Single cell genomics indicates horizontal gene transfer and viral infections in a deep subsurface Firmicutes population

Frontiers in Microbiology

Volumn 6, Issue APR, 2015, Pages

  Labonté, Jessica M ^a   Field, Erin K ^a   Lau, Maggie ^b   Chivian, Dylan ^c   Van Heerden, Esta ^d   Wommack, K Eric ^e   Kieft, Thomas L ^f   Onstott, Tullis C ^b   Stepanauskas, Ramunas ^a  

^a BIGELOW LABORATORY FOR OCEAN SCIENCES   (United States)

^b PRINCETON UNIVERSITY   (United States)

^c LAWRENCE BERKELEY NATIONAL LABORATORY   (United States)

^d UNIVERSITY OF THE FREE STATE   (South Africa)

^e UNIVERSITY OF DELAWARE   (United States)

^f NEW MEXICO INSTITUTE OF MINING AND TECHNOLOGY   (United States)

Author keywords

Desulforudis; Evolution; Horizontal gene transfer (HGT); Population genomics; Single cell genomics; Terrestrial deep subsurface; Transposable phages; Transposases

Indexed keywords

RESTRICTION ENDONUCLEASE; TRANSPOSASE;

ARTICLE; BACTERIAL GENOME; BACTERIUM; CARBON DIOXIDE FIXATION; CLUSTERED REGULARLY INTERSPACED SHORT PALINDROMIC REPEAT; CONTROLLED STUDY; DESULFORUDIS AUDAXVIATOR; DNA TRANSPOSITION; FIRMICUTES; GENE SEQUENCE; GENETIC RECOMBINATION; GENETIC VARIABILITY; HORIZONTAL GENE TRANSFER; MARINE ENVIRONMENT; METAGENOMICS; MICROBIAL COMMUNITY; NITROGEN FIXATION; NONHUMAN; NUCLEOTIDE SEQUENCE; PHYLOGENY; POLYMERASE CHAIN REACTION; PROPHAGE; SINGLE AMPLIFIED GENOME; SINGLE CELL ANALYSIS; STRUCTURAL GENOMICS; VIRUS INFECTION; VIRUS MORPHOGENESIS;

FIRMICUTES; PROKARYOTA;

EID: 84930946066     PISSN: None     EISSN: 1664302X     Source Type: Journal    
DOI: 10.3389/fmicb.2015.00349     Document Type: Article

References (82)

