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(12) United States Patent (Io) Patent No.: US 7,189,556 B2 Venkateswaran Et Al

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(12) United States Patent (Io) Patent No.: US 7,189,556 B2 Venkateswaran Et Al https://ntrs.nasa.gov/search.jsp?R=20080009496 2019-08-30T03:39:08+00:00Z (12) United States Patent (io) Patent No.: US 7,189,556 B2 Venkateswaran et al. (45) Date of Patent: Mar. 13,2007 (54) BACILLUS ODYSSEYI ISOLATE Reisenman, P.J. and Nicholson, W.L. (2000) Role of the spore coat layers in Bacillus subtilis spore resistance to hydrogen peroxide, (75) Inventors: Kasthuri Venkateswaran, Arcadia, CA artificial UV-C, UV-B, and solar UV radiation. Appl Environ (US); Myron Thomas La Duc, Microbiol 66: 620-626. Arcadia, CA (US) Ruger, H. J., Fritze, D., and Sproer, C. (2000) New psychrophilic and psychrotolerant Bacillus marinus strains from tropical and polar deep-sea sediments and emended description of the species. Int J (73) Assignee: California Institute of Technology, Syst Evol Microbiol 50: 1305-1313. Pasadena, CA (US) Schaeffer, P., Millet, J. & Aubert, J.-P. (1965) Catabolic repression of bacterial sporulation. Proc Natl Acad Sci 54:, 704-711. ( * ) Notice: Subject to any disclaimer, the term of this Swofford, D. (1990) PAUP: phylogenetic analysis using parsimony, patent is extended or adjusted under 35 version 3.0. Computer program distributed by the Illinois Natural U.S.C. 154(b) by 0 days. History Survey, Champaign, IL. Venkateswaran, K., Kempf, M., Chen, F., Satomi, M., Nicholson, (21) Appl. No.: 10/759,327 W., & Kem. R. (2003) Bacillus nealsonii sp. nov., isolated from a spacecraft assembly facility, whose spores are gamma-radiation (22) Filed: Jan. 17, 2004 resistant. Int J Syst Evol Microbiol 53: 165-172. Venkateswaran, K., Satomi, M., Chung, S., Kern, R., Koukol, R., (65) Prior Publication Data Basic, C. & White, D. (2001) Molecular microbial diversity of a spacecraft assembly facility. Syst Appl Microbiol 24: 3 11-320. US 200410158042 A1 Aug. 12, 2004 Yoon, J.-H., Lee, K.-C., Weiss, N., Kho, Y. H., Kang, K. H. & Park, Y.-H. (2001) Sporosarcina aquimarina sp. nov., a bacterium iso- Related U.S. Application Data lated from seawater in Korea, and transfer of Bacillus globisporus Provisional application No. 601440,790, filed on Jan. (Larkin and Stokes 1967), Bacilluspsychrophilus (Nakamura 1984) (60) and Bacillus pasteurii (Chester 1898) to the genus Sporosarcina as 17. 2003. Sporosarcina globispora comb. nov., Sporosarcina psychrophila comb. nov. and Sporosarcina pasteurii comb. nov., and emended (51) Int. C1. description of the genus Sporosarcina . Int J Syst Evol Microbiol5 1: Cl2N 1/2 0 (2006.01) 1079-1086. (52) U.S. C1. ................................. 435/252.5; 424193.46 Ash, C, Wallbanks, S. and Collins, M. D. (1991) Phylogenetic (58) Field of Classification Search ..................... None heterogeneity of the genus Bacillus as revealed by comparative See application file for complete search history. analysis of small-subunit-ribosomal RNA sequence, Lett Appl Microbiol 13: 202-206. (56) References Cited Ezaki, T., Hashimoto, Y and Yabuuchi, E. (1989) Fluorometric deoxyribonucleic acid-deoxyribonucleic acid hybridization in OTHER PUBLICATIONS microdilution wells as an alternative to membrane filter hybridiza- La Duc, M. et a1 (Characterization of microbes intimately associated tion in which radioisotopes are used to determine genetic related- with the Mars Odyssey Orbiter and it’s assembly facility, Abstracts ness among bacterial strains. Int J Syst Bacteriol 39: 224-229. of the general meeting of the American Society for Microbiology, * cited by examiner 2002; 102: 389).* Venkateswaran, K. et a1 (Bacillus nealsonii sp. nov., isolated from Primary Examiner-Irene Marx a spacecraft-assembly facility, whose spores are g-radiation resis- (74) Attorney, Agent, or Firm-Tope-McKay & Associates tant, International Journal of Systematic and Evolutionary Microbiology, Jul. 2002; 53: 165-172).* (57) ABSTRACT Allen et al, Section VI, Isolation and Examination of Anaerobic Bacteria, Manual of Clinical Microbiology, Fourth Edition, 1985, p. 413-14.* The present invention relates to discovery and isolation of a Anonymous, (1980) NASA Standard Procedures for the Microbio- biologically pure culture of a Bacillus odysseyi isolate with logical Examination of Space Hardware, NHB 5340.1B, 1980, Jet high adherence and sterilization resistant properties. B. Propulsion Latoratory communication, National Aeronautics and Space Administration, pp. 2-26. odysseyi is a round spore forming Bacillus species that La Duc M.T., Nicholson W., Kern R., and K. Venkateswaran (2003) produces an exosporium. This novel species has been char- Microbial Characterization of the Mars Odyssey Spacecraft and Its acterized on the basis of phenotypic traits, 16s rDNA Encapsulation Facility. Environ Microbiol, Oct. 5, 2003 p. 977 sequence analysis and DNA-DNA hybridization. According Abstract only; entire article not provided. to the results of these analyses, this strain belongs to the Nicholson, W. L., Munakata, N., Horneck, G., Melosh, H. J. and genus Bacillus and the type strain is 34hs-lT (=ATCC Setlow, P. (2000) Resistance of Bacillus endospores to extreme PTA-4993T=NRRL B-30641T=NBRC 100172T). The Gen- terrestrial and extraterrestrial environments. Microbiol Mol Biol Bank accession number for the 16s rDNA sequence of strain Rev 64: 548-572. 