The Effect of Modeled Microgravity on Microbial Gene Expression

University of Houston • University of Houston-Clear Lake • ISSO Annual Report Y2005 • 23-26

George E. Fox, Richard C. Willson, Duane L. Pierson, Viktor Stepanov

Abstract--The effect of low-shear modeled microgravity (LSMMG) on microbial gene expression and physiology is being examined using functional genomics, survival assays, and molecular techniques in Escherichia coli. Reproducible changes in transcription were seen but no direct response to changes in the gravity vector was identified. Instead, absence of shear and a randomized gravity vector appeared to cause local extra-cellular environmental changes, which elicited reproducible cellular responses. In minimal media, the majority of the significantly up- or down-regulated genes of known function were associated with the cell envelope. Comparison with earlier studies of Salmonella enterica serovar Typhimurium conducted under similar growth conditions revealed essentially no similarity in genes, which were significantly up- or down-regulated. Given the substantial overlap in gene content between these closely related organisms, this result clearly demonstrates that different organisms may dramatically differ in their responses to medically significant low-shear and space environments. The project is part of a larger effort to characterize the types of organisms that may inhabit space craft environments and to understand how they might be affected by the space environment.

Bacteria are capable of living in and adapting to a far larger range of environmental conditions than are normally encountered in usual laboratory environments. Even with full knowledge of an organism's gene content, it is currently impossible to predict how expression patterns will change in different situations. Thus, usual laboratory growth conditions may not invoke key aspects of an organism's potential response. Consequently, such studies may conceal behaviors that in a different environment may contribute to undesirable phenomena such as pathogenesis. One such case is the low-shear, low-turbulence environments present in utero, at the brush border microvilli of epithelial cells, and in other medically important host environments.¹ Another example is the space environment characterized by microgravity and high background radiation. In this case, the absence of gravity also produces a low-shear environment, which likely results in microorganisms having difficulty removing themselves from immediate surroundings that have been nutrient depleted and that have received waste products.²

In order to adapt to life in a low-shear world, bacteria likely express different combinations of genes than they do in more usual laboratory environments and may ultimately make evolutionary adaptations as well. Thus, a particular bacterium may exhibit properties such as antibiotic resistance, biofilm formation, or virulence that are not generally associated with it. It is therefore not certain which organisms may be problematic. 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. Thus, there are two related objectives needing detailed study before embarking on detailed missions in space. The first is to better understand the effect of the microgravity environment on bacterial behavior. Second, given that organisms may behave in unanticipated ways, it will be important to develop technology to determine which organisms are causing an unanticipated problem without prior knowledge of what the causal organisms might be. The former is being directly addressed in the work supported here by ISSO and the latter in parallel efforts being conducted primarily with other funding sources.

Methodology
Wild type E. coli MG1655 (CGSC7740) was grown aerobically at 37°C in rich (LB) and minimal MOPS plus glucose medium in low-shear modeled microgravity (LSMMG) and in a normal gravity vector control environment. They were compared with each other as well as control experiments conducted with a normal gravity vector. Thirteen separate minimal MOPS and sixteen LB medium growth experiments were performed. Total RNA was extracted from cells and, following removal of ribosomal RNA, radioactively labeled. Whole genome transcriptional assays were performed using matched pairs of PCR-product DNA macroarrays (Panorama E. coli Gene Arrays, Sigma-Genosys, Houston, TX). The hybridized membrane phosphorimages were imported into image analysis software. The data was normalized by reporting each spot as a percentage of the sum of intensities of all spots on the array image. Significant changes in gene expression were identified based on three previously documented criteria: (1) an overall p-value of < 0.05 which implies a 95% probability that a change in expression between strains or media was significant, (2) a log ratio of gene expression which differed from the mean of the log ratios by > 3.0 standard deviations giving a 99.9% confidence in gene expression, and (3) similar gene expression in all three biological replicates.