1. Altschul, S., Gish, W., and Miller, W. (1990). Basic local alignment search tool. J. Mol. Biol. 215, 403-410. doi: 10.1016/S0022-2836(05)80360-2.
2. Aziz, R. K., Breitbart, M., and Edwards, R. A. (2010). Transposases are the most abundant, most ubiquitous genes in nature. Nucleic Acids Res. 38, 4207-4217. doi: 10.1093/nar/gkq140.
3. Borgonie, G., García-Moyano, A., Litthauer, D., Bert, W., Bester, A., van Heerden, E., et al. (2011). Nematoda from the terrestrial deep subsurface of South Africa. Nature 474, 79-82. doi: 10.1038/nature09974.
4. Brazelton, W. J., and Baross, J. A. (2009). Abundant transposases encoded by the metagenome of a hydrothermal chimney biofilm. ISME J. 3, 1420-1424. doi: 10.1038/ismej.2009.79.
5. Brüssow, H., Canchaya, C., and Hardt, W. (2004). Phages and the evolution of bacterial pathogens: from genomic rearrangements to lysogenic conversion. Microbiol. Mol. Biol. Rev. 68, 560-602. doi: 10.1128/MMBR.68.3.560-602.2004.
6. Camacho, C., Coulouris, G., Avagyan, V., Ma, N., Papadopoulos, J., Bealer, K., et al. (2009). BLAST+: architecture and applications. BMC Bioinformatics 10:421. doi: 10.1186/1471-2105-10-421.
7. Canchaya, C., Proux, C., Fournous, G., Bruttin, A., and Brüssow, H. (2003). Prophage genomics prophage genomics. Microbiol. Mol. Biol. Rev. 67, 238-276. doi: 10.1128/MMBR.67.2.238-276.2003.
8. Chivian, D., Brodie, E., Alm, E., Culley, D., Dehal, P., DeSantis, T., et al. (2008). Environmental genomics reveals a single-species ecosystem deep within Earth. Science 322, 275-278. doi: 10.1126/science.1155495.
9. Cole, J. R., Wang, Q., Fish, J. A., Chai, B., McGarrell, D. M., Sun, Y., et al. (2014). Ribosomal Database Project: data and tools for high throughput rRNA analysis. Nucleic Acids Res. 42, D633-D642. doi: 10.1093/nar/gkt1244.
10. Colwell, F. S., and D'Hondt, S. (2013). Nature and extent of the deep biosphere. Rev. Mineral. Geochem. 75, 547-574. doi: 10.2138/rmg.2013.75.17.
11. Coombs, J. M. (2009). Potential for horizontal gene transfer in microbial communities of the terrestrial subsurface. Horiz. Gene Transf. 532, 413-433. doi: 10.1007/978-1-60327-853-9_24.
12. Cozzarelli, I. M., Weiss, J. V, Hurst, C. J., Crawford, R. L., Garland, J. L., Lipson, D. A., et al. (2007). "Biogeochemistry of aquifer systems," in Manual of Environmental Microbiology, eds C. J. Hurst, R. L. Crawford, J. L. Garland, D. A. Lipson, A. L. Mills, and L. D. Stetzenbach (Washington, DC: ASM Press), 843-859.
13. Darriba, D., Taboada, G. L., Doallo, R., and Posada, D. (2012). jModelTest 2: more models, new heuristics and parallel computing. Nat. Methods 9, 772. doi: 10.1038/nmeth.2109.
14. Delcher, A. L., Phillippy, A., Carlton, J., and Salzberg, S. L. (2002). Fast algorithms for large-scale genome alignment and comparison. Nucleic Acids Res. 30, 2478-2483. doi: 10.1093/nar/30.11.2478.
15. Dupont, C. L., Rusch, D. B., Yooseph, S., Lombardo, M.-J., Richter, R. A., Valas, R., et al. (2012). Genomic insights to SAR86, an abundant and uncultivated marine bacterial lineage. ISME J. 6, 1186-1199. doi: 10.1038/ismej.2011.189.
16. Edgar, R. C. (2004). MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 32, 1792-1797. doi: 10.1093/nar/gkh340.
17. Engelhardt, T., Sahlberg, M., Cypionka, H., and Engelen, B. (2013). Biogeography of Rhizobium radiobacter and distribution of associated temperate phages in deep subseafloor sediments. ISME J. 7, 199-209. doi: 10.1038/ismej.2012.92.
18. Field, E. K., Sczyrba, A., Lyman, A. E., Harris, C. C., Woyke, T., Stepanauskas, R., et al. (2015). Genomic insights into the uncultivated marine Zetaproteobacteria at Loihi Seamount. ISME J. 9, 857-870. doi: 10.1038/ismej.2014.183.
