Current planetary protection policies require that spacecraft targeted to sensitive solar system bodies be assembled and readied for launch in controlled cleanroom environments. A better understanding of the distribution and frequency at which high-risk contaminant microbes are encountered on spacecraft surfaces would significantly aid in assessing the threat of forward contamination. However, despite a growing understanding of the diverse microbial populations present in cleanrooms, less abundant microbial populations are probably not adequately taken into account due to technological limitations. This novel approach encompasses a wide spectrum of microbial species and will represent the true picture of spacecraft cleanroom-associated microbial diversity.
All of the current microbial diversity assessment techniques are based on an initial PCR amplification step. However, a number of factors are known to bias PCR amplification and jeopardize the true representation of bacterial diversity. PCR amplification of a minor template appears to be suppressed by the amplification of a more abundant template. It is widely acknowledged among environmental molecular microbiologists that genetic biosignatures identified from an environment only represent the most dominant populations. The technological bottleneck overlooks the presence of the less abundant minority population and may underestimate their role in the ecosystem maintenance.
DNA intercalating agents such as propidium monoazide (PMA) covalently bind with DNA molecules upon photolysis using visible light, and make it unavailable for DNA polymerase enzyme during polymerase chain reaction (PCR). Environmental DNA samples will be treated with suboptimum PMA concentration, enough to intercalate with 90–99% of the total DNA. The probability of PMA binding with DNA from abundant bacterial species will be much higher than binding with DNA from less abundant species. This will increase the relative DNA concentration of previously “shadowed” less abundant species available for PCR amplification. These PCR products obtained with and without PMA treatment will then be subjected to downstream diversity analyses such as sequencing and DNA microarray. It is expected that PMA-coupled PCR will amplify the “minority population” and help in understanding microbial diversity spectrum of an environmental sample at a much deeper level.
This new protocol aims to overcome the major potential biases faced when analyzing microbial 16S rRNA gene diversity. This study will lead to a technological advancement and a commercial product that will aid microbial ecologists in understanding microbial diversity from various environmental niches. Implementation of this technique may lead to discoveries of novel microbes and their functions in sustenance of the ecosystem.
This work was done by Parag A. Vaishampayan and Kasthuri J. Venkateswaran of Caltech for NASA’s Jet Propulsion Laboratory. NPO-48200
This Brief includes a Technical Support Package (TSP).
Molecular Technique to Reduce PCR Bias for Deeper Understanding of Microbial Diversity
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Overview
The document presents a study conducted by researchers at NASA's Jet Propulsion Laboratory (JPL) focused on a novel molecular technique designed to reduce PCR bias, thereby enabling a deeper understanding of microbial diversity in soil samples. The research highlights the complexity and significance of microbial communities, which consist of approximately 10^8 to 10^9 microorganisms from thousands of different species that interact closely within soil environments.
The study employs a technique involving Propidium Monoazide (PMA) treatment to selectively inactivate non-viable DNA, allowing for a more accurate assessment of viable microbial populations. The results indicate that the control soil sample, which was not treated with PMA, revealed a total of 5011 distinct operational taxonomic units (OTUs). In contrast, applying PMA-mediated inactivation showed varying results: 50% inactivation yielded 4665 OTUs, while 90% inactivation resulted in 5150 OTUs, marking a 3% increase compared to the control. Notably, a 99% inactivation led to the detection of 5989 OTUs, representing a 19% increase and revealing 2174 new OTUs not present in the control sample. This suggests that the novel technique is effective in uncovering the 'minority population' of microbes, which are often overlooked in traditional assessments.
The implications of this research extend beyond academic interest; the findings are poised to advance technological applications in microbial ecology. The technique is particularly relevant for NASA's planetary protection efforts, as it can help assess microbial diversity in spacecraft assembly clean rooms, thereby mitigating the risk of forward contamination during space missions. The study emphasizes the potential for discovering novel microbes and understanding their ecological functions, which could have significant implications for ecosystem sustainability.
The document also mentions plans for high-throughput sequencing (454 sequencing) to validate the results obtained through the PhyloChip analysis, indicating a commitment to rigorous scientific methodology. Overall, this research represents a significant step forward in microbial ecology, offering new tools and insights that can enhance our understanding of microbial diversity across various environmental niches.
