Evolution of an ionizing and desiccation resistant organism
Ionizing resistant organism:
Ionizing radiation resistant populations of Escherichia coli have been created through 20 iterative cycles of radiation and outgrowth. The sequencing of 7 isolates from the final population revealed 40 to 71 single nucleotide polymorphisms (SNPs) in each of the isolates and a total of 378 mutations (Harris et al., 2009). These mutations may have arisen random and their pattern of evolution cannot be revealed by sequencing the final isolates. Our aim has been to use real time PCR based assay, TaqMAMA assay and deep coverage population sequencing to separate the beneficial mutations from the non-beneficial mutations necessary for radioresistance.
Desiccation resistant organism:
The stresses from both ionizing radiation and desiccation produce similar damage in cells in the form of double-stranded breaks. Many bacteria found on Earth that are ionizing radiation resistant are also desiccation tolerant. Perhaps similar mechanisms are involved in the resistance to both stresses. Our lab is focused on creating a desiccation tolerant organism and characterizing the mutations that accumulate to discover mechanisms that lead to desiccation tolerance.
Selective capture technique: Reconstruction of microbial communities
Metagenomics is an emerging field in molecular biology and is best defined as the extraction and sequencing of DNA from environmental samples. The process begins with the collection of organisms and metadata from the environment. The DNA is extracted and sequenced through a variety of platforms which include Sanger dye terminator, 454 pyrosequencing, Illumina Genome Analyzer, Ion Torrent, and Applied Biosystems SOLiD. After sequencing, the fragments are aligned using computer assembly programs. Whole genome sequences are put together this way however, many times it is difficult to retrieve complete genomes from environmental samples. Gaps and spaces appear frequently throughout the assembly process. Solutions to this issue include resequencing the DNA and/or primer walking to fill in the gaps; both processes are tedious. Another issue for metagenomic studies are environments with low biomass. Some environments do not have enough biomass to meet the standards of some current sequencing platforms that require microgram (µg) to miligram (mg) amounts. In some cases, gathering more genomic DNA is not an option. An additional issue is attempting to define the community structure in environmental communities. Microbial ecologists use other methods (alone or in conjunction with metagenomic sequencing) to define community structure from an environmental sample. The 16S rRNA gene is a highly conserved locus found in all prokaryotes. The sequence of this locus is commonly used to define taxa during phylogenetic studies. Typically, isolated environmental DNA will be amplified using the polymerase chain reaction (PCR) directed by conserved sequences within the 16S rRNA gene. Conceptually, amplification and isolation of the gene of interest reduces the amount of sequence that must be obtained to define the community, but there are limitations when performing PCR. Organism identification depends on successful amplification of their 16S gene and a number of taxon-specific factors can negatively affect the amplification of these identifying sequences. The primer binding sites are not well-conversed in all taxa, resulting in poor or unequal amplification Large differences in GC content can also alter the success of PCR amplification for certain sequences. Chimeras can also be introduced during PCR amplification between similar molecules of DNA. These PCR biases can result in an overestimation of the actual microbial diversity within an environment.
Our lab has developed a technique to help alleviate some of these problems associated with current metagenomic studies. Our method, called "Selective capture", pulls out specific DNA sequences from complex and/or low biomass environments. To date, we have pulled out the 16S rDNA from a mock community consisting of 21 bacterial species at varying concentrations. We have also targeted and pulled out genes specific for methanogenesis from genomic DNA isolated from the basal ice of Taylor Glacier.
Another objective of this laboratory are to define the subset of proteins required for the ionizing radiation resistance of Deinococcus radiodurans R1, and characterize the activities of those proteins. D. radiodurans, a non-sporeforming bacterium, has extraordinary tolerance for ionizing radiation. D. radiodurans , a non-sporeforming bacterium, has extraordinary tolerance for ionizing radiation. Exponential phase cultures of D. radiodurans R1 survive 500,000 Rad (5000 Gray) of γ radiation without loss of viability or evidence of mutation. In terms of DNA damage, 5000 Gy γ radiation introduces approximately 200 DNA double strand breaks, over 3000 single strand breaks, and greater than 1000 sites of base damage per genome. This organism does not passively protect its genome from the incident radiation. Instead all available evidence argues that D. radiodurans efficiently and accurately repairs DNA damage. The most recent work from this laboratory is an outgrowth of a DOE-funded project in which a D. radiodurans-specific DNA microarray was used to identify gene products potentially involved in ionizing radiation resistance. D. radiodurans R1’s transcriptional response to a sub-lethal dose of ionizing radiation (IR) was characterized, and 72 genes identified that are up-regulated three fold or higher during the first hour post-irradiation. This expression profile was compared with R1 cultures recovering from desiccation and with an isogenic irrE defective strain exposed to IR. Two key results have come from these comparisons. First, this organism’s remarkable ability to repair IR-induced DNA damage does not appear to be related to massive alterations in gene expression, or large magnitude changes in transcript abundance. Second, we establish that the irrE gene product is a transcriptional activator that regulates at least 23 downstream targets, 16 of which are part of this species’ common response to IR and desiccation. This work focused our attention on a limited number of genes that should confer radiotolerance on D. radiodurans, including a number of genes that encode proteins of unknown function. The five hypothetical genes that were induced to highest level in response to ionizing radiation and desiccation were deleted, and the radioresistance of the resulting strains compared to the R1 parent. All five have been shown to contribute to the ionizing radiation resistance of this species, unequivocally establishing that D. radiodurans’ radioresistance is, at least in part, due to the action of novel proteins.