19. Fuhrman, J. A. (1999). Marine viruses and their biogeochemical and ecological effects. Nature 399, 541-548. doi: 10.1038/21119.
20. Gogarten, J., Doolittle, W., and Lawrence, J. (2002). Prokaryotic evolution in light of gene transfer. Mol. Biol. Evol. 19, 2226-2238. doi: 10.1093/oxfordjournals.molbev.a004046.
21. Guindon, S., Dufayard, J.-F., Lefort, V., Anisimova, M., Hordijk, W., and Gascuel, O. (2010). New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0. Syst. Biol. 59, 307-321. doi: 10.1093/sysbio/syq010.
22. Heesakkers, V., Murphy, S., and Reches, Z. (2011). Earthquake rupture at focal depth, Part I: structure and rupture of the pretorius fault, TauTona Mine, South Africa. Pure Appl. Geophys. 168, 2395-2425. doi: 10.1007/s00024-011-0354-7.
23. Hoehler, T. M., and Jørgensen, B. B. (2013). Microbial life under extreme energy limitation. Nat. Rev. Microbiol. 11, 83-94. doi: 10.1038/nrmicro2939.
24. Huson, D. H., Mitra, S., Ruscheweyh, H., Weber, N., and Schuster, S. C. (2011). Integrative analysis of environmental sequences using MEGAN4. Genome. Res. 21, 1552-1560. doi: 10.1101/gr.120618.111.
25. Itävaara, M., Nyyssönen, M., Kapanen, A., Nousiainen, A., Ahonen, L., and Kukkonen, I. (2011). Characterization of bacterial diversity to a depth of 1500 m in the Outokumpu deep borehole, Fennoscandian Shield. FEMS Microbiol. Ecol. 77, 295-309. doi: 10.1111/j.1574-6941.2011.01111.x.
26. Iverson, V., Morris, R. M., Frazar, C. D., Berthiaume, C. T., Morales, R. L., and Armbrust, E. V. (2012). Untangling genomes from metagenomes: revealing an uncultured class of marine Euryarchaeota. Science 335, 587-590. doi: 10.1126/science.1212665.
27. Jørgensen, B. B., and D'Hondt, S. (2006). Ecology. A starving majority deep beneath the seafloor. Science 314, 932-934. doi: 10.1126/science.1133796.
28. Jungbluth, S. P., Grote, J., Lin, H.-T., Cowen, J. P., and Rapp, É, M. S. (2013). Microbial diversity within basement fluids of the sediment-buried Juan de Fuca Ridge flank. ISME J. 7, 161-172. doi: 10.1038/ismej.2012.73.
29. Kallmeyer, J., Pockalny, R., Adhikari, R. R., Smith, D. C., and D'Hondt, S. (2012). Global distribution of microbial abundance and biomass in subseafloor sediment. Proc. Natl. Acad. Sci. U.S.A. 109, 16213-16216. doi: 10.1073/pnas.1203849109.
30. Kearse, M., Moir, R., Wilson, A., Stones-Havas, S., Cheung, M., Sturrock, S., et al. (2012). Geneious Basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics 28, 1647-1649. doi: 10.1093/bioinformatics/bts199.
31. Koonin, E. (2001). Horizontal gene transfer in prokaryotes: quantification and classification. Annu. Rev. Microbiol. 55, 709-742. doi: 10.1146/annurev.micro.55.1.709.
32. Krzywinski, M., Schein, J., Birol, I., Connors, J., Gascoyne, R., Horsman, D., et al. (2009). Circos: an information aesthetic for comparative genomics. Genome Res. 19, 1639-1345. doi: 10.1101/gr.092759.109.
33. Kyle, J. E., Eydal, H. S. C., Ferris, F. G., and Pedersen, K. (2008). Viruses in granitic groundwater from 69 to 450 m depth of the Aspö hard rock laboratory, Sweden. ISME J. 2, 571-574. doi: 10.1038/ismej.2008.18.
34. Lampson, B. C., Inouye, M., and Inouye, S. (2005). Retrons, msDNA, and the bacterial genome. Cytogenet. Gen. Res. 110, 491-499. doi: 10.1159/000084982.
35. Lau, M. C. Y., Cameron, C., Magnabosco, C., Brown, C. T., Schilkey, F., Grim, S., et al. (2014). Phylogeny and phylogeography of functional genes shared among seven terrestrial subsurface metagenomes reveal N-cycling and microbial evolutionary relationships. Front. Microbiol. 5, 1-17. doi: 10.3389/fmicb.2014.00531.
