University of Houston • University of Houston-Clear Lake • ISSO Annual Report Y2002—pp. 21-25

George E. Fox (UH), Richard Willson (UH), Duane Pierson (NASA-JSC), and Don L. Tucker (UH)

Abstract
The effect of low-shear modeled microgravity on microbial gene expression and physiology is under study using functional genomics and molecular techniques in Escherichia coli.

Contrary to popular belief, bacterial virulence does not decrease in response to microgravity. In fact, recent evidence indicates increased Salmonella virulence in response to modeled microgravity. It is possible that bacterial virulence, resistance to antibiotics, and cell survival may increase in response to the microgravity of space. Employment of a high aspect rotating vessel (HARV) bioreactor has allowed investigators to study bacterial physiology in modeled microgravity. Using culture physiology and DNA macroarray technology, researchers are attempting to identify changes in bacterial (E. coli) gene transcription and regulation in response to the low-shear modeled microgravity (LSMMG) environment experienced in HARV bioreactors.

A number of LSMMG-regulated genes have been identified that respond in a significant way. These include genes that are substantially up-regulated as well as those that are down-regulated. Although many are genes of unknown function, a number have known or putative functions. Among the up-regulated genes are those associated with cell motility, acid tolerance response, and the chaperone hdeA. Research with HARV grown cultures is continuing to determine whether these and other genes are responding to the low-shear environment or the simulated microgravity in the HARV bioreactors.

THE ABILITY TO ADAPT TO ADVERSE ENVIRONMENTS IS extremely important to pathogenic bacteria such as E. coli. One entirely unique environment is the microgravity experienced by bacteria during space flight. A large body of whole organism-based research has demonstrated that prolonged exposure to microgravity has significant effects at a basic, cellular level.¹ The analysis of bacteria under microgravity has received considerably less attention because of the expense and difficulty of performing in-flight experiments onboard the shuttle or space station. In order to overcome this limitation, investigators have taken advantage of the partial simulation of microgravity obtained by growing bacterial cultures in High Aspect Rotating Vessels (HARV) developed by NASA.² For example, a recent study³ showed that, Salmonella enterica serovar typhimurium grown under low-shear modeled microgravity (LSMMG) appeared to have increased virulence potential in a murine model system. A follow-up study⁴ revealed that a significant number of the genes are transcriptionally regulated in response to LSMMG. In addition to immediate changes in behavior, it is also possible that bacterial strains will evolve during long-term space flight, potentially modifying virulence, resistances, and rate of culture survival.Our goal is to develop a more general and deeper understanding of LSMMG on bacterial gene expression.

Technical Plan and Equipment
The HARV bioreactor was originally developed to minimize fluid motion for tissue culture differentiation, while maintaining culture aeration through a gas permeable membrane at the base of the HARV. The device’s rotation also has the effect of randomizing the gravity vector, by rotating in the plane of gravity, producing the LSMMG environment. To obtain this environment, the HARV device must be rotated at a speed sufficient to maintain cell suspension in the media and be completely filled so that gas bubbles cannot cause solution turbulence (i.e., shear). HARV apparatus approximates the physiological and transcriptional changes occurring in space flight attributed to microgravity, while allowing Earth-based culturing. Used in conjunction with commercially available functional genomics technology (Panorama Gene Arrays, Sigma-Genosys), the HARV makes it possible to study microbial gene expression on a genome-wide basis under LSMMG.

Experimental Activity
The availability of the complete genomic sequence, commercially produced genomic arrays and the well-characterized knowledge of its metabolism and gene regulation, led to the choice of E. coli as our first model system for the initial bacterial functional genomic (gene expression) analysis in the HARV’s LSMMG environment. We have compared LSMMG gene expression in minimal glucose media with control cultures (1 x g HARV, static flasks, and 250 rpm shaken cultures) under aerobic conditions. The 1 x g control HARV is treated identically to the LSMMG HARV (no bubbles, same rotation speed), but the angle of rotation is perpendicular to gravity, allowing the effects of gravity to act on the culture. More recently, the facultative anaerobic characteristics of E. coli have been employed to compare culture physiology and functional genomics in the absence of oxygen as the terminal electron acceptor. In addition, proteomics, RT-PCR, and molecular biology techniques are being employed for comparison to the functional genomic results for further identification and elucidation of the genes and operons regulated by LSMMG. These methods of analysis will be repeated on a number of overexpressed LSMMG genes and gene knockouts in E. coli to further elucidate their roles in modeled microgravity transcription and growth.

