Microorganisms are inevitable companions in human space exploration with the consequence risk of human disease. This risk is enhanced by the observation that astronauts in space may have suppressed immune systems making them more susceptible to bacterial infection (Nefedov et al., 1978, Nickerson & Curtis, 1997). Bacterial populations can impact manned missions in other ways too. For example, buildups of biofilms may damage or interfere with the performance of hardware and changes in bacterial populations in advanced life support systems may interfere with biodegradation of waste or food production. The background radiation levels encountered on the International Space Station are approximately 70-100 times those seen on the Earth and the gravity vector approaches zero. A key risk is that such a doubly stressed environment may select for significant changes in the microorganisms themselves over the lifetime of an extended mission.

In order to begin to define the microbial risk associated with human space missions, efforts have been underway for some time to directly characterize the populations encountered in space. This was first attempted on the US Space Shuttle and the Russian Mir Space Station using culture based techniques (LaRocco and Pierson, 1999). More recently, detailed identification of culturable bacteria isolated from air, water and surfaces on the International Space Station was undertaken using 16S rRNA sequencing (Castro et al., 2004). The general observation is that Gram-positive bacteria such as Staphylococcus, Micrococcus, and Bacillus are most common in air and surface samples whereas Gram negative bacteria are dominant in water samples. The surveys illustrate that the organisms most likely to be present are those that are routinely associated with humans and those which are found in spacecraft assembly environments. This is perhaps expected, but not comforting; as these are the organisms most likely to be able infect a human host if they develop novel virulent properties.

Preliminary Results

We have successfully employed a high aspect rotating vessel (HARV) bioreactor in the Pierson lab to study the response of Escherichia coli to modeled microgravity. The device minimizes fluid motion while maintaining culture aeration through a gas permeable membrane. The rotation also has the effect of randomizing the gravity vector, by rotating in the plane of gravity, producing a low shear modeled microgravity (LSMMG) environment. To obtain this environment, the HARV device is rotated at a speed sufficient to maintain cell suspension in the media and must be completely filled so that gas bubbles cannot cause solution turbulence (i.e. shear).

Our expression studies to date have revealed a substantial number of genes that are either up-regulated or down-regulated relative to controls in replicate experiments. While many of these genes are currently of unknown function, some of the genes with increased transcription in response to LSMMG are involved in the E. coli acid tolerance response system (transcriptional gene regulators [yhiE, yhiF] and the chaperones [hdeA, hdeB and hdeD), or are involved in cell motility (many flg and fli genes) or are chemotaxis regulating genes (cheZ and tar). The induction of acid tolerance response genes could indicate their involvement in a general E. coli stress response pathway. Increased transcription of the flagellar and chemotaxis genes in LSMMG, increasing cell mobility, may indicate that zones of low nutrients and high waste are occurring in the LSMMG HARV similar to those theorized to occur in space. These identified changes in bacterial LSMMG gene expression could lead to increased cell survival, virulence, and antibiotic resistances, indicating serious potential problems during long-term space flight.

Specific Aim

It is hypothesized that long term simultaneous exposure to microgravity and elevated background radiation will lead to changes in gene and protein expression patterns in common bacteria. We will seek to determine in ground-based studies the extent to which this occurs and what genes are affected in a representative Gram-negative and Gram-positive bacteria and whether or not the changes are likely to be consequential with regard to other cellular activities.