36. Lennon, J. T., and Jones, S. E. (2011). Microbial seed banks: the ecological and evolutionary implications of dormancy. Nat. Rev. Microbiol. 9, 119-130. doi: 10.1038/nrmicro2504.
37. Leplae, R., Hebrant, A., Wodak, S. J., and Toussaint, A. (2004). ACLAME: a CLAssification of Mobile genetic Elements. Nucleic Acids Res. 32, D45-D49. doi: 10.1093/nar/gkh084.
38. Lerm, S., Westphal, A., Miethling-Graff, R., Alawi, M., Seibt, A., Wolfgramm, M., et al. (2013). Thermal effects on microbial composition and microbiologically induced corrosion and mineral precipitation affecting operation of a geothermal plant in a deep saline aquifer. Extremophiles 17, 311-327. doi: 10.1007/s00792-013-0518-8.
39. Lin, L.-H., Wang, P.-L., Rumble, D., Lippmann-Pipke, J., Boice, E., Pratt, L. M., et al. (2006). Long-term sustainability of a high-energy, low-diversity crustal biome. Science 314, 479-482. doi: 10.1126/science.1127376.
40. Lippmann, J., Stute, M., Torgersen, T., Moser, D. P., Hall, J. A., Lin, L., et al. (2003). Dating ultra-deep mine waters with noble gases and 36Cl, Witwatersrand Basin, South Africa. Geochim. Cosmochim. Acta 67, 4597-4619. doi: 10.1016/S0016-7037(03)00414-9.
41. Lippmann-Pipke, J., Erzinger, J., Zimmer, M., Kujawa, C., Boettcher, M., Heerden, E., et al. (2011). Geogas transport in fractured hard rock-correlations with mining seismicity at 3.54km depth, TauTona gold mine, South Africa. Appl. Geochem. 26, 2134-2146. doi: 10.1016/j.apgeochem.2011.07.011.
42. Lollar, B. S., Lacrampe-Couloume, G., Slater, G. F., Ward, J., Moser, D. P., Gihring, T. M., et al. (2006). Unravelling abiogenic and biogenic sources of methane in the Earth's deep subsurface. Chem. Geol. 226, 328-339. doi: 10.1016/j.chemgeo.2005.09.027.
43. Lollar, B. S., Lacrampe-Couloume, G., Voglesonger, K., Onstott, T. C., Pratt, L. M., and Slater, G. F. (2008). Isotopic signatures of CH4 and higher hydrocarbon gases from Precambrian Shield sites: a model for abiogenic polymerization of hydrocarbons. Geochim. Cosmochim. Acta 72, 4778-4795. doi: 10.1016/j.gca.2008.07.004.
44. Lollar, B. S., Onstott, T. C., and Ballentine, C. J. (2014). The contribution of the Precambrian continental lithosphere to global H2 production. Nature 516, 379-382. doi: 10.1038/nature14017.
45. Lomstein, B. A., Langerhuus, A. T., D'Hondt, S., Jørgensen, B. B., and Spivack, A. J. (2012). Endospore abundance, microbial growth and necromass turnover in deep sub-seafloor sediment. Nature 484, 101-104. doi: 10.1038/nature10905.
46. Magnabosco, C., Tekere, M., Lau, M. C. Y., Linage, B., Kuloyo, O., Erasmus, M., et al. (2014). Comparisons of the composition and biogeographic distribution of the bacterial communities occupying South African thermal springs with those inhabiting deep subsurface fracture water. Front. Microbiol. 5:679. doi: 10.3389/fmicb.2014.00679.
47. Markowitz, V. M., Chen, I.-M. A., Palaniappan, K., Chu, K., Szeto, E., Grechkin, Y., et al. (2012). IMG: the Integrated Microbial Genomes database and comparative analysis system. Nucleic Acids Res. 40, D115-D122. doi: 10.1093/nar/gkr1044.
48. Markowitz, V. M., Mavromatis, K., Ivanova, N. N., Chen, I.-M. A., Chu, K., and Kyrpides, N. C. (2009). IMG ER: a system for microbial genome annotation expert review and curation. Bioinformatics 25, 2271-2278. doi: 10.1093/bioinformatics/btp393.
49. Marraffini, L. A., and Sontheimer, E. J. (2010). CRISPR interference: RNA-directed adaptive immunity in bacteria and archaea. Nat. Rev. Genet. 11, 181-190. doi: 10.1038/nrg2749.