Results
Our analysis of the E. coli LSMMG culture physiology has encompassed growth rate, final culture yield, cell morphology, and the length of lag and exponential phases. Interestingly, little change in culture physiology has been observed between the LSMMG, 1 x g, and static cultures. Only when comparisons are made to 250 rpm shaken cultures are there significant differences. The shake flask cultures exhibit an increased growth rate and greater final culture density compared to the other cultures. To an extent, this was expected due to the increased aeration, availability of nutrients, and removal of wastes present in the shaken cultures.

The expression studies conducted on E. coli grown under LSMMG revealed a substantial number of genes that were either up-regulated or down-regulated relative to controls in replicate experiments. While the majority 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 [ykiE, yhiF] and the chaperone [hdeA]), or are involved in cell motility (many flg and fli genes). The yhi and hde genes identified are possibly involved in a general E. coli stress response system which is also activated by LSMMG. 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.

Although growth in the HARV bioreactor has a significant effect on bacterial gene expression in multiple strains, it remains to be determined to what extent the effects seen correlate with the different properties of the HARV environment. The transcriptional regulation that is seen may be induced by the modeled microgravity (i.e. randomized gravity vector) of the HARV or the low shear fluid effects present in the rotating bioreactor. This question will be addressed in the future by growing E. coli cultures in HARVs with perturbed solution flows in the vessels. This perturbation of the low-shear phenomenon will be achieved through the addition of a few small beads to the HARV culture media or introduction of small air bubbles. Functional genomic analysis of these samples will hopefully help in separating the low shear effects from the randomized gravity vector (modeled microgravity) present in the HARV.

It is interesting to note that while Salmonella and E. coli are closely related, there is limited comparability between the LSMMG expressed genes reported in Salmonella and those we have identified in E. coli. The reasons for these differences in gene expression are currently unknown and will require additional research. In this regard, it will be of special interest to replicate the media and other environmental conditions used in the earlier Salmonella work as exactingly as possible in order to increase the comparability of the results. In addition, the LSMMG environment will be extended to the Gram-positive, spore forming bacterium Bacillus subtilus.

Acknowledgments
We would like to thank Dr. Yuriy Fofanov in the Department of Computer Science at the University of Houston and the various members of his group (Tong-Bin Li, Chetan Belapurkar, Lulu Shi, R. Luo, and J. Wang) for advice and assistance in performing statistical analyses on the microarray data.

References
¹J. M. Jessup and N. R. Pellis. "NASA Biotechnology: Cell Science in Microgravity," In Vitro Cell Dev. Biol. Anim. 37 (2001): 61-63.
²T. L. Prewett, T. J. Goodwin, and G. F. Spaulding. "Three-Dimensional Modeling of T24 Human Bladder Carcinoma Cell Line: A New Simulated Microgravity Culture Vessel," J Tissue Cult Methods 15 (1993): 29-36.
³C. A. Nickerson, C. M. Ott, S. J. Mister, B. J. Morrow, L. Burns-Kelihe r, and D. L. Pierson. "Microgravity as a Novel Environmental Signal A ffecting Salmonella enterica serovar typhimurium Virulence," Infect. Immun. 68 (2000): 3147-52.
⁴J. W.Wilson, 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., 99 (2002): 13807-12.

Publications
Balan, S., J. C. Murphy, I. Galaev, A. Kumar, G. E. Fox, B. Mattiasson, and R. C. Willson. "Metal Chelate Affinity Precipitation of Nucleic Acids," Biotechnology Letters. (Accepted.)
Kourentzi, K. D., G. E. Fox, and R. C. Willson. "Hybridization-Sensitive Fluorescent DNA Probes Containing the Adenine Analog 2-AminoPurine," Analytical Biochemistry. (Accepted.)
Kourentzi, K. D., G. E. Fox, and R. C. Willson. "Microbial Identification by Immunohybridization Assay of Artificial RNALabels," J. Microbiol. Methods 49 (2002): 301-06.
Larios-Sanz, M., K. Kourentzi, D. L. Pierson, R. C. Willson, and G. E. Fox. "Monitoring Bacterial Contaminants in Space Environments using 16S Ribosomal RNA," J. Microbiological Methods. (Submitted.)
Nagaswamy, U. and G. E. Fox. "RNA Ligation and the Origin of tRNA," Origins Life & Evol. Biosphere 33 (2003): 199-209.
Starikov, D., C. Boney, N. Medelci, J-W. Um, M. Larios-Sanzs, G. E. Fox, and A. Bensaoula. "Experimental Simulation of Integrated Optoelectronic Sensors Based on III Nitrides, J. Vacuum Science & Technology B 20 (2002): 1815-20.
Zhang, Z., R. C. Willson, and G. E. Fox. "Identification of Characteristic Oligonucleotides in the 16S Ribosomal RNA Sequence Dataset," Bioinformatics 18 (2002): 244-50.

