Buildups of significant numbers of microorganisms during space travel can cause disease and other problems such as corrosion. Moreover, the effect of long-term exposure of microorganisms to high radiation levels and weightlessness is unknown, but there is evidence that organisms under these conditions may exhibit altered properties that could affect their pathogenicity and/or sensitivity to antibiotics. A team headed by Dr. George E. Fox is developing in-flight systems to monitor microbial populations in space to ensure the quality of spacecraft air and water. Fox is also studying the effects of weightlessness on microorganisms. This research will lead to an alternate identification system that will not only test for organisms that are known to be problematic but would also identify the genetic affinity of an unknown microorganism that proves to be problematic. This research will provide the means to understand microbial ecosystems in the space environment and lead to a detection system for harmful microorganisms that can be used in space and on Earth.
George E. Fox, Ph.D.
University of Houston
Our work in the past year was focused on four major project areas. We have completed development of a set of 16S rRNA targeted probes that will indicate the presence of major problem and indicator bacteria in flight samples. These probes can be used with similar efficiency under a standard set of operating conditions. The utility of these probes was demonstrated on several samples isolated from various systems including Mars and Moon soil simulants. We also completed the development of a computational algorithm that allows identification of short 16S rRNA subsequences that are highly characteristic of various phylogenetic groupings. In principle, an array of appropriately designed probes based on these signature sequences could be used to determine the genetic affinity (nearest known relatives) in the absence of any prior knowledge of what the problematic organism might be. In practice, the signature sequences themselves tend to be short (15 nucleotides or less) and hence not ideal for use in arrays. We are currently developing an extension to the original algorithm that will allow us to identify very long (30-60 nucleotides) signature fragments whose sequences, despite occasional mismatches, are highly characteristic of various phylogenetic groupings.
An effective set of probes may have utility in a large variety of formats. It is likely that actual implementation in space flight will be driven by mission instrumentation capabilities. Although there is considerable interested in development of array-based instruments other approaches may be preferable, especially for International Space Station applications. We therefore have focused attention on several alternative formats as well. In this regard, several probes for organisms of primary interest have been successfully implemented in a molecular beacon format. Homogenous solution assays of this type would require minimal sample processing and could be readily conducted by astronauts in flight with results signaled by the presence or absence of color changes. It was found that "red-shifted" beacons have minimal contributions from sample autofluorescence. In addition, we demonstrated the potential utility of fluorescent nucleotides such as 2-aminopurine in molecular beacon applications. Efforts were also initiated late in Year 2 to assess the possibilityof identifying signature oligonucleotides with mass spectrometry.
Regardless of the assay system ultimately chosen, rapid and simplified systems for sample processing in space will be required. During the past year, we provided further evidence that RNA/DNA purification using compaction agents eliminates the need for preprocessing steps and that the same agents can be used to enhance the adsorption capacity of anion exchangers. Also in the past year we developed further evidence that immobilized metal affinity chromatography (IMAC) which is widely used with proteins is also effective with nucleic acids. Most recently, we have begun to look at novel ways of obtaining rapid final purification of specific RNAs such as 16S rRNA.
There is preliminary evidence that the microgravity environment seen in space effects bacteria in non-obvious ways with such possible outcomes as altered drug resistance or pathogenicity. In order to explore this possibility further, we are examining the response of E. coli cells grown in simulated microgravity. In order to do this, we are using modern proteomics technology to examine the expression levels of each and every gene in E. coli when cells are grown in a low shear modeled microgravity (LSMGG) environment. Various kinetic controls have been completed and initial hybridizations with organisms grown under simulated microgravity have been made. These initial studies point to several interesting clusters of genes that appear to be part of a specific response to the LSMMG. In the coming year, we will complete replicates of these initial experiments and examine the results in a multiorganism context.
Overall, Year 2 of the project was very productive. During the past year, four-peer review papers were published, three more are in press, and five additional papers have been submitted. In addition, a book chapter is in press.