Bacillus Pumilus SAFR-032L: A Model for Planetary Protection Research

University of Houston • University of Houston-Clear Lake • ISSO Annual Report Y2006 • 40-42

Bacillus Pumilus SAFR-032L: A Model for Planetary Protection Research

George E. Fox

ABSTRACT—In order to prevent forward contamination of Mars, it is necessary to minimize the bio-burden on spacecraft that will contact the Martian surface. Bacillus pumilus strains whose spores are unusually resistant to ultraviolet light and other means of sterilization have been consistently found in the clean rooms used in space craft assembly as well as on space craft. In order to better understand the origin of these organisms and to develop effective means for eliminating them, the complete genome of Bacillus pumilus SAFR-032 is being sequenced in collaboration with the Baylor College of Medicine Human Genome Sequencing Center (HGSC). The sequence is in the finishing stage, and annotation is nearly complete. Comparisons of the genes involved in the sporulation process and UV repair are being intercompared in detail to identify genomic features that might be responsible for the unusual resistances that this organism exhibits.

A primary objective of the space sciences is to search for evidence of living systems in the universe. The most tractable target for such efforts has been and for the near future will continue to be the planet Mars. Having ratified the 1967 Outer Space Treaty, the United States is obliged to avoid harmful contamination of celestial bodies that might harbor life. Spacecraft that land on Mars but are not equipped with life-detection experiments must minimize the bio-burden they bring to the planet. Thus, the components are subjected to rigorous cleaning and must be assembled in a Class 100,000 clean room or better. If the mission contains life-detection experiments, a sterilization process must be applied as good as or better than that applied to the landers used in the 1967 Viking missions.

In preparing that protocol, researchers argued that the organisms most likely to be resistant to sterilization would be endospore-forming bacteria of the genera Bacillus and Clostridium. Hence, a spore assay was developed to evaluate the effectiveness of the sterilization process using Bacillus subtilis as the model organism.

Given this background, modern planetary protection research focuses on two issues: (1) development of cleaning and sterilization technologies and better methods of evaluating their success and (2) the assessment of which terrestrial microorganisms are most likely to survive cleaning and sterilization and hence possibly confound tests for life done on the Martian surface or contaminate returned samples.

Spacecraft are assembled in clean rooms which employ sterilants including vapor-phase hydrogen peroxide (VHP), a strong oxidizing agent, and UV, in addition to physical containment to obtain and maintain the necessary pristine surfaces. While conducting a microbial census of NASA spacecraft assembly facilities, Venkateswaran and his colleagues discovered that strict contamination control measures did not provide an absolute barrier to microbial contamination but, instead, established a series of selective "bottlenecks" which are sufficient to prevent the penetration or survival of all but the hardiest microbes into the interior assembly area.¹ The predominant isolates repeatedly found to have penetrated deepest within the cleanest parts of the spacecraft assembly facilities were spore-forming bacteria, especially strains of Bacillus pumilus, B. nealsonii,^2,3 and a closely related grouping recently named B. odysseyi because it was first isolated from the surface of the Mars Odyssey spacecraft.⁴ B. pumilus strains have also regularly been isolated from space craft including hardware from the International Space Station (ISS). In comparison with the model organism B. subtilis, which is used in the standard spore assay, B. odysseyi spores had survival times that were 3 times, 10 times and 6 times longer when exposed to UV, gamma radiation, and hydrogen peroxide. The most resistant strain isolated to date, B. pumilus SAFR-032 (Space Craft Assembly Facility Resistant Isolate 32) has at least a 10-fold increase in its resistance to Mars UV radiation conditions than the standard B. subtilis.

In view of these findings, it is clear that assays for cleanliness and survival potential will be more meaningful if they are based on the organisms that are likely to be problematic such as B. pumilus and B. oddysseyi. Of the various resistant Bacillus strains that were isolated, the SAFR-032 isolate is the most resistant and has been selected for two key follow-up studies. First, a flight experiment was proposed and has been selected for a study of the effect of radiation resistance on this organism. This experiment will be carried out aboard the ISS using the European Technology Exposure Platform and Experiment Facility (EXPOSE). Second, the complete sequence of the genome of SAFR-032 is currently being determined by investigators at the Baylor College of Medicine Human Genome Sequencing Center (HGSC) with funding from the National Science Foundation (NSF).

