Claire M. Fraser, Jeannine D. Gocayne, Owen White, Mark D. Adams, Robert D. Fleischmann, Rebecca Clayton, Kenneth F. Bott**, Hamilton O. Smith***, Clyde A. Hutchison III**, and J. Craig Venter
The Institute for Genomic Research, Gaithersburg, MD, Johns Hopkins University, Baltimore, MD and University of North Carolina, Chapel Hill, NC.
Mycoplasmas are small wall-less bacteria that parasitize a wide range of hosts including humans, animals, plants, insects, and tissue culture,[1,2] and are believed to represent a minimalist life form, having yielded to selective pressure to reduce genome size to eliminate unnecessary genes. M. genitalium currently has the smallest genome of any free-living organism (580 kb) and is believed to have evolved from ancestors common to higher gram positive organisms.[3,4] A new approach[5] for genome analysis based on whole chromosome shotgun sequencing and assembly has been successfully applied to obtain the complete nucleotide sequence (580,070 bp) of the genome of M. genitalium. A small insert (2 kb) M. genitalium genomic DNA library was constructed in pUC18 vector. A total of 5760 double-stranded DNA templates were prepared with a modified "boiling bead" method from AGTC (Gaithersburg, MD). Templates were sequenced from both ends using M13 forward (M13-21) and M 13 reverse (M 13RP1) primers; a total of 8472 good sequences were obtained. Assembly of the M. genitalium genome was accomplished with the TIGR Assembler which simultaneously clusters and assembles the genome. The 8472 sequences assembled into 39 contigs that ranged in size from 705 to 84,124 bp. For closure, the edited length of the sequences at the ends of the contigs was increased and the contigs were searched against each other. This process closed 11 gaps. The remaining 28 sequence gaps were closed by a single primer walk across the gaps. Analysis of putative open reading frames suggests that the M. genitalium genome contains approximately 470 genes. A complement of genes involved in DNA maintenance, repair, transcription, translation, and cellular transport are present; however, no pathways for amino acid, fatty acid, purine or pyrimidine biosynthesis were identified. Comparison of the M. genitalium genome to that of Haemophilus influenzae5 suggests that differences in genome content are reflected as profound differences in physiology and metabolic capacity between these two organisms.
* Supported in part by a Department of Energy Cooperative Agreement DE-FC02-95ER61962.A000
** University of North Carolina, Chapel Hill, NC.
*** Johns Hopkins University, Baltimore, MD.
[1]S. Razin, Microbiol. Rev. 49, 419 (1985).
[2]J. Maniloff, Mycoplasmas: molecular biology and pathogenesis, J. Maniloff et al., Eds. (American Society for Microbiology, Washington, D.C., 1992), pp.549-559.
[3]S.D. Colman, et al., Mol. Microbiol. 4, 683 (1990)
[4]C. Su and J.B. Baseman, J. Bacteriol. 172, 4705 (1990).
[5]R. Fleischmann et al., Science 269, 496 (1995).