Online databases were used for gene nomenclature, gene location and orientation, putative co-transcription, product function, and presence in S. Typhimurium. The Colibiri WWW Server v3.1 <http://genolist.pasteur.fr/Colibri/genome.cgi> was used to determine individual gene locations and orientations in theE. coli genome as well as possible co-transcription with other expressed genes. EcoCyc (Institute for Genomic Research, University of California; San Diego, CA <http://ecocyc.org/>) and EcoSearch (University of Miami School of Medicine; Miami, FL <http://bmb.med.miami.edu/search.htm>) were used in determining gene names/synonyms and gene product function. The coliBase Web site <http://colibase.bham.ac.uk/> was used to identify genes significantly expressed in this study that are present or have an orthologue in S. Typhimurium.

Separately, following growth in the LSMMG environment, the cultures were subjected to antibiotic sensitivity and stress, resistance studies to determine if their response had been changed by the exposure to the LSMMG environment. Antibiotics employed were ampicillin, kanamycin, polymyxin E, chloramphenicol and rifampicin. The stress conditions included acidic and basic conditions, oxidative stress, osmotic stress, alcohol stress, and heat shock. Culture survival was compared to controls that had not been grown under LSMMG conditions.

Equipment/Special Technology
The project takes advantage of the high aspect rotating vessel (HARV) bioreactor which was originally developed by NASA scientists⁵ to minimize fluid motion for tissue culture differentiation, while maintaining culture aeration through a gas permeable membrane. The HARV'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 is rotated at a speed sufficient to maintain cell suspension in the media and completely filled, thereby preventing gas bubbles from causing solution turbulence (i.e., shear). The HARV apparatus approximates the physiological and transcriptional changes occurring in space flight due 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.

Results
Statistical analysis identified 19 up-regulated and 24 down-regulated genes in LSMMG compared to the control during the mid-log phase of growth in minimal MOPS medium. Of these, 1 up-regulated and 12 down-regulated genes were coded for hypothetical proteins. Among the up-regulated genes of known or putative function in LSMMG were genes involved in the E. coli acid tolerance response (transcriptional regulator gadE, the putative chaperone hdeA and associated genes hdeB, hdeD and dctR, flg and fli genes involved in cell motility, chemotaxis regulating genes cheY, cheZ and tar, and the phage related gene ydfD).

Among the MOPS LSMMG down-regulated genes were five genes involved in heavy metal efflux (CusCFBA and copA). Other LSMMG down-regulated genes included five putative bacteriaphage lambda homologs, four genes involved in various stress responses, the drug resistance gene emrE, the acetylCoA carboxylase subunit accB, and the putative NAD(P) binding enzyme ybeM. Two of the MOPS LSMMG down-regulated genes (cpxP and yfiA) were regulators. CpxP serves as repressor of the Cpx envelope/extracytoplasmic toxicity stress response system, protects the cell from toxic, transitory stresses, is involved in adhesion and virulence of pathogenic E. coli, and may also act as a periplasmic chaperone. Yfia stabilizes 30S rRNA under cold shock conditions. Possible co-transcribed genes of putative operons were identified based on genomic location and orientation. Physical mapping of the LSMMG MOPS-regulated genes found 34 of 43 genes in four gene clusters. Similar analysis of the LB LSMMG cultures identified seven down-regulated genes in LSMMG. These genes were mostly involved in biosynthesis and energy utilization. No significant change in response relative to the controls was seen with any of the antbiotics or stresses that were tested.

Salmonella Typhimurium is a very close evolutionarily relative of E. coli, and its response to LSMMG had been studied previously.⁴ In fact, the majority of the E. coli MG1655 LSMMG up- and down-regulated genes have homologues or orthologues in S. Typhimurium. We therefore reduced the statistical stringency of our analysis so that a direct comparison could be made with the earlier Salmonella results. When individual genes were intercompared, it was abundantly clear that the vast majority of genes affected by LSMMG in E. coli MG1655 and S. Typhimurium were not affected in the same manner in the other organism.

