirty replicate lineages, representing populations with diverse thermal histories, have been adapted to 20°C for 2000 generations and efforts are in progress to examine adaptation to even lower temperatures (12-14°C). Significantly, initial analysis of the 2000 generation evolved populations indicates that adaptation to 20°C is frequently associated with a loss of competitive fitness at 40°C. Thus, a tradeoff associated with low-temperature adaptation may be a loss of fitness for growth at high temperatures. A genetic analysis of the evolved strains indicates that deletion events are more common than gene duplication events in the low temperature-evolved populations. Determining whether these changes in the genome contribute to the increase in competitiveness at low temperature is now an important goal. Adaptation to freezing conditions is also being investigated as part of the Bacterial Adaptation project. The first question that we have addressed is whether there is a possibility that E. coli has the capacity to evolve increased fitness to freezing conditions. The results to date suggest that it may. In particular, we have found that lines of E. coli that have been adapted for 20,000 generations to growth at 37°C have a greater kill-rate when subjected to daily freeze-thaw cycles than do non-adapted populations that have not been selected for greater fitness at high temperature. Thus, E. coli can display genetic variation in freezing tolerance suggesting that selection for increased freezing tolerance may result in the evolution of strains with increased fitness for freezing. One of the objectives in our Field Truth project, Indigenous Bacteria of Arctic and Antarctic Permafrost, is to increase our understanding of the phylogenetic diversity of the bacteria that inhabit the Arctic and Antarctic permafrost and to assess how much of that diversity can be successfully captured upon culturing from the permafrost. In these studies, 16S rDNA genes were amplified by PCR (polymerase chain reaction) using “universal” bacterial primers and template DNA isolated directly from permafrost soil samples. High throughput DNA sequencing was performed on the cloned PCR products to obtain 16S rDNA gene sequences. The soils tested to date had either been kept frozen since initial isolation or had been incubated under either aerobic or anaerobic conditions at 10°C for 8 weeks. DNA sequences for over 2,000 rrn genes have been determined and compared with sequences in the Ribosomal Database Project (RDP) <http://rdp.cme.msu.edu/> by automated software that we designed. The results indicate significant microbial diversity in all samples, including the Antarctic as well as the Arctic soils. Furthermore, microbial growth at 10°C was observed in most samples indicating the presence of living microbes. Members of the following phylogenetic Divisions were found: Green_Non-Sulfur Bacteria, Leptospirillum-Nitrospira, Nitrospina, Flexibacter-Cytophaga-Bacteroides, Planctomyces and relatives, Proteobacteria, Fibrobacter, and Acidobacterium. Among the Gram Positive Bacteria were members of the Thermotogales, Cyanobacteria, Anaerobaculum and two Divisions known only by environmental clones, OPB80 and WCHB1-31. To our knowledge, this is the highest resolution sampling of soil bacterial diversity in existence for any habitat. While the above results indicate which microbial groups are present and that some are able to grow in the permafrost soil when the temperature was raised to 10°C, they do not indicate whether the microbes are active at the in situ temperature and water activity. Hence, additional experiments were undertaken in the tundra zone of the Kolyma Lowland near the East Siberian Sea coast (70°N, 160°E). Here, the permafrost sediments are up to 800 m thick and have a mean annual temperature of -9 to -11°C. One sample investigated was a peaty-loam of Holocene age origin (2920 ±40 years old), located at a depth of 80 cm below the permafrost surface. Metabolic activity was determined by analysis of hydrogenotrophic and acetoclastic rates of 14__4 production. The results indicated methane formation from H14CO3- and 14CH3CO2- at temperatures down to -16.5°_. From this sample, a methanogenic psychrophilic bacterium, Methanosarcina sp. JL01 (16S rrn sequence AF 519802), was isolated. The second sample was collected from the lenses of overcooled brines (170-300 g/l) situated at depths of 40-50 m within permafrost sandy soils of marine origin that date back to Wisconsin glaciation (100-120 thousand years BP). These so-called “cryopegs” are the only hydrologic system on the Earth that are characterized by permanent temperatures below zero, high salinity, and isolation from external factors throughout their geologic history. The metabolic activity of the indigenous cryopeg microorganisms was detected down to -15°_ as measured by monitoring the incorporation of [14C]-labeled D-glucose into microbial cells. From this sample, a psychrophilic bacterium, designated Psychrobacter 2pS (16S rrn sequence AF177557), was isolated. It is believed that extraterrestrial life is only possible in the presence of water. The majority of planets and their satellites in the solar system are distinguished by low temperatures, at which water remains unfrozen only if the solute concentration is high. The evidence that there is free water on Mars implies that its water is saline. Thus, Martian free water may occur as brine lenses formed when Mars lost its atmosphere and became dry and cold. The Arctic cryopegs, together with the inhabiting microorganisms, may serve as a model for low temperature exobiological econiches on Mars and other planets and satellites in our solar system. Director, Dr. Michael F. Thomashow <thomash6@msu.edu>
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