Mycoplasma mobile Project Information

Contents

Additional information

Who are we?

The Mycoplasma mobile genome project is a collaboration between George Church and the Broad Institute.

George Church, Professor at the Department of Genetics and Director of the Harvard-Lipper Center for Computational Genetics at Harvard Medical School (Cambridge, Massachusetts) leads an inter-disciplinary team of experts in the fields of biology, biophysics, computer science, mathematics, and technology development. Dr. Church's research group focuses on integrating biosystems-modeling with high-throughput data for haplotypes, RNA arrays, proteomics, and metabolites. The ultimate goal is to achieve more accurate and automated genomic biomedical and ecological engineering.

The Eli and Edythe L. Broad Institute is a partnership among MIT, Harvard and affiliated hospitals and the Whitehead Institute for Biomedical Research. Its mission is to create the tools for genomic medicine and make them freely available to the world and to pioneer their application to the study and treatment of disease.

Questions about the project should be directed to annotation-info@broad.mit.edu.

What is Mycoplasma mobile?

Mycoplasmas are members of the class Mollicutes, a large group of bacteria that lack a cell wall and have a characteristically low G+C content (Razin et al. 1998). These diverse organisms are parasites in a wide range of hosts, including humans, animals, insects, plants, and cells grown in tissue culture (Razin et al. 1998). Aside from their role as potential pathogens, Mycoplasmas are of interest because they evolved from Gram-positive eubacteria by a drastic reduction of genome size, resulting in the loss of many biosynthetic abilities. With genome sizes smaller than 1 Mb, they have been described as the "smallest free-living organisms", and are considered to be the best representatives for the concept of a minimal cell (Mushegian and Koonin 1996).

When this project first started, the complete nucleotide sequences of seven Mycoplasma genomes were known: Mycoplasma genitalium (Fraser et al. 1995), Mycoplasma pneumoniae (Himmelreich et al. 1996), Ureaplasma urealyticum (Glass et al. 2000), Mycoplasma pulmonis (Chambaud et al. 2001), Mycoplasma penetrans (Sasaki et al. 2002), Mycoplasma mycoides subsp. mycoides (Westberg et al. 2004) and M. gallisepticum (Papazisi et al. 2003). Additional species were already subject to extensive sequencing efforts. Global transposon mutagenesis had been used with M. genitalium and M. pneumoniae, two (relatively) closely related Mycoplasma species, to identify nonessential genes in an effort to find the minimal set of genes required to sustain independent life under laboratory growth conditions (Hutchison et al. 1999).

Why do we sequence the Mycoplasma mobile genome?

We have chosen Mycoplasma mobile as the next genome to be added to the growing list of sequenced Mycoplasmas because (a) it represents a model system for genetics and proteomics analysis of pathogens and for comparative genomics, and (b) it is a platform for studying minimal genomes. M. mobile has the following attractive features:

  • M. mobile is a non-pathogenic organism, yet shares many of the simple features found in the pathogenic species. Research on this organism can be performed without the additional regulatory and safety issues.
  • M. mobile has a unique environmental preference: Isolated from a fresh-water fish, the tench, it is the first instance of a Mycoplasma which colonizes an aquatic organism.
  • M. mobile glides much faster and more robustly than any other Mycoplasma known to date. The availability of its genome sequence provides a powerful tool for comparative genomics targeting the identification of genes necessary for locomotion.
  • M. mobile is not too closely related with M. genitalium and M. pneumoniae, the two species which were used as basis for the definition of the minimal genome (Hutchison et al. 1999), but it is a member of the same genus. Its genome sequence, combined with other Mycoplasma genome sequences, would be extremely valuable for the identification of a consensus minimal genome.

Sequencing and assembling the Mycoplasma genome

Our strategy involved whole genome shotgun (WGS) sequencing, in which sequence from the entire genome is generated and reassembled.

Plasmid libraries with average insert sizes of 2 kb, 4 kb, 6 kb, 8 kb and 10 kb, respectively, were prepared from randomly sheared and size-selected DNA. During the initial phase (shotgun phase) of this WGS project, we sequenced both ends from these cloned inserts to a sequence coverage of greater than 10X. Assemblies were generated with a variety of combinations of read types and assembly parameters using ARACHNE, a software package developed at the Broad Institute, and the optimal assembly was selected for finishing. The optimal assembly was generated with a total of 16,376 reads derived from the 2 kb, 4 kb, 6 kb, 8 kb and 10 kb inserts and yielded approximately 13.4-fold sequence coverage of the Mycoplasma genome (12.0-fold coverage in bases with a PHRED quality of >=20), with 10 gaps spanned by plasmid clones and 8 unspanned gaps. To supplement regions of low coverage and to obtain sequence for the unspanned gaps, additional paired-end reads from all 5 plasmid libraries were incorporated into the genome.