Discussion
The primary differences between the LSMMG environment and the control are the randomized gravity vector and low shear present in LSMMG. In attempting to interpret the differences seen, one must consider that they might be due to either or both of these factors or as an indirect effect of one or both. In minimal MOPS medium, E. coli chemotactic and flagellar genes as well as genes involved in the acid tolerance response were up-regulated in LSMMG. It is attractive to theorize that the LSMMG up-regulation of flagellar and chemotactic genes in minimal medium is related to a cellular requirement for relocation away from zones of local nutrient depletion and excreted waste hypothesized to occur in the low mixing environment of space.⁶ The majority of minimal medium LSMMG down-regulated genes are involved in metal or drug transport, cell lysis, or in regulating cellular stress responses, which alludes to the importance of the cell envelope in regulating the LSMMG response in minimal medium grown E. coli MG1655. More generally, all of the LSMMG up-regulated genes and a majority of the down-regulated genes of known function are present in or involved with regulation of the cellular envelope. This suggests that the cell envelope is superlative in sensing changes in its local environment and able to rapidly respond to the changes in a multifaceted way. Future time course studies of the LSMMG response to minimal media in cells preadapted to the HARV control environment may allow detailed study of how the genes involved are coordinated.

S. Typhimurium appears to be responding to LSMMG by activating genes involved in associated adhesion in an attempt to promote colonization in the low-shear environment. These are the same genes usually associated with pathogenicity. In contrast, E. coli MG1655 is a commensal that lacks many of these genes; hence, adhesion in preparation for colonization is apparently not its preferred response to LSMMG.

Conclusions
Bacteria, having lived on the Earth for billions of years, have not typically encountered microgravity and, hence, it would seem unlikely that genes governing a direct response to variations in gravity would have evolved. With specific reference to the LSMMG environment, then, we would anticipate that low-shear is more important in the bacterial transcriptional response than a direct effect of the randomized gravity vector. Experiments to date have confirmed this conclusion. In addition, our studies have reinforced the notion that the cell envelope is superlative in sensing changes in its local environment and able to rapidly respond to the changes in a multifaceted way. Future time course studies of the LSMMG response to minimal media in cells preadapted to the HARV control environment may allow detailed study of how the genes involved are coordinated. However, the dramatically different response to LSMMG that is observed between E. coli MG1655 and S. Typhimurium emphasizes that different species can respond to LSMMG in very different ways. This is a frustrating conclusion for those seeking to ascertain what the effect of exposure to low-shear or the space environment will be for microorganisms in general.

Acknowledgments
We would like to thank Dr. Yuriy Folfanov in the Department of Computer Science at the University of Houston for his advice and assistance in performing statistical analyses on the microarray data.

References
¹U. A. Stock and J. P. Vacanti, "Cardiovascular Physiology During Fetal Development and Implications for Tissue Engineering," Tissue Eng. 7 (2001): 1-7.
²J. M. Jessup and N. R. Pellis, "NASA Biotechnology: Cell Science in Microgravity," In Vitro Cell Dev. Biol. Anim. 37 (2001): 61-63.
³C. A. Nickerson, 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.
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. U. S. A. 99 (2002): 13807-12.
⁵T. L. Prewett, T. J. Goodwin, and G. F. Spaulding, "Three-Dimensional Modeling of T-24 Human Bladder Carcinoma Cell Line: A New Simulated Microgravity Culture Vessel," J. Tiss. Cult. Methods 15 (1993): 29-36.
⁶P. Todd and D. M. Klaus, "Theories and Models on the Biology of Cells in Space," Adv. Space Res. 17 (1996): 3-10.