Proposed Studies

We will monitor the immediate gene expression response to LSMMG in two additional cases. The first will be E. coli cells grown under anaerobic conditions using nitrate as the terminal electron acceptor. An initial study has actually been run already but further replicates are needed to allow statistical verification of the results. The second will focus on the Gram positive model organism, Bacillus subtilis. This spore forming organism is the best characterized Gram positive bacterium. Its complete genome sequence is available as are genomic expression arrays. These studies will allow us to compare the genes affected in three organisms (e.g. also Salmonella (Wilson et al., 2002)) in four distinct environments. (2) The gene expression response of LSMMG grown E. coli populations (aerobic and anaerobic) to a variety of stresses (acid, base, heat, antibiotic, H₂O₂, NaCl, and Sucrose) will be examined and compared to the 1 x g HARV controls to gain insight as to whether the types of changes induced by LSMMG are likely to effect other cellular responses, e.g. pathogenesis. (3) We will conduct long-term (3-6 months) studies to determine if E. coli cells continuously exposed to LSMMG (but not continuously growing-i.e. regular periods of nutrient depletion will be included to allow a range of selection conditions) evolve a different gene expression pattern. At the end of 3 months and 6 months we will reexamine the response to acid and temperature shock. (4) A parallel set of long term (3-6 months) experiments will be conducted using flask cultures continuously exposed to low levels of ionizing radiation. Bacteria will again be grown intermittently, e.g. with periods of nutrient depletion. At the 3 month and 6 month time points, the global patterns of gene expression will be determined and the responses to acid and temperature shock monitored. (5) Finally the experiments in (4) will be conducted with the simultaneous presence of LSMMG and radiation. The experiments in (4) and (5) will likely be run simultaneously.

Timeline

During the first six months anaerobic studies on E. coli will be completed. The second six months will focus on B. subtilis and pilot studies of the procedures to be used in the radiation studies. Year two will focus on simultaneous exposure to the stress of LSMMG and radiation and the effect of adaptation to such a double stressed environment on cellular response to common stimuli such as heat shock, acid tolerance etc.

JSC Facilities

The fellow will work in the laboratory of Dr. Duane Pierson at JSC and will utilize HARV bioreactors which are routinely utilized in that laboratory. A JSC Cesium-137 source will be utilized in the initial radiation studies.

References

Castro, V. A., A. N. Thrasher, M. Healy, C. M. Ott, and D. L. Pierson. "Microbial diversity aboard space craft: Evaluation of the Intn. Space Station," Micro Ecol (2004). (In press.)
LaRocco, M. T. and D. L. Pierson. “Deep space exploration: will we be ready?” ASM News. 65 (1999): 817-82.
Nefedov, Y. U. G., A. V. Yeremin, V. I. Drozdova, A. S. Skryabin, O. A. Gueseva, and N. N. Mukhina. "Immunological reactivity and prediction of allergic complications in the crew of the second expedition of Salyut 4," Kosm Biol I Avikosm Med 12 (1978): 15-29.
Nickerson, C. A. and R. Curtis, III. "Role of sigma factor RpoS in initial stages of Salmonella typhimurium infection," Infect Immun 65 (1997): 1814-23.
Nickerson, C. A., C. M. Ott, S. J. Mister, B. J. Morrow, L. Burns-Keliher, and D. L. Pierson. "Microgravity as a novel environmental signal affecting Salmonella enterica serovar typhimurium virulence," Infect Immun 68 (2000): 3147-52.
Wilson, J. W., R. Ramamurthy, S. Porwollik, M. McClelland, T. Hammond, P. Allen, C. M. Ott, D. L. Pierson, and C. A. Nickerson. "Microarray analysis identifies Salmonella genes belonging to the low-shear modeled microgravity regulon," Proc. Natl. Acad. Sci. U. S. A. 99 (2002): 13807-12.

Desired Academic Background

The ideal candidate will have a strong interest in the Space Sciences with research experience in molecular biology or microbiology and familiarity with the bioinformatics tools used in bacterial genomics. Prior experience with expression arrays or knockout mutagenesis would be significant additions to the applicant's qualifications.