Presentations
Fox, G. E. and R. C. Willson. "Designing Signature Probes for Hybridization Arrays," Environmental Sentinels 2002, Houston, TX, Sept. 17-18, 2002.
Kourentzi, K., M. Larios-Sanz, Z. Zhang, G. E. Fox, and R. C. Willson. "Microbial Identification Targeting Ribosomal RNA and Artificial RNA Labels," 224th American Chemical Society National Meeting, Boston, MA, Aug. 18-22, 2002.
Larios-Sanz, M., K. D. Kourentzi, G. E. Fox, and R. C. Willson. "Microbial Identification Using Signature Probes," Annual Meeting American Chemical Society, New Orleans, LA, March 23-27, 2003.
Larios-Sanz, M., K. Kourentzi, D. Warmflash, R. C. Willson, D. L. Pierson, and G. E. Fox. "Microbial Contaminants in Crew Habitat Modules During Exploration Class Human Space Missions: Monitoring Potential Pathogens," 73rd Annual Meeting, Aerospace Medical Association, Montreal, Canada, May 5-10, 2002.
Larios-Sanz, M., K. Kourentzi,, Z. Zhang, R. C. Willson , D. L. Pierson, and G. E. Fox. "Molecular Tools To Monitor Microbial Contaminants During Long-Term Exploration Class Missions," World Space Congress, 34th Scientific Assembly of the Committee on Space Research (COSPAR) and the 53rd International Astronautical Congress (IAC) of the International Astronautical Federation (IAF), International Academy of Astronautics (IAA), and International Institute of Space Law (IISL), Houston, TX, Oct. 10-19, 2002.
Larios-Sanz, M., K. Kourentzi, Z. Zhang, R. C. Willson, D. L. Pierson, and G. E. Fox. "A Ribosomal RNA-Based System To Monitor Microbial Contaminants in Space Environments," ASM Texas Branch Meeting, Austin, TX, Nov. 7-9, 2002.
Larios-Sanz, M., K. Kourentzi, Z. Zhang, R. C. Willson, D. L. Pierson, D. L. Tucker, and G. E. Fox. "Molecular Tools To Monitor Microbial Ecosystems During Long-Term Exploration Class Missions," 103rd General Meeting of the American Society for Microbiology,Washington, D.C., May 18-22, 2003.
Nagaswamy, U. and G. E. Fox. "Transfer RNA May Have Arisen by RNA Ligation," NASA Astrobiology General Meeting 2003, Phoenix, AZ, Feb. 10-12, 2003.
Shi, L., T.-B. Li, D. L. Tucker, F. Karouia, R. C. Willson, G. E. Fox, and Y. Fofanov. "A Pair-Wise Correlation Analysis Applied to Gene Expression Data from Two Escherichia coli Strains," 20th Annual Meeting, Houston Society for Engineering in Medicine and Biology, Houston, TX, April 3-4, 2003.
Tucker, D. L. "Functional Genomic Identification of a Novel E. coli Stress Response Regulon," Department of Molecular Biology and Biochemistry, Rutgers University/Cook College, New Brunswick, NJ, April 4, 2003.
Tucker, D. L., M. C. Ott, D. L. Pierson, R. C. Willson, and G. E. Fox. "Functional Genomic Analysis of E. coli in a Low-Shear Modeled Microgravity Environment," 103rd General Meeting, American Society Microbiology, Washington, D.C., May 18-22, 2003.
Wang J., Y. Luo, T.-B. Li, D. L. Tucker, F. Karouia, G. E. Fox, R. C. Willson, and Y. Fofanov. "Image Analyzer: A Flexible Application for Microarray Image Analysis," 8th Annual Structural Biology Symposium, Sealy Center for Structural Biology, Galveston, TX, May 2-4, 2003.
Warmflash, D., M. Larios-Sanz, G. E. Fox, and D. S. McKay. "Progress in the Use of Rapid Molecular Techniques To Detect Life Forms in Soil: Implications for Interplanetary Astrobiology Missions," 33rd Lunar & Planetary Science Conference, Houston, TX , March 11-15, 2002.
Warmflash, D., M. Larios-Sanz, G. E. Fox, and D. S. McKay. "Technologies for Microbial Detection To Support Both Vehicle/Habitat Medical Monitoring and Planetary Astrobiology During Human Surface Exploration of Mars," 73rd Annual Meeting, Aerospace Medical Association, Montreal, Canada, May 5-10, 2002.
Warmflash, D. M., D. S. McKay, C. C. Allen, G. E. Fox, A. Steele, M. Schweitzer, S. Pincus, J. Perez-Mercader, V. P. Garcia, L. C. Briones, and K. L. Thomas-Keprta. "Detection of Biomarkers of Viable Life-Using Immunoassay," Proc., SPIE Conference, Instruments, Methods and Missions for Astrobiology V, Waikoloa, HI, Aug. 22-23, 2002.