Methodology
The SAFR-032 genome is essentially complete, and the HGSC group is currently conducting finishing studies in order to finalize the assembly of several very large reliable contigs. In addition, partial data were obtained for a second strain, F036B. A high-quality manual annotation is in progress using the HGSC-developed CONAN interface. Possible open reading frames are initially predicted by Glimmer⁵ and GeneMarkS⁶ and then automatically examined for similarity to known COGS, protein domains and other annotated genes (i.e., pre-run BLAST searches). The annotator looks at all the data and in some cases literature references before assigning a name and if possible a likely function for the open reading frame.⁷ Each gene is annotated separately by two annotators and the resulting annotations compared automatically. Differences are subsequently resolved by conference between the annotators with input from the group as a whole when needed.

The initial analysis will identify genes that are shared or not shared with Bacillus subtilis⁸ and other Bacillus genomic sequences. Of special interest will be genes known to be involved in spore resistance,^9-12 DNA repair,^13-15 and sporulation in general.^16,17 Examination of shared genes will allow us to assess whether regulatory signals, e.g., promoter sequences for various sigma factors, are changed or not. In particular, genes known to be associated with particular sigma factors can be used to define recognition sites in SAFR-032. With this knowledge in place, genes that are uniquely present or absent in SAFR-032 will be broken into two groups, those associated with known sporulation genes and regulons and those not so associated. The leader regions of unique genes not associated with known sporulation operons will be further examined to see if they are likely to be transcribed by any of the sigma factors associated with sporulation.¹⁸ A second analysis will be a detailed comparison of the SAFR-032 gene organization with the well established operon structure of B. subtilis¹⁹ to identify possible regulatory differences. The most promising changes here will likely be operons whose structure is uniquely changed in SAFR-032 relative to the other known Bacillus genomes, all lacking resistant spores.

Results
As a first step toward understanding the biology of these unique strains, the whole genome sequence of one isolate, B. pumilus SAFR-032, was determined. The B. pumilus SAFR-032 genome is 3.62 MB in size with approximately 3950 genes. Sequence alignment and subsequent construction of a tree of phylogenetic relationship for multiple housekeeping genes revealed that among published complete genomes, SAFR-032 is most closely related to B. subtilis and B. licheniformis, thereby rendering these organisms of greatest relevance for comparison. Since the sporulation machinery of B. subtilis has been extensively studied, this is an especially favorable outcome. A similar analysis of the F036B strain of B. pumilus revealed that it is much closer to SAFR-032 than the latter is to B. subtilis or B. licheniformis. A comparison of gene order between SAFR-032 and B. licheniformis revealed substantial co-linearity. This is important because one can scan local clusters for gene additions or losses that may correlate with changes in regulation.

Discussion
Despite the substantial similarity to B. licheniformis and B. subtilis, there are, nevertheless, many coding regions in SAFR-032 with no obvious homolog in these other organisms. Genes known to be involved in the sporulation processes, including regulation, spore protection, and germination, were compared with their homologs in B. subtilis and B. licheniformis in order to identify unusual absences and changes in gene order. The largest number of such changes are associated with genes involved in the generation of the spore coat protein which has likely been redesigned in B. pumilus relative to its sister species. Genes encoding the small acid soluble proteins, which are not only crucial for spore DNA protection but also known to be highly conserved within and across species, have interesting sequence variation, as compared to those of other Bacillus strains. Further, detailed comparative analysis revealed that several proteins annotated as hypothetical in B. subtilis/B. licheniformis, could actually be classified as functional genes (such as families of transporters, transcriptional regulator proteins etc.), genes unique to B. pumilus. Researchers found also that gene clusters encoding the polyketide pathway have substantial sequence variation from those of other Bacillus strains.

