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It has been repeatedly found that species of extremely resilient, spore-forming bacteria belonging to genus Bacillus are the most highly represented organisms in samples collected from spacecraft and spacecraft assembly facility surfaces (La Duc et al., 2003; 2004; Puleo et al., 1977). The extremely oligotrophic, low-humidity, temperature-controlled conditions of spacecraft assembly facilities appear to select for microbes able to withstand such unfavorable surroundings. While monitoring the microbial diversity of spacecraft associated environments over a period of 5 years (1999 to 2004), Bacillus pumilus was found to be the second most dominant species among aerobic spore-forming bacteria (the predominant being B. licheniformis; La Duc et al., 2004). B. pumilus isolates showed resistance to H2O2 ( Kempf et al., 2005) and are thus considered problematic microbes since H2O2 is recommended for use in bioreduction of spacecraft components.
B. pumilus, is a ubiquitous Gram-positive, aerobic, rod-shaped endospore-forming bacteria that can be isolated from a wide variety of soils, plants and environmental surfaces, and even from the interior of Sonoran desert basalt (Benardini et al., 2003). B. pumilus isolates were also recently recovered aboard the International Space Station from hardware surfaces and air particles. It is likely that B. pumilus isolates were present in spacecraft assembly facilities as metabolically dormant spores. Bacillus spores are notoriously resistant to unfavorable conditions such as low or no nutrient availability, extreme desiccation, H2O2, UV, gamma-radiation, or chemical disinfection (Nicholson et al., 2000). What is astounding is the elevated resistance observed in B. pumilis spores when compared to the spores of any other Bacillus species in the spacecraft assembly facilities.
While characterizing the microbial diversity of a spacecraft assembly facility H2O2 resistant bacterial strains were repeatedly isolated from various surface locations. H2O2 is a possible sterilant for spacecraft hardware because it is a low-temperature process and is compatible with various modern-day spacecraft materials, electronics and components. Both conventional biochemical testing and molecular analyses identified these strains as B. pumilus. This Bacillus species was found in both unclassified (entrance floors, ante-room, and air-lock) and classified (floors, cabinet tops, and air) locations. Both vegetative cells and spores of several B. pumilus isolates were exposed to 5% liquid H2O2 for 60 min. Spores of each strain exhibited higher resistance than their respective vegetative cells to liquid H2O2. Results indicate that the H2O2 resistance observed in both vegetative cells and spores is strain-specific, as certain B. pumilus strains were 2 to 3 times more resistant than a standard B. subtilis dosimetry strain. An example of this trend was observed when the type strain of B. pumilus, ATCC 7061, proved sensitive, whereas several environmental strains exhibited varying degrees of resistance to H2O2 (Kempf et al., 2005). Repeated isolation of H2O2-resistant strains of B. pumilus in a clean-room is a concern because their persistence might potentially compromise life-detection missions, which have very strict cleanliness and sterility requirements for spacecraft hardware.
Spore-forming microbes recovered from spacecraft surfaces and assembly facilities were exposed to simulated Mars UV irradiation. The effects of UVA (315-400 nm), UVA+B (280-400 nm), and full spectrum (200-400 nm) at intensities, expected to strike Mars, on the survival of microorganisms showed that spores of Bacillus species isolated from spacecraft associated surfaces were more resistant than a standard dosimetric strain, B. subtilis 168. Among all Bacillus species tested, spores of a strain of B. pumilus SAFR-032 showed the highest resistance to all three UV bandwidths as well as the total spectrum. Although the elevated resistance to simulated Mars UV irradiation was strain-specific, B. pumilus exhibited more resistance compared to other species tested. The presence of organisms like B. pumilus SAFR-032 on the spacecraft associated surfaces and its elevated survival (6 times) compared to that of the standard dosimetric strains should be considered when determining the ability of the Martian UV environment to sanitize landed spacecraft (Newcombe et al., 2005; personal communication).
Key questions include: What are the genetic mechanism(s) behind high resistance to VHP and UV? Does B. pumilus produce "antioxidants" to protect the bacteria from oxidative agents? Are these compounds medically or biotechnologically important? Do the B. pumilus strains possess "universal" common mechanism(s) used by Bacillus species as protection from unfavorable conditions and are useful to mankind? With its genome sequenced, B. pumilis will be an invaluable experimental system to address such questions because it will be possible to focus expression and proteomics studies on sporulation using strain SAFR-032 in conjunction with B. pumilis strains that have normal spore resistances.
The Bacillus pumilus genome is currently being sequenced at BCM-HGSC.
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References for Bacillus pumilus:
Benardini JN, Sawyer J, Venkateswaran K, & Nicholson WL. (2003). Spore UV and acceleration resistance of endolithic Bacillus pumilus and B. subtilis isolates obtained from Sonoran desert basalt: implications for lithopanspermia. Astrobiology 3: 709-717.
Dickinson DN, La Duc MT, Satomi M, Winefordner JD, Powell DH, & Venkateswaran K. (2004). MALDI TOFMS compared with other polyphasic taxonomy approaches for the identification and classification of Bacillus pumilus spores. J Microbiol Methods 58:1-12.
Kempf MJ, Quigley MS, Chen F, Satomi M, Kern R, & Venkateswaran K. (2005). Isolation and characterization of hydrogen peroxide resistant spores of Bacillus pumilus from a Spacecraft Assembly Facility. Astrobiology, in press.
Link L, Sawyer J, Venkateswaran K, & Nicholson W. (2004). Extreme spore UV resistance of Bacillus pumilus isolates obtained from an ultra-clean Spacecraft Assembly Facility. Microbial Ecology, 47: 159-163.
Newcombe DA, Schuerger AC, Benardini JN, Dickinson D, Tanner R, & Venkateswaran K. Survival of Spacecraft- Associated Microorganisms under Simulated Martian UV Irradiation. Appl Environ Microbiol (submitted)
Priest FG. Systematics and ecology of Bacillus. In Losick R, Hoch JA, & Sonenshein AL (eds) Bacillus subtilis and other Gram-positive bacteria: biochemistry, physiology, and molecular genetics. ASM Press, Washington DC. pp. 3-16 (1993).
Valadez VA, Thrasher AN, Ott CM, & Pierson DL. (2002). Evaluation of Bacterial Diversity aboard the International Space Station. Abstracts 102nd General Meeting of the American Society of Microbiology, Salt Lake City, UT.