Professional Biographies of Investigators

Dr. George E. Fox, Professor, Dept. Biology & Biochemistry
University of Houston, Houston, TX 77204-5001

Dr. Fox received his Ph.D. in chemical engineering in 1974 from Syracuse University. He subsequently was a postdoctoral research associate in microbiology at the University of Illinois with Dr. Carl Woese at the University of Illinois from 1974-1977 during which time he was a co-discover of the archaebacteria. He subsequently joined the University of Houston in 1977 and following several promotions is now a Full Professor. Dr. Fox's research interests include the effect of space environments on bacteria, molecular phylogeny, RNA structure, function and evolution and the origin of life. He has over 100 peer review publications, many of which relate to NASA interests and programs. His current research funding includes grants from NASA's Exobiology Program and NASA's Office of Biological and Physical Research. He is an elected fellow of the American Academy of Microbiology, the American Association for the Advancement of Science and the American Institute of Medical and Biological Engineering. He is a past member of the National Research Council - Space Science Board Committee on Planetary Biology and Chemical Evolution and the Exobiology peer review panel. Dr. Fox is currently a member of the University Space Research Association (USRA) Division of Space Life Sciences Advisory Council and an active participant in workshops relating to Planetary Protection.

Dr. Richard C. Willson, Dept. Chemical Engineering
University of Houston, Houston, TX 77204-4004

Dr. Willson received his Ph.D. in biochemical engineering at the Massachusetts Institute of Technology under the direction of Dr. Charles Cooney in 1988. Subsequently, he was a Research Associate in the laboratory of Dr. Jonathan King in the Biology Department at the Massachusetts Institute of Technology. He joined the University of Houston in 1989 and will be a Full Professor beginning in the Fall of 2004. Dr. Willson’s research interests focus on bioseparations and biosensors and his numerous peer review publications and patents include many related to microbial detection in the space environment. His current research funding includes a grant from NASA’s Office of Biological and Physical Research. He is an elected fellow of the American Institute of Medical and Biological Engineering and a past recipient of the 3M Young Faculty Award and the NSF Presidential Young Investigator Award. He has twice served on the peer review panel for the joint NIH/NASA molecular diagnostics program. He is currently President of the International Society for Molecular Recognition and an Editorial Board member of multiple journals including; International Journal of Biochromatography, International Journal of Biological Physics and Chemistry, Biotechnology Progress (a joint publication of AIChE and ACS), Journal of Molecular Recognition Applied Biochemistry and Biotechnology.

Dr. Duane L. Pierson, Chief Microbiologist
NASA Johnson Space Center 

Dr. Pierson received his Ph.D. in biochemistry at Oklahoma State University in 1971. Following several years as an Assistant Professor at the Baylor College of Medicine he the Biomedical Operations Research Branch at the Johnson Space Center as the Toxicology Lab technical monitor in 1984. He was subsequently Deputy Chief, of the Biomedical Operations and Research Branch before obtaining his present position as Chief Microbiologist. Over the years he has maintained his connections with academia as well as is reflected in the fact he is an Adjunct Professor of Pathology at University of Texas Medical Branch, Galveston and Adjunct Assistant Professor in the Department of Microbiology and Immunology, Baylor College of Medicine. In addition to managing aspects of NASA operations relating to astronaut health, Dr. Pierson maintains an independent research program which regularly competes for and receives external funding from various NASA programs with results published in key journals. His research interests include all aspects of microbiology as they relate to space flight and he is an internationally recognized authority as is reflected in numerous invited reviews and chapters on various aspects of the subject. Recent efforts have included pioneering studies on the effect of low shear modeled microgravity on gene expression in Salmonella typhimurium. Dr. Pierson's research has been recognized by his election to membership in the American Academy of Microbiology. His contributions to the Space Program have been repeatedly recognized and the awards received include the NASA Medal for Exceptional Scientific Achievement and the NASA Medal for Exceptional Service

Contact Information

Dr. George E. Fox
Dept. Biology and Biochemistry
University of Houston
Houston, TX 77204-5001
713-743-8363
713-743-8351 (FAX)
fox@uh.edu
http://prion.bchs.uh.edu/1/