Publications
Chumakov, S., C. Putoni, B. M. Pettitt, G. E. Fox, R. C. Willson, and Y. Fofanov. "Using Statistical Properties of Short Subsequences in Microbial Identification," Proc., Intl. Conf. on Mathematics and Engineering Techniques in Medicine and Biological Sciences, Las Vegas, NV, June 2004. 363-67.
Chumakova, S., C. Bepapurkara, C. Putoni, T. B. Li, B. M. Pettitt, G. E. Fox, R. C. Willson, and Y. Fofanov. "Theoretical Basis for Universal Identification Systems for Bacteria and Viruses," J. Biol. Phys & Chem. 5 (2006): 121-28.
Larios-Sanz, M., K. E. Kourentzi, D. Warmflash, J. Jones, R. C. Willson, D. L. Pierson, and G. E. Fox. "16S rRNA Beacons for Bacterial Monitoring in Craft and Habitat Modules in Human Space Missions," Aviation. Sci. Environ. Med. (In press.)
Putonti, C., G. E. Fox, R. C. Willson, B. M. Pettitt, and Y. Fofanov. "DNA Microbial and Viral Identification Using Ultraspecific Probes 'Blind' to Host and Background DNA," Proc., Interface, Baltimore, MD, 2004.
Tucker, D. L., F. Karouia, J. Wang, Y. Luo, T. B. Li, R. C. Willson, Y. Fofanov, and G. E. Fox. "The Effect of an Artificial RNA Marker on Gene Expression in Escherichia coli," Appl. Environ. Microbiol. 71L (2005): 4156-59.
Zhang, Z., G. W. Jackson, G. E. Fox, and R. C. Willson. "Microbial Identification by Mass Cataloging," BMC- Bioinformatics. (In press.)
Zhu, D., Y. Fofanov, R. C. Willson, and G. E. Fox. "ProkProbePicker (PPP): A Fast Program to Extract 16S rRNA-Targeted Probes for Prokaryotes," Proc., Intl. Conf. on Mathematics and Engineering Techniques in Medicine and Biological Sciences, Las Vegas, NV, June 2005. 41-47.

Presentations
Fox, G. E., G. W. Jackson, and R. C. Willson. "Identification of Unknown Bacteria by Mass Spectroscopy," Planetary Biology Workshop titled Mars Genetic Inventory of Spacecraft Analysis, JPL, Pasadena, CA, Feb. 28-March 1, 2006.
Jackson, G. W., Z. Zhang, G. E. Fox, and R. C. Willson. "Rapid Microbial Identification by MALDI-TOF Mass Spectrometry of 16S Ribosomal RNA," 22nd Annual Meeting, Houston Society for Engineering in Medicine and Biology, Houston, TX, Feb. 11, 2005.
Potty, A., S. Balan, G. E. Fox, and R. C. Willson. "Neutral Additive Effects on Metal Affinity Adsorption of Nucleic Acids," 229th National Meeting American Chemical Society, San Diego, CA, March 13-17, 2005.
Warmflash, D., J. Miller Jr., D. S. McKay, G. E. Fox, and N. Keerthi. "In Situ Detection of Extant Microbial Life in Extraterrestrial Settings Using Dielectric Spectroscopy," Biennial Meeting of the NASA Astrobiology Institute, Univ. of Colorado, Boulder, CO, April 10-14, 2005. Astrobiology 5.2 (2005): Abstract 641.
Zhu, D., Y. Fofanov, R. C. Willson, and G. E. Fox. "ProkProbePicker (PPP): a Fast Program to Extract 16S rRNA-Targeted Probes for Prokaryotes," Intl. Conf. on Mathematics and Engineering Techniques in Medicine and Biological Sciences, Las Vegas, NV, June 20-23, 2005.

Funding and Proposals
Fofanov, Y., R. C. Willson, and G. E. Fox. "Tools for Ultraspecific Probe/Primer Design" Homeland Security Advanced Research Projects Agency, March 15, 2005-March 14, 2006, $286,000. (Current funding.)
Fox, G. E. "Chiral-Selective Planetary Chemistries as a Marker for Life." NASA-ASTID Program, Aug. 1, 2005-July 31, 2007, $429,920. (Current funding.)
Fox, G. E. and K. Venkateswaran. "Comparative Genome Analysis and the Resistance Properties of Various Bacillus Species," NASA-Planetary Protection Program, Feb. 1, 2005-Jan. 31, 2008, UH costs: $226,723.
Fox, G. E. and R. C. Willson. "Microorganisms in the Spacecraft Environment," NASA-Office of Exploration Research, June 7, 2004-Sept. 30, 2006, $904,809. (Current funding.)
Miller, J. H., Jr. and G. E. Fox "Biosensors Based on Dielectric Response: A Non-Geocentric Approach for In Situ Life Detection," NASA-ASTEP Program, Jan. 1, 2005-Dec. 31, 2008, $794,864. (Not funded.)

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