Browsing by Subject "16S rRNA Gene Sequencing"

The Genome Encyclopedia of Spacecraft Associated Microbes (GESAM) project seeks to increase our knowledge of spacecraft associated microbes surviving in the extreme environments of spacecraft associated clean rooms. Planetary Protection researchers at NASA’s Jet Propulsion Laboratory have been collecting and archiving bacterial isolates from NASA Mars missions since the 1970s, however, only a small fraction of the entire bacterial collection has been taxonomically identified and even fewer have been functionally characterized. Previous efforts to taxonomically identify microbes employed 16S rRNA gene sequencing techniques, however, this method alone failed to provide the resolution required to consistently identify these isolates at a species and strain level, many of which are members of taxonomic groups further challenging identification. Samples collected from spacecraft surfaces and within cleanrooms throughout assembly testing and launch operations were subjected to NASA’s heat shock assay and stored for future analysis. In this thesis, for the first time, Matrix Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS) has been used to identify bacterial strains from the Mars Odyssey and Mars Pathfinder missions. Due to its clinically biased design framework, the development of an in-house database of mass spectrum profiles complemented with 16S rRNA gene sequencing-based taxonomic IDs was necessary to identify this project’s isolates which reside in extreme environments of spacecraft assembly cleanrooms and are not well represented in existing shared spectral databases. MALDI-TOF MS was found to be a fast, reliable, and powerful method of identifying bacterial isolates at species and strain level. Despite MALDI-TOF’s high resolving power, its ability to identify space associated microbes residing in spacecraft assembly cleanrooms is hindered by the reliance of mass spectrum profile creation on 16S rRNA gene taxonomic identification, and the lack of shared spectrum data and publicly available databases. The resolution of taxonomic groups via composite correlation index analysis is paramount to the development of spectra libraries and the utilization of MALDI to its full potential. While MALDI is well suited to identifying the proxy representation of cultivatable spore-forming bacteria produced by NASA’s Standard Assay, a surprising number of non-spore-forming have been identified and many more are expected to be uncultivatable suggesting the need for less selective omics-based approaches in order to establish a more comprehensive idea of the communities present in spacecraft assembly cleanroom.