Conclusions
Results obtained here will in the short term generate multiple candidate genomic features that may separately or in combination be responsible for the unusual resistance associated with B. pumilus spores. In the immediate future it will be of interest to explore these alternative possibilities by comparing gene expression patterns in resistant and non-resistant strains. Funding for this purpose will be sought from the National Science Foundation and other federal agencies. In the longer view, the processes by which non-resistant strains readily evolve resistance to UV will be a useful model system for studying evolution.

References
¹M. J. Kempf, F. Chen, R. Kern, and K. Venkateswaran, "Recurrent Isolation of Hydrogen Peroxide-Resistant Spores of Bacillus pumilus from a Spacecraft Assembly Facility," Astrobiology 5 (2005): 391-405.
²K. M. Venkateswaran, M. Satomi, S. Chung, R. Kern, R. Koukol, C. Basic, and D. White. "Molecular Microbial Diversity of a Spacecraft Assembly Facility," Systematic and Applied Microbiology, 24 (2001): 311-20.
³K. Venkateswaran, M. J. Kempf, F. Chen, W. Nicholson, and R. Kern, "Description of Bacillus nealsonii sp., Nov., Isolated from Spacecraft Assembly Facility, Whose Spores Are Radiation Resistant," Intl. J. of Systematic and Evolutionary Microbiology 53 (2003): 165-72.
⁴M. T. La Duc, M. Satomi, and K. Venkateswaran, "Bacillus odysseyi Sp. Nov., a Round-Spore-Forming Bacillus Isolated from the Mars Odyssey Spacecraft," Intl. J. of Systematic and Evolutionary Microbiology 54 (2004): 195-201.
⁵A. L. Delcher, D. Harmon, S. Kasif, O. White, and S. L. Salzberg, "Improved Microbial Gene Identification with GLIMMER," Nucleic Acids Research 27 (1999): 4636-41.
⁶J. Besemer J., A. Lomsadze, and M. Borodovsky, "GeneMarkS: A Self-Training Method for Regulatory Regions," Nucleic Acids Research 29 (2001): 2607-18.
⁷M. P. McLeod, S. E. Karpathy, J. Gioia, X. Qin, S. K. Highlander, G. E. Fox, T. Z. McNeil, H. Jiang, D. Muzny, L. S. Jacob, A. C. Hawes, E. Sodergren, R. Gill, J. Hume, M. Morgan, C. Hong, X. Yu, D. H. Walker, and G. M. Weinstock, "The Complete Genome of Rickettsia Typhi and Comparison with R. Prowazekii and R. Conorii," J. of Bacteriology 186 (2004): 5842-55.
⁸I. Moszer, L. M. Jones, S. Moreira, C. Fabry, and A. Danchin, "SubtiList: The Reference Database for the Bacillus subtilis Genome,"" Nucleic Acids Research 30 (2002): 62-5.
⁹P. Setlow, "Spores of Bacillus subtilis: Their Resistance to and Killing by Radiation, Heat, and Chemicals," J. Appl. Microbiol. 101.3 (2006): 514-25.
¹⁰T. A. Slieman, and W. L. Nicholson, "Role of Dipicolinic Acid in Survival of Bacillus subtilis Spores Exposed to Artificial and Solar UV Radiation," Appl. and Environmental Microbiology, 67 (2001): 1274-79.
¹¹P. Setlow, "Resistance of Spores of Bacillus Species to Ultraviolet Light," Environmental Molecular Mutagenesis 38 (2002): 97-104.
¹²P. Setlow, "Spores of Bacillus subtilis: Their Resistance to and Killing by Radiation, Heat and Chemicals," J. of Applied Microbiology 101 (2006): 514-25.
¹³Y.M. Xue and W. L. Nicholson, "The Two Major Spore DNA Repair Pathways, Nucleotide Excision Repair and Spore Photoproduct Lyase, Are Sufficient for the Resistance of Bacillus subtilis Spores to Artificial UV-C and UV-B but not to Solar Radiation," Appl. Environ. Microb. 62.7 (1996): 2221-7.
¹⁴M. A. Ross, and P. Setlow, "The Bacillus subtilis HBsu Protein Modifies the Effects of Alpha/beta-Type, Small Acid-Soluble Spore Proteins on DNA," J. of Bacteriology 182 (2000): 1942-48.
¹⁵C. S. Hayes and P. Setlow, "An Alpha/Beta-Type, Small, Acid-Soluble Spore Protein Which Has Very High Affinity for DNA Prevents Outgrowth of Bacillus subtilis Spores," J. of Bacteriology 183 (2001): 2662-66.
¹⁶D. W. Hilbert and P. J. Piggot,"Compartmentalization of Gene Expression during Bacillus subtilis Spore Formation," Microbiology and Molecular Biology Reviews 68 (2004): 234-62.
¹⁷Makita, Y., M. Nakao, N. Ogasawara, and K. Nakai, "DBTBS: Database of Transcriptional Regulation in Bacillus subtilis and Its Contribution to Comparative Genomics," Nucleic Acids Research 32 (2004): D75-77.
¹⁸D. W. Hilbert and P. J. Piggot, 2004, 234-62.
¹⁹M. J. De Hoon, S. Imoto, K. Kobayashi, N. Ogasawara, and S. Miyano, "Predicting the Operon Structure of Bacillus subtilis Using Operon Length, Intergene Distance and Gene Expression Information," Pacific Symposium on Biocomputing (2004): 276-87.

