The minimal number of genes required by a saprophytic microbe living in a nutrient-rich environment was estimated from minimal requirements for metabolism, genome expression, and other essential cellular functions (Panel 1 presenters Fraenkel, Riley); comparisons of sequenced genomes (Lawrence); and the genetic capacity of the smallest known genome, that of Mycoplasma genitalium (Moore, Lawrence). There was general agreement among panelists and discussants that approximately 250 to 450 genes compose the set of minimal essential genes. This is strikingly consistent with the known composition of the genome of M. genitalium, which contains approximately 470 genes, not all of which are essential. On the other hand M. genitalium is an obligate parasite, lacking functions required for independent, saprophytic life. Thus, the upper limit of 450 genes is likely to be conservative.
In the discussion period, questions were raised about possible ways to reduce the genome size even further. Dr. Riley asked whether a cell wall was essential. The role of some kind of relatively rigid cell wall in preventing osmotic lysis and in maintaining cell shape was cited. Respondents noted that mycoplasmas (and bacterial L-forms) lack a rigid cell wall, but that these cells are pleomorphic and are sensitive to osmotic lysis to a greater or lesser degree. However, a cell wall is clearly not essential for cell division, and "naked" cells should be able to exist in an osmotically protective environment. However, as Dr. Ferris commented, if an organism is not spherical (or pleomorphic), it must have some kind of wall or skeletal structure to confer and maintain a defined shape (e.g., rod, spiral).
Drs. Osborn and Fraenkel wondered whether a reduction in the number of ribosomal proteins might be possible, not only eliminating additional genes, but also yielding a significantly smaller, "minimal" ribosome. However, as discussed further below, this was deemed unlikely by panelists.
Dr. de Duve estimated the contribution of the genome to the dry weight of a cell the size of E. coli to be on the order of 4 to 6%. These modest values, however, are almost certain to be underestimated. They should be almost doubled if only one of the two DNA strands is taken to be coding and must be increased further to the extent that the genome contains non-coding DNA. Thus, values of 10 to 15% of dry weight would seem to be acceptable for the E. coli genome.
Dr. Moore's presentation emphasized that, as cell volume decreases, the fraction of volume occupied by the genome increases greatly, and eventually becomes a major determinant of minimal cell size. Calculated as a fraction of cell volume, the E. coli genome represents a negligible contribution (0.013 g/ ml, ca. 1%). However, that fraction rises to nearly 10% of cell volume (0.10 g/ml) in M. genitalium. Dr. Osborn asked at what density of DNA packing the transcription and replication machinery can no longer function. Dr. Moore responded that T4 phage DNA, which is functionally inert, occupies 65% of the available volume. A question was raised as to whether a single-stranded RNA genome might occupy a relatively smaller volume; however, the sense of the panelists was that the complex three-dimensional structures formed by intramolecular base-pairing would not be likely to offer significant advantage in this regard.
Dr. de Duve initially emphasized the importance of the ribosome as a major determinant of minimal cell size, noting that even a single ribosome, if surrounded by membrane and wall, would occupy a sphere of 50 to 60 nm in diameter. Dr. Riley noted that, although E. coli , growing in rich medium, has some 30,000 ribosomes per cell, the number of ribosomes is highly dependent on growth rate. Thus, if one allows the "minimal cell" to have a very long doubling time, the necessary number of ribosomes can