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Project Information

Who Are We?

The Mesoplasma florum genome project is a collaboration between Tom Knight, Senior Research Scientist at the MIT Artificial Intelligence Laboratory and the MIT Electrical Engineering and Computer Science Department, and the Broad Institute of MIT and Harvard. Dr. Knight, a computer architect and electrical engineer, has a strong interest in biologically oriented projects. Recent research projects include implementation of digital logic gates in living bacterial cells, standardized biological component fabrication and testing techniques, novel genetic circuits, biological design and simulation tools, direct design and synthesis of long DNA fragments, and engineering of reduced complexity living cells.

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(at)broad.mit.edu.

What is Mesoplasma florum?

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 of their remarkable evolution:

  1. Extremely small genome size

    Mycoplasmas evolved from Gram-positive eubacteria by multiple reductions in genome size, resulting in the loss of many biosynthetic abilities and the need to acquire many nutrients from their hosts. With genome sizes smaller than 1 Mb, they have been described as the smallest self-replicating organisms, and are considered to be the best representatives for the concept of a minimal cell (Mushegian and Koonin 1996).

  2. Unusual genetic code

    The usual genetic code has been altered: In Mycoplasmas, the codon UGA (= stop codon in the standard genetic code) is translated as tryptophan.

  3. Fast evolution

    Mycoplasmas have high mutation rates, and lack part of the SOS response as well as a considerable number of the DNA repair proteins that exist in E. coli and B. subtilis. This allows rapid adaptation to sudden environmental changes and to host defense mechanisms.

To date, the complete nucleotide sequences of six Mycoplasma genomes have been determined by whole-genome sequencing and assembly: 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), and Mycoplasma penetrans (Sasaki et al. 2002) and Mycoplasma mycoides subsp. mycoides (Westberg et al. 2004). Additional species are subject to extensive sequencing efforts. Global transposon mutagenesis has 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 Mesoplasma florum genome?

Mesoplasma florum represents a model system for genetic and proteomic analysis of pathogens and for comparative genomics, and is a platform for studying minimal genomes.

Attractive features of Mesoplasma florum are:

  1. Nonpathogenic model

    M. florum is a nonpathogenic 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.

  2. Nonmotile species

    M. florum is a nonmotile Mycoplasma species. Thus, its genome sequence, combined with the sequences of M. mobile and other motile Mycoplasmas can aid in the identification of genes that are essential for motility.

  3. Minimal genome

    M. florum is not closely related to M. genitalium and M. pneumoniae, the two species used as basis for the definition of a minimal genome. Therefore, 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 Mesoplasma 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, and a Fosmid library (40 kb inserts) were prepared from randomly sheared and size-selected DNA. During the initial phase (shotgun phase) of this 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 22,631 reads derived from the 2 kb, 6 kb and 8 kb inserts and yielded approximately 18-fold sequence coverage of the Mesoplasma florum genome (16.6-fold coverage in bases with a PHRED quality of >20).

    In the second phase of sequencing (finishing phase) additional sequence was obtained from specific genome regions that were missing from the original assembly or were recognized to be of low quality in the consensus. The final product of this phase is the complete and finished DNA sequence of the Mesoplasma florum genome.

    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

    The finished genome sequence of Mesoplasma florum has been annotated. The complete sequence and annotation is available for BLAST search and sequence download.

    References

    • 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.
    • 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 Uhl?n, 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.

Photo Captions and Credits

The images on the home page are, from left to right:

  1.  Flower of a lemon tree (Citrus limon), from which Mesoplasma florum L1 (ATCC 33453, originally deposited as Acholeplasma florum) was initially recovered. Mesoplasma is thought to be associated with plant insect vectors, although its primary vector is as yet unidentified. Courtesy of Tom Knight
  2. Two agar colonies of Mesoplasma florum exhibiting typical "fried-egg" morphology. Courtesy of Tom Knight
  3. A single slice of a three-dimensional reconstruction of a whole Mesoplasma florum cell, quick-frozen in its growth media across a lacey carbon film without any fixative or stain. The reconstruction was calculated from a series of tilted images recorded in a cryo-electron microscope. Courtesy of Grant Jensen
  4. + 5.  Four frozen-hydrated Mesoplasma florum cells, suspended in vitreous ice around a lacey carbon support; cryo-electron microscopy. California Institute of Technology, Jensen Lab