Funding and proposals
Fox, G. E. "Evolution of Genomes and Cellular Processes in Astronomically Reasonable Environments," NASA Astrobiology Institute, June 1, 2003-May 31, 2008, $7,188,349 (submitted). (The project involves 15 laboratories at five institutions.)
Fox, G. E. and R. C. Willson. "Microorganisms in the Spacecraft Environment," National Space Biomedical Research Institute, Oct. 1, 2000-Dec. 31, 2003, $926,040.00.

UH PDAF: Gary E. Schultze, Jr., Ph.D.
Ph.D.: College of William and Mary,
Gloucester Point, VA

ISSO-Title: Post Doctoral Aerospace Fellow
Dept. Marine Science, Texas A&M University at Galveston
5007 Avenue U, Galveston, TX 77551
Phone: (713) 790-9264
E-mail: schultzg@tamug.tamu.edu

UH PDAF: Karine Maillard, Ph.D.
Section of Molecular Carcinogenesis
Institute of Cancer Research, Haddow Laboratories
15 Cotswold Rd., Sutton, Surrey, SM2 5NG Great Britain
Phone: (0181) 643-8901 x4671; Fax: (0181) 643-0257
E-mail: kim@icr.ac.uk

Publications
Clark J, S. Edwards, M. John, P. Flohr, T. Gordon, K. Maillard, I. Giddings, C. Brown, A. Bagherzadeh, C. Campbell, J. Shipley, R. Wooster, and C. S. Cooper. "Identification of Amplified and Expressed Genes in Breast Cancer by Comparative Hybridization onto Microarrays of Randomly Selected cDNA Clones," Genes Chromosomes Cancer 34 (2002): 104-14.
Hung, C. C., L. Guo, G. E. Schultz Jr., J. L. Pinckney, and P. H. Santschi. "Production and Flux of Carbohydrate Species in the Gulf of Mexico," Global Biogeochemical Cycles. (In press.)
Lee Y. F., M. John, S. Edwards, J. Clark, P. Flohr, K. Maillard, M. Edema, L. Baker, D. C. Mangham, R. Grimer, R. Wooster. J. M. Thomas, C. Fisher, I. Judson, and C. S. Cooper. "Classification of Synovial Sarcomas, Leiomyosarcomas and Malignant Fibrous Histiocytomas by Gene Expression Profiling," Br. J. Cancer 88 (2003): 510-15.
Santschi, P .H., C. C. Hung, G. E. Schultz, Jr., N. L. Alvarado-Quiroz, J. Guo, J. Pinckney, and I. Walsh. "Control of Acid Polysaccharide Production and 234Th and POC Export Fluxes by Marine Organisms," Geophysical Research Lett. (In press.)
Schultz, Jr., G. E., E. D. White III, and H. W. Ducklow. "Bacterioplankton Dynamics in the York River Estuary: Primary Influence of Temperature and Freshwater Inputs," Aquatic Microbial Ecology 30 (2003): 135-48.

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Institute for Space Systems Operations - Y2002 Annual Report
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