A combination of standard finishing methods including transposon-mediated sequencing and PCR was applied to close gaps and to resolve regions of low sequence quality. The finished M. mobile genome assembly was validated by PCR, by pulsed-field gel electrophoresis of genomic DNA which has been digested with AvrII, BamHI, BsmBI and BstEII, respectively, and by comparison of virtual restriction enzyme digests (ApaI, BamHI, MluI and NruI) of the assembly to a previously generated physical map of the Mycoplasma mobile genome (Bautsch 1988).

Our immediate goal was to produce a highly accurate whole genome-based analysis of this organism. The availability of this sequence in an annotated form will promote discovery of genes, permit reconstruction of pathways and enable comparative genomic approaches to analysis.

Releases

Release 1:

The finished genome sequence of Mycoplasma mobile has been annotated. The complete sequence and annotation is available for BLAST search and sequence download. Our sequence and annotation data has been deposited in GenBank under accession number AE017308.

References

Bautsch, W. 1988. Rapid physical mapping of the Mycoplasma mobile genome by two-dimensional field inversion gel electrophoresis techniques. Nucleic Acids Res. 16: 11461-11467.

Chambaud, I., Heilig, R., Ferris, S., Barbe, V., Samson, D., Galisson, F., Moszer, I., Dybvig, K., Wroblewski, H., Viari, A., Rocha, E.P.C., and Blanchard, A. 2001. The complete genome sequence of the murine respiratory pathogen Mycoplasma pulmonis. Nucleic Acids Res. 29: 2145-2153.

Fraser, C.M., Gocayne, J.D., White, O., Adams, M.D., Clayton, R.A., Fleischmann, R.D., Bult, C.J., Kerlavage, A.R., Sutton, G., Kelley, J.M., Fritchman, J.L., Weidman, J.F., Small, K.V., Sandusky, M., Fuhrmann, J., Nguyen, D., Utterback, T.R., Saudek, D.M., Phillips, C.A., Merrick, J.M., Tomb, J.-F., Dougherty, B.A., Bott, K.F., Hu, P.-C., Lucier, T.S., Peterson, S.N., Smith, H.O., Hutchison III, C.A., and Venter, C. 1995. The minimal gene complement of Mycoplasma genitalium. Science 270: 397-403.

Glass, J.I., Lefkowitz, E.J., Glass, J.S., Heiner, C.R., Chen, E.Y., and Cassell, G.H. 2000. The complete sequence of the mucosal pathogen Ureaplasma urealyticum. Nature 407: 757-762.

Himmelreich, R., Hilbert, H., Plagens, H., Pirkl, E., Li, B.-C., and Herrmann, R. 1996. Complete sequence analysis of the genome of the bacterium Mycoplasma pneumoniae. Nucleic Acids Res. 24: 4420-4449.

Hutchison III, C.A., Peterson, S.N., Gill, S.R., Cline, R.T., White, O., Fraser, C.M., Smith, H.O., and Venter, J.C. 1999. Global transposon mutagenesis and a minimal Mycoplasma genome. Science 286: 2165-2169.

Mushegian, A.R. and Koonin, E.V. 1996. A minimal gene set for cellular life derived by comparison of complete bacterial genomes. Proc. Natl. Acad. Sci. USA 93: 10268-10273.

Papazisi, L., T.S. Gorton, G. Kutish, P.F. Markham, G.F. Browning, D.K. Nguyen, S. Swartzell, A. Madan, G. Mahairas, and S.J. Geary. 2003. The complete genome sequence of the avian pathogen Mycoplasma gallisepticum strain R(low). Microbiology 149: 2307-2316.

Razin, S., Yogev, D,, and Naot, Y. 1998. Molecular Biology and Pathogenicity of Mycoplasmas. Microbiol. Mol. Biol. Rev. 62: 1094-1156.

Sasaki, Y., Ishikawa, J., Yamashita, A., Oshima, K., Kenri, T., Furuya, K., Yoshino, C., Horino, A., Shiba, T., Sasaki, T. and Hattori, M. 2002. The complete genomic sequence of Mycoplasma penetrans, an intracellular bacterial pathogen in humans. Nucleic Acids Res. 30: 5293-5300.

Westberg, J., Persson, A., Holmberg, A., Goesmann, A., Lundeberg, J., Johansson, K.-E., Pettersson, B., and UhlÚn, M. (2004). The Genome Sequence of Mycoplasma mycoides subsp. mycoides SC Type Strain PGT, the Causative Agent of Contagious Bovine Pleuropneumonia (CBPP). Genome Research 14: 221-227.