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Global Transposon Mutagenesis and a Minimal Mycoplasma Genome

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Global Transposon Mutagenesis and a Minimal Mycoplasma Genome R EPORTS thermore, while immunosubunit precursors- stained positive, whereas no staining of PA28Ϫ/Ϫ purchased from PharMingen (San Diego, CA). 25D1.16 containing 15S proteasome assembly inter- cells was observed. (10) and a PA28a-specific antiserum (2) were kindly 11. S. C. Jameson, F. R. Carbone, M. J. Bevan, J. Exp. Med. provided by A. Porgador and M. Rechsteiner, respective- mediates were detected in wild-type cells, 177, 1541 (1993). ly. Metabolic labeling, immunoprecipitation, immuno- under identical conditions 15S complexes 12. T. Preckel and Y. Yang, unpublished results. blotting, and sodium dodecyl sulfateÐpolyacrylamide were hardly detectable in PA28Ϫ/Ϫ cells (Fig. 13. M. Ho, Cytomegalovirus: Biology and Infection (Ple- gel electrophoresis (SDS-PAGE) were performed as de- 4B). Notably, PA28 was found to be associ- num, New York, ed. 2, 1991). scribed (3). 14. P. Borrow and M. B. A. Oldstone, in Viral Pathogenesis, 19. T. Flad et al., Cancer Res. 58, 5803 (1998). ated with the immunosubunit precursor-con- N. Nathanson, Ed. (Lippincott-Raven, Philadelphia, 20. M. W. Moore, F. R. Carbone, M. J. Bevan, Cell 54, 777 taining 15S complexes, suggesting that PA28 1997), pp. 593Ð627; M. J. Buchmeier and A. J. Zajac, (1988). is required for the incorporation of immuno- in Persistent Virus Infections, R. Ahmed and I. Chen, 21. A. Vitiello et al., Eur. J. Immunol. 27, 671 (1997). Eds. ( Wiley, New York, 1999), pp. 575Ð605. subunits into proteasomes. 22. H. Bluthmann et al., Nature 334, 156 (1988). 15. G. Niedermann et al., J. Exp. Med. 186, 209 (1997); 23. W. Jacoby, P. V. Cammarata, S. Findlay, S. H. Pincus, It has been demonstrated that immunosub- M. Groettrup et al., J. Biol. Chem. 270, 23808 (1995). J. Invest. Dermatol. 83, 302 (1984). units are required for efficient antigen process- 16. M. Conconi, L. Djavadi-Ohaniance, W. Uerkvitz, K. B. 24. All animal experiments were conducted in accordance ing (1) and that, in vitro, PA28 does not pref- Hendil, B. Friguet, Arch. Biochem. Biophys. 362, 325 with institutional guidelines. We thank S. Sutton, J. (1999). Culver and S. Courtney for technical assistance and J.-F. erentially activate immunoproteasomes and 17. P. C. Ramos, J. Hockendorff, E. S. Johnson, A. Var- Huang, and G. Schoenhals for critical reading. The tech- does not alter proteasome substrate specificity shavsky, R. J. Dohmen, Cell 92, 489 (1998). nical assistance of the DNA, vivarium, and peptide (15). Thus, our findings strongly suggest that 18. Antisera specific to the proteasome subunit C9, LMP2, synthesis facilities of the R. W. Johnson Pharmaceutical the inability to generate immune responses in PA28a, and PA28b have been described (3). Antibodies Research Institute is gratefully acknowledged. Ϫ Ϫ specific to CD4, CD8, CD3-e, TCR-ab, CD23, CD25, PA28 / mice was a consequence of a de- CD28, CD45, CD69, class I, and class II molecules were 2 July 1999; accepted 2 November 1999 creased cellular level of immunoproteasomes. Taken together with the fact that, in PA28- expressing cells, immunoproteasomes have greater stability than proteasomes not contain- Global Transposon Mutagenesis ing immunosubunits (3, 12) and that PA28 interacts with the ␣-subunit ring of the protea- and a Minimal Mycoplasma some (1), we hypothesize that, by inducing conformational changes of the proteasome ␣-subunit ring (3, 16), PA28 promotes immu- Genome noproteasome assembly. PA28 might facilitate Clyde A. Hutchison III,1,2* Scott N. Peterson,1*† Steven R. Gill,1 recruitment and assembly of the immunosub- Robin T. Cline,1 Owen White,1 Claire M. Fraser,1 unit-containing proteasome ␤-subunit ring onto Hamilton O. Smith,1‡ J. Craig Venter1‡§ the ␣-subunit ring, which serves as a scaffold for the proteasome ␤-subunit ring formation Mycoplasma genitalium with 517 genes has the smallest gene complement of and Ump1p-mediated maturation (17). As a any independently replicating cell so far identified. Global transposon mu- consequence of increased cellular level of im- tagenesis was used to identify nonessential genes in an effort to learn whether munoproteasomes, MHC class I antigen pro- the naturally occurring gene complement is a true minimal genome under cessing and presentation are greatly enhanced laboratory growth conditions. The positions of 2209 transposon insertions in by PA28. the completely sequenced genomes of M. genitalium and its close relative M. pneumoniae were determined by sequencing across the junction of the trans- poson and the genomic DNA. These junctions defined 1354 distinct sites of References and Notes 1. K. L. Rock and A. L. Goldberg, Annu. Rev. Immunol. 17, insertion that were not lethal. The analysis suggests that 265 to 350 of the 480 739 (1999); K. Fru¬h and Y. Yang, Curr. Opin. Immunol. protein-coding genes of M. genitalium are essential under laboratory growth 11, 76 (1999). conditions, including about 100 genes of unknown function. 