34hs-lTis AF526913. Priest, F. G., Goodfellow, M. and Todd, C. (1988) A numerical classification of the genus Bacillus, J Gen Microbiol 234: 1847- 1882. 1 Claim, 5 Drawing Sheets U.S. Patent Mar. 13,2007 Sheet 1 of 5 US 7,189,556 B2 FIGURE 1 FIGURE 2 U.S. Patent Mar. 13,2007 Sheet 2 of 5 US 7,189,556 B2 FIGURE 3 FIGURE 4 U.S. Patent Mar. 13,2007 Sheet 3 of 5 US 7,189,556 B2 FIGURE 5 FIGURE 6 U.S. Patent Mar. 13,2007 Sheet 4 of 5 US 7,189,556 B2 Characteristic 1 2 3 4 5 6 Substrate oxidized Acetate Pyruvate D - Hydroxy butyra t e /I-Hydroxybutyrate Methyl p).rumte L-Alanine Glycyl t-glummate Adenosine 2' -Deoxj.;ldenosine Inosine AMP ULLf P Growth in 5% NaCl 165 rDXA sequence sirnitariry (96)' FIGURE 7 FIGURE 8 U.S. Patent Mar. 13,2007 Sheet 5 of 5 US 7,189,556 B2 Similarity (%) t !abekd DNA from: 1 2 3 7 100 18 17 17 23 100 17 15 17 18 100 15 11 ND NB 11 10 Ne, ND 16 7 ND ND 15 7 Nb ND 100 7 ND ND 8 FIGURE 9 US 7,189,556 B2 1 2 BACILLUS ODYSSEYI ISOLATE odysseyi could be used in sunscreens or to prolong the lives of convertible tops, tents, etc. as a W-ray retardant spray. PRIORITY CLAIM BRIEF DESCRIPTION OF THE DRAWINGS This application is a non-provisional application, claim- 5 ing the benefit of priority to provisional application No. The objects, features and advantages of the present inven- 601440,790, filed in the United States on Jan. 17, 2003, tion will be apparent from the following detailed descrip- entitled “Bacterial spore-forming species that is extremely tions of the various aspects of the invention in conjunction resistant to various sterilization methods.” with reference to the following drawings, where: 10 FIG. 1 is a light microscopy image of sporulating veg- GOVERNMENT RIGHTS etative cells showing terminal swelling of mother cells; FIG. 2 is a microscopy image of a purified spores showing This invention was made with Government support under an intact spore with exosporium; Contract NAS7-1407 awarded by NASA. The Government FIG. 3 is a microscopy image of a longitudinal section of has certain rights in the invention. 15 an untreated spore, showing the exosporium, spore coat, cortex and spore core; FIELD OF INVENTION FIG. 4 is a microscopy image of spores after being exposed to gamma radiation; The present invention relates to an isolated biologically FIG. 5 is a microscopy image of spores after being pure culture of a novel spore forming Bacillus species, and 20 exposed to H,O,; more particularly, to a Bacillus odysseyi isolate with high FIG. 6 is a microscopy image of spores after being adherence and sterilization resistant properties. exposed to both gamma radiation and H,O,; FIG. 7 is a table illustrating characteristics for differen- BACKGROUND OF INVENTION tiating B. odysseyi 34-hsl from related species; 25 FIG. 8 is a chart illustrating a phylogenetic tree of Several physiologically and phylogenetically distinct round-spore forming Bacillus and other species closely microorganisms have been encountered while examining related to strain 34-hsl based on maximum likelihood and microbial contamination of spacecraft surfaces. Some of parsimony analysis of 16s rDNA nucleotide sequences; and these micro-organisms form round, exosporium-bearing FIG. 9 is a table illustrating DNA-DNA hybridization spores, whose exosporia might be responsible for adaptation 30 between B. odysseyi sp. Nov. 34-hsl and related species. to the extreme clean conditions of, and direct adhesion to, spacecraft surfaces. DETAILED DESCRIPTION Such biofouling is a concern in not only space travel, but in a number of industries. Isolation, identification and under- The present invention relates to an isolated biologically standing of the highly resistant and adhesive micro-organ- 35 pure culture of a novel spore forming Bacillus species, and isms could be of significant use in industry, where biofouling more particularly, to Bacillus odysseyi. is a major cause of reduction in productivity (resulting in a The following description, taken in conjunction with the loss of over $6.5 billion in marine industries alone), and in referenced drawings andor tables, is presented to enable one medicine, where bacterial adhesion is often a primary step in of ordinary skill in the art to make and use the invention. human disease. In addition, purified exosporium compo- 40 Various modifications will be readily apparent to those nents (proteins, lipids, etc.) could possibly be used in other skilled in the art, and the general principles defined herein ways, such as in sunscreens or to prolong the lives of may be applied to a wide range of aspects. Thus, the present convertible tops, tents, etc.
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