The minimal cell genome: "on being the right size"

‘‘On being the right size’’ is in quotation marks in the title because it has been used twice before–in each case to discuss the size of biological systems in terms of interest at that time. J. B. S. Haldane (1) originally used this title in the 1920s, for a paper describing the relationship between animal size and constraints like gravity, surface tension, and food and oxygen consumption. In the 1970s, N. W. Pirie (2) (acknowledging Haldane) used the title for a paper analyzing how factors like membrane properties, water structure, and the volume needed for ribosomes and other macromolecules set lower limits on cell size. Now, in the 1990s, the title is used to discuss efforts to define the minimal genome content necessary and sufficient for a living cell. The search for the ‘‘smallest autonomous self-replicating entity,’’ which subsequently became the search for the smallest cell genome, was begun in the late 1950s by Harold Morowitz and coworkers (for review, see ref. 3). This led to studies of the mycoplasmas, showing that these microorganisms have the smallest reported cell (4) and genome (5, 6) sizes. The DNA sequence of the smallest known mycoplasma genome, that of Mycoplasma genitalium, recently was determined (7). Now, Mushegian and Koonin (8), in a paper in the current issue of the Proceedings, have carried out an intensive comparative analysis of the M. genitalium sequence with that of Haemophilus influenzae (9), the only other complete bacterial genome sequence presently available, to try to identify the minimal cell gene set and to suggest how this set might be reduced even further to reconstruct the genetic content of the primordial ancestral cell. M. genitalium and H. influenzae provide a unique opportunity for such an analysis because both microorganisms evolved by repeated genome reductions from bacteria with larger genomes, M. genitalium from Gram-positive bacteria and H. influenzae from Gram-negative bacteria. Mushegian and Koonin (8) note that these genome losses and the fact that the Gram-positive and Gram-negative bacteria arose from an ancient phylogenetic divergence [at least 3200 million years (Myr) ago; ref. 10] suggest the genes conserved in both microorganisms should approximate the minimal gene set for a living cell. Using sequence data for the 469 putative protein genes encoded in theM. genitalium genome (8) and 1703 genes in the H. influenzae sequence (11), Mushegian and Koonin (8) constructed a minimal gene set by identifying genes with similar amino acid sequences and functions in both microorganisms (233 genes), adding in genes needed to fill gaps in essential metabolic pathways and not encoded in similar sequences in both microorganisms (23 genes), and subtracting functionally redundant and host-specific genes (6 genes). The resulting 250 genes are presented as an approximation of the minimal gene set necessary and sufficient for a ‘‘modern-type’’ cell. This corresponds to a genome size about 315 kb, using 1.25 kb per gene for small genomes based on theM. genitalium data (7). Mushegian and Koonin (8) also present a strategy for reducing the minimal gene set further, to try to reconstruct the genetic content of the last common ancestor of the Archaea, Bacteria, and Eucarya. The Mushegian and Koonin (8) paper is the latest of several recent efforts to define a minimal genome. In considering these studies, it is important to remember that organisms with small genomes have arisen by two vastly different evolutionary pathways, one ‘‘top down’’ and the other ‘‘bottom up.’’ ‘‘Top down’’ genomes evolved in organisms with increasing metabolic requirements. At each metabolic level, there can be a minimal genome: from photoautotrophs, able to grow in a medium of only CO2, light, and inorganic salts; to simple heterotrophs, for which growth requires a basic medium containing an organic carbon and energy source (like glucose) and inorganic salts; to fastidious heterotrophs, for which growth requires a complex medium, frequently containing undefined components (like serum); to obligate intracellular parasites, only able to grow within eukaryotic host cells; and, finally, to organelles (i.e., mitochondria and chloroplasts) that have lost almost all bacterial genes and become eukaryotic cytoplasmic organelles. ‘‘Bottom up’’ genomes refer to the genetic contents of the ‘‘organisms,’’ postulated by different investigators, to have arisen during the origin and evolution of cells. In the absence of a plausible alternative, small RNA oligonucleotides are believed to have been the primordial informational macromolecules (for review, see refs. 12 and 13). The first ‘‘organism’’ in the RNA world probably contained a population of similar, but not identical, self-replicating RNA oligonucleotides with relatively broad catalytic specificity. This presumably led to an organism in which genetically encoded RNA directed synthesis of a protein–designated the ‘‘breakthrough organism’’ by Steven Benner and coworkers (14). Subsequently, selection must have produced organisms with increasing numbers of different RNAs and proteins, but inaccurate replication and translation systems would have limited the size of these macromolecules to minigenes (perhaps 50–100 nt) and miniproteins (perhaps 20 residues or shorter) (12). This organism, containing populations of heterogeneous minigenes and miniproteins subject to natural selection, probably corresponds to the ‘‘progenote’’ hypothesized by Carl Woese (15): ‘‘a theoretical construct, an entity that, by definition, has a rudimentary, imprecise linkage between its genotype and phenotype.’’ Eventually, more accurate replication and translation systems must have evolved, allowing evolution of larger DNA genes and proteins, comparable in size to modern organisms. An organism containing these larger DNA genes and proteins would have been the last common ancestor of the Archaea, Bacteria, and Eucarya–designated the ‘‘protogenote’’ by Steven Benner and Andrew Ellington (16) and the ‘‘ancestral cell’’ by Christian de Duve (12). It is not evident exactly when DNA would have arisen during this progression. Assembly of minigenes into longer genes, and eventually genomes, can be visualized most easily in an RNA world in which RNA splicing could be utilized. DNA biochemistry may have been present as early as the breakthrough organism but not selected for until the transition from progenote to ancestral cell. As noted by Christian de Duve (12): ‘‘In other words, life may have played with DNA for a long time in what might be called a halfhearted way and have adopted it definitely only when the need for it made itself felt through selective benefits.’’