50. Martinez-Garcia, M., Swan, B. K., Poulton, N. J., Gomez, M. L., Masland, D., Sieracki, M. E., et al. (2012). High-throughput single-cell sequencing identifies photoheterotrophs and chemoautotrophs in freshwater bacterioplankton. ISME J. 6, 113-123. doi: 10.1038/ismej.2011.84.
51. Molin, S., and Tolker-Nielsen, T. (2003). Gene transfer occurs with enhanced efficiency in biofilms and induces enhanced stabilisation of the biofilm structure. Curr. Opin. Biotechnol. 14, 255-261. doi: 10.1016/S0958-1669(03)00036-3.
52. Moser, D. P., Gihring, T., Fredrickson, J. K., Brockman, F. J., Balkwill, D., Dollhopf, M. E., et al. (2005a). Desulfotomaculum spp. and Methanobacterium spp. dominate 4-5 km deep fault. Appl. Environ. Microbiol. 71, 8773-8783. doi: 10.1128/AEM.71.12.8773-8783.2005.
53. Moser, D. P., Gihring, T. M., Brockman, F. J., Fredrickson, J. K., Balkwill, D. L., Michael, E., et al. (2005b). Desulfotomaculum and Methanobacterium spp. dominate a 4-to 5-kilometer-deep fault. Appl. Environ. Microbiol. 71, 8773-8783. doi: 10.1128/AEM.71.12.8773.
54. Moser, D. P., Onstott, T. C., Fredrickson, J. K., Brockman, F. J., Balkwill, D. L., Drake, G. R., et al. (2003). Temporal shifts in the geochemistry and microbial community structure of an ultradeep mine borehole following isolation. Geomicrobiol. J. 20, 517-548. doi: 10.1080/713851170.
55. Narasingarao, P., Podell, S., Ugalde, J. A., Brochier-Armanet, C., Emerson, J. B., Brocks, J. J., et al. (2012). De novo metagenomic assembly reveals abundant novel major lineage of Archaea in hypersaline microbial communities. ISME J. 6, 81-93. doi: 10.1038/ismej.2011.78.
56. Ochman, H., Lawrence, J., and Groisman, E. (2000). Lateral gene transfer and the nature of bacterial innovation. Nature 405, 299-304. doi: 10.1038/35012500.
57. Onstott, T. C., Lin, L.-H., Davidson, M., Mislowack, B., Borcsik, M., Hall, J., et al. (2006). The origin and age of biogeochemical trends in deep fracture water of the Witwatersrand Basin, South Africa. Geomicrobiol. J. 23, 369-414. doi: 10.1080/01490450600875688.
58. Onstott, T. C., Magnabosco, C., Aubrey, A. D., Burton, A. S., Dworkin, J. P., Elsila, J. E., et al. (2014). Does aspartic acid racemization constrain the depth limit of the subsurface biosphere? Geobiology 12, 1-19. doi: 10.1111/gbi.12069.
59. Phelps, T., Murphy, E., Pfiffner, S., and White, D. (1994). Comparison between geochemical and biological estimates of subsurface microbial activities. Microb. Ecol. 335-349. doi: 10.1007/BF00662027.
60. Pruesse, E., Peplies, J., and Glöckner, F. O. (2012). SINA: accurate high-throughput multiple sequence alignment of ribosomal RNA genes. Bioinformatics 28, 1823-1829. doi: 10.1093/bioinformatics/bts252.
61. Richter, M., and Rosselló-Móra, R. (2009). Shifting the genomic gold standard for the prokaryotic species definition. Proc. Natl. Acad. Sci. U.S.A. 106, 19126-19131. doi: 10.1073/pnas.0906412106.
62. Scott, K. (2002). The role of conjugative transposons in spreading antibiotic resistance between bacteria that inhabit the gastrointestinal tract. Cell. Mol. Life Sci. C 59, 2071-2082. doi: 10.1007/s000180200007.
63. Setlow, B., Loshon, C. A., Genest, P. C., Cowan, A. E., Setlow, C., and Setlow, P. (2002). Mechanisms of killing spores of Bacillus subtilis by acid, alkali and ethanol. J. Appl. Microbiol. 92, 362-375. doi: 10.1046/j.1365-2672.2002.01540.x.
64. Setlow, P. (1995). Mechanisms for the prevention of damage to DNA in spores of Bacillus species. Annu. Rev. Microbiol. 49, 29-54. doi: 10.1146/annurev.mi.49.100195.000333.
65. Shapiro, J. A. (2010). Mobile DNA and evolution in the 21st century. Mob. DNA 1:4. doi: 10.1186/1759-8753-1-4.