2. C. Realini et al., J. Biol. Chem. 272, 25483 (1997); X. Song, J. Von Kampen, C. A. Slaughter, G. N. De- martino, J. Biol. Chem. 272, 27994 (1997). One important question posed by the avail- quence revealed 480 protein-coding genes 3. K. Ahn et al., J. Biol. Chem. 271, 18237 (1996); Y. ability of complete genomic sequences (1–3) plus 37 genes for RNA species (2). Yang, K. Fru¬h, K. Ahn, P. A. Peterson, J. Biol. Chem. is how many genes are essential for cellular The fraction of nonminimal genomes that 270, 27687 (1995). 4. T. P. Dick et al., Cell 86, 253 (1996); M. Groettrup et life. We are now in a position to approach this is essential for cell growth and division has al., Nature 381, 166 (1996). problem by rephrasing the question “What is been experimentally measured in yeast (12%) 5. Y. Li et al., Immunogenetics 49, 149 (1998). life?” in genomic terms: “What is a minimal and in the bacterium Bacillus subtilis (9%) 6. Supplemental Web figures are available at www. set of essential cellular genes?” (6). The indispensable portion of the B. sub- sciencemag.org/feature/data/1043186.shl. 7. J. R. Knowlton et al., Nature 390, 639 (1997). Interest in the minimal cellular genome tilis genome was estimated to be 562 kb, 8. M. R. Jackson, E. S. Song, Y. Yang, P. A. Peterson, Proc. predates genome sequencing [for a review, close to the size of the M. genitalium genome. Natl. Acad. Sci. U.S.A. 89, 12117 (1992). see (4)]. The smallest known cellular genome Theoretical approaches to defining a minimal 9. Splenocytes were metabolically labeled for 60 min followed by a 60-min chase. NP-40 cell lysates were (5) is that of Mycoplasma genitalium, which gene set have also been attempted. With the incubated at 31¡, 37¡, and 43¡C for 45 min before is only 580 kb. This genome has been com- availability of the first two complete genome being subjected to immunoprecipitation with anti-Kb pletely sequenced, and analysis of the se- sequences (Haemophilus influenzae and M. b or D . The amounts of class I molecules were quan- genitalium) and the assumption that genes tified. It was found that while an equivalent amount of class I molecules was present in wild-type and 1The Institute for Genomic Research, 9712 Medical conserved across large phylogenetic distanc- Ϫ Ϫ PA28 / splenocytes before temperature challenge, Center Drive, Rockville, MD 20850, USA. 2Department es are likely to be essential, a minimal gene Ϫ Ϫ the amount of class I molecules in PA28 / cells of Microbiology and Immunology, University of North ϳ set of 256 genes was proposed (7). after the temperature challenge decreased to 80% Carolina at Chapel Hill, Chapel Hill, NC 27599, USA. of the wild-type levels. Mycoplasma pneumoniae is the closest 10. A. Porgador, J. W. Yewdell, Y. Deng, J. R. Bennink, R. N. *These authors contributed equally to this report. known relative of M. genitalium, with a ge- Germain, Immunity 6, 715 (1997). Ovalbumin-loaded †To whom reprint requests should be addressed. nome size of 816 kb, 236 kb larger than that LPS blasts from wild-type and PA28Ϫ/Ϫ mice were ‡Present address: Celera Genomics, 45 West Gude stained with 25D1.16 and analyzed by flow cytom- Drive, Rockville, MD 20850, USA. of M. genitalium (3). Comparison of the two etry. It was found that ϳ13% of the wild-type cells §To whom correspondence should be addressed. genomes indicates that M. pneumoniae in- www.sciencemag.org SCIENCE VOL 286 10 DECEMBER 1999 2165 R EPORTS cludes orthologs of virtually every one of the (1.8 hits/kb) was about 5.5 times that found in plasma genome, Ureaplasma urealyticum 480 M. genitalium protein-coding genes, plus the portion common to both species (0.33 (http://genome.microbio.uab.edu), consistent an additional 197 genes (8). There is a sub- hits/kb). This result supports our assumption with the idea that they are not essential. stantial evolutionary distance between or- that the M. pneumoniae–specific portion of One approach to estimating the total num- thologous genes in the two species, which the genome is fully dispensable. In addition ber of nonessential M. genitalium genes is to share an average of only 65% amino acid to the species-specific insertions in M. pneu- determine the number of dispensable or- sequence identity. The existence of these two moniae, insertions were observed widely dis- thologs of these genes in M. pneumoniae (see species with overlapping gene content pro- tributed throughout the shared regions of both Table 2). We obtain similar estimates of non- vided an experimental paradigm to test genomes. The conspicuous absence of trans- essential M. genitalium orthologs whether we whether the 480 protein-coding genes shared poson insertions into certain regions expected use data from all genes disrupted in M. pneu- between the species were already close to a to be essential—for example, the region con- moniae (ϳ121 orthologs) or just from those minimal gene set.
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