Publications
Gioia, J., X. Qin, H. Jiang, K. Clinkenbeard, R. Lo, Y. Liu, G. E. Fox, M. P. McLeod, T. Z. McNeil, L. Hemphill, E. Sodergren, G. M. Weinstock, and S. K. Hightower. "The Genome Sequence of Mannheimia Haemolytica A1: Insights into Virulence, Natural Competence and Pasteurellaceae Phylogeny," J of Bacteriology 188 (2006): 7257-66.
Petrosino, J.F., Q. Xiang, S. E. Karpathy, H. Jiang, S. Yerrapragada, Y. Liu, J. Gioia, L. Hemphill, A. Gonzalez, T. M. Raghaven, A. Suman, G. E. Fox, S. K. Highlander, M. Reichard, R. J. Morton, K. D. Clinkenbeard, and G. W. Weinstock. "Chromosome Rearrangements and Diversification of Francisella Tularensis Revealed by the Type B (OSU18) Genome Sequence," J. of Bacteriology 188 (2006): 6977-85.
Viswanath, L., Y. Lu, and G. E. Fox. "Genome Display Tool: Visualizing Possible Correlations in Complex Data Sets," Source Code for Biology and Medicine (2007). (In Press.)

Presentations
Fox, G. E. "Overview of the B. Pumilus Genome Sequencing Project," Planetary Biology Workshop Entitled Mars Genetic Inventory of Spacecraft Analysis, JPL, Pasadena, CA, February 28-March 1, 2006.
Tirumalai, M. R., Y. Yerrapragada, J. Gioia, I. DaGupta, L. Bokhetache, Y. Liu, P. E. Moorthy, B. D. McWilliams, J. Siefert, F. Karouia, A. A. Olowu, K. D. Clinkenbeard, A. Verma, P. Buzombo, H. Zwiya, O. Igboeli, A. Suman, X. Qin, H. Jiang, S. K. Highlander, K Venkateswaran, G. E. Fox, and G. M. Weinstock. "Whole Genome Sequence of a Bacillus pumilus strain isolated from a Spacecraft Assembly Facility," 107th American Society of Microbiology General Meeting, May 21-25, 2007, Toronto, Canada. (Forthcoming).

Funding and Proposals
Venkateswaran, Kasthuri, JPL. "Microbial Ecological Perspectives of Space-Exposed Microbes: A Genetic Approach," NASA: Human Support Technology, April 1, 2005-March 31,2008. UH Subcontract Total Costs: $120,000. (Not funded.)
Fox, G. E. "Comparative Genome Analysis and the Resistance Properties of Various Bacillus Species," NASA-Planetary Protection Program: Feb. 1, 2005-Jan. 31, 2008. UH Total Costs: $226,723. (Not funded.)

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