66. Simon, D. M., and Zimmerly, S. (2008). A diversity of uncharacterized reverse transcriptases in bacteria. Nucleic Acids Res. 36, 7219-7229. doi: 10.1093/nar/gkn867.
67. Stepanauskas, R. (2012). Single cell genomics: an individual look at microbes. Curr. Opin. Microbiol. 15, 613-620. doi: 10.1016/j.mib.2012.09.001.
68. Stepanauskas, R., and Sieracki, M. E. (2007). Matching phylogeny and metabolism in the uncultured marine bacteria, one cell at a time. Proc. Natl. Acad. Sci. U.S.A. 104, 9052-9057. doi: 10.1073/pnas.0700496104.
69. Stevens, T. O., and McKinley, J. P. (1995). Lithoautotrophic microbial ecosystems in deep basalt aquifers. Science 270, 450-455. doi: 10.1126/science.270.5235.450.
70. Swan, B. K., Martinez-Garcia, M., Preston, C. M., Sczyrba, A., Woyke, T., Lamy, D., et al. (2011). Potential for chemolithoautotrophy among ubiquitous bacteria lineages in the dark ocean. Science 333, 1296-1300. doi: 10.1126/science.1203690.
71. Swan, B. K., Tupper, B., Sczyrba, A., Lauro, F. M., Martinez-Garcia, M., Gonzalez, J. M., et al. (2013). Prevalent genome streamlining and latitudinal divergence of planktonic bacteria in the surface ocean. Proc. Natl. Acad. Sci. U.S.A. 110, 11463-11468. doi: 10.1073/pnas.1304246110.
72. Takai, K. E. N., Moser, D. P., Flaun, M. D. E., Onstott, T. C., and Fredrickson, J. K. (2001a). Archaeal diversity in waters from deep South African gold mines. Society 67, 5750-5760. doi: 10.1128/AEM.67.21.5750-5760.2001.
73. Takai, K., Moser, D. P., Onstott, T. C., Spoelstra, N., Pfiffner, S. M., Dohnalkova, A., et al. (2001b). Alkaliphilus transvaalensis gen. nov., sp. nov., an extremely alkaliphilic bacterium isolated from a deep South African gold mine. Int. J. Syst. Evol. Microbiol. 51, 1245-1256. doi: 10.1099/00207713-51-4-1245.
74. Tatusov, R. L., Galperin, M. Y., Natale, D. A., and Koonin, E. V. (2000). The COG database: a tool for genome-scale analysis of protein functions and evolution. Nucleic Acids Res. 28, 33-36. doi: 10.1093/nar/28.1.33.
75. Taylor, C. D., and Wirsen, C. O. (1999). Rapid microbial production of filamentous sulfur mats at hydrothermal vents. Appl. Environ. Microbiol. 65, 2253-2255.
76. Toussaint, A. (2013). Transposable Mu-like phages in Firmicutes: new instances of divergence generating retroelements. Res. Microbiol. 164, 281-287. doi: 10.1016/j.resmic.2013.01.008.
77. Wang, P. W., Chu, L., and Guttman, D. S. (2004). Complete sequence and evolutionary genomic analysis of the pseudomonas aeruginosa transposable bacteriophage D3112. J. Bacteriol. 186, 400-410. doi: 10.1128/JB.186.2.400-410.2004.
78. Wang, Q., Garrity, G. M., Tiedje, J. M., and Cole, J. R. (2007). Naive Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy. Appl. Environ. Microbiol. 73, 5261-5267. doi: 10.1128/AEM.00062-07.
79. Ward, J. A., Slater, G. F., Moser, D. P., Lin, L. H., Lacrampe-Couloume, G., Bonin, A. S., et al. (2004). Microbial hydrocarbon gases in the Witwatersrand Basin, South Africa: implications for the deep biosphere. Geochim. Cosmochim. Acta 68, 3239-3250. doi: 10.1016/j.gca.2004.02.020.
80. Whitman, W. B., Coleman, D. C., and Wiebe, W. J. (1998). Perspective Prokaryotes: the unseen majority. Proc. Natl. Acad. Sci. U.S.A. 95, 6578-6583.
81. Whittle, G., Shoemaker, N., and Salyers, A. (2002). The role of Bacteroides conjugative transposons in the dissemination of antibiotic resistance genes. Cell. Mol. Life Sci. C 59, 2044-2054. doi: 10.1007/s000180200004.
82. Williamson, S. J., Cary, S. C., Williamson, K. E., Helton, R. R., Bench, S. R., Winget, D., et al. (2008). Lysogenic virus-host interactions predominate at deep-sea diffuse-flow hydrothermal vents. ISME J. 2, 1112-1121. doi: 10.1038/ismej.2008.73.