Based on the conjoint analysis of several computational and experimental strategies designed to define the minimal set of protein-coding genes that are necessary to maintain a functional bacterial cell, we propose a minimal gene set composed of 206 genes. Such a gene set will be able to sustain the main vital functions of a hypothetical simplest bacterial cell with the following features.
(i) A virtually complete DNA replication machinery, composed of one nucleoid DNA binding protein, SSB, DNA helicase, primase, gyrase, polymerase III, and ligase. No initiation and recruiting proteins seem to be essential, and the DNA gyrase is the only topoisomerase included, which should perform both replication and chromosome segregation functions.
(ii) A very rudimentary system for DNA repair, including only one endonuclease, one exonuclease, and a uracyl-DNA glycosylase.
(iii) A virtually complete transcriptional machinery, including the three subunits of the RNA polymerase, a σ factor, an RNA helicase, and four transcriptional factors (with elongation, antitermination, and transcription-translation coupling functions). Regulation of transcription does not appear to be essential in bacteria with reduced genomes, and therefore the minimal gene set does not contain any transcriptional regulators.
(iv) A nearly complete translational system. It contains the 20 aminoacyl-tRNA synthases, a methionyl-tRNA formyltransferase, five enzymes involved in tRNA maturation and modification, 50 ribosomal proteins (31 proteins for the large ribosomal subunit and 19 proteins for the small one), six proteins necessary for ribosome function and maturation (four of which are GTP binding proteins whose specific function is not well known), 12 translation factors, and 2 RNases involved in RNA degradation.
(v) Protein-processing, -folding, secretion, and degradation functions are performed by at least three proteins for posttranslational modification, two molecular chaperone systems (GroEL/S and DnaK/DnaJ/GrpE), six components of the translocase machinery (including the signal recognition particle, its receptor, the three essential components of the translocase channel, and a signal peptidase), one endopeptidase, and two proteases.
(vi) Cell division can be driven by FtsZ only, considering that, in a protected environment, the cell wall might not be necessary for cellular structure.
(vii) A basic substrate transport machinery cannot be clearly defined, based on our current knowledge. Although it appears that several cation and ABC transporters are always present in all analyzed bacteria, we have included in the minimal set only a PTS for glucose transport and a phosphate transporter. Further analysis should be performed to define a more complete set of transporters.
(viii) The energetic metabolism is based on ATP synthesis by glycolytic substrate-level phosphorylation.
(ix) The nonoxidative branch of the pentose pathway contains three enzymes (ribulose-phosphate epimerase, ribose-phosphate isomerase, and transketolase), allowing the synthesis of pentoses (PRPP) from trioses or hexoses.
(x) No biosynthetic pathways for amino acids, since we suppose that they can be provided by the environment.
(xi) Lipid biosynthesis is reduced to the biosynthesis of phosphatidylethanolamine from the glycolytic intermediate dihydroxyacetone phosphate and activated fatty acids provided by the environment.
(xii) Nucleotide biosynthesis proceeds through the salvage pathways, from PRPP and the free bases adenine, guanine, and uracil, which are obtained from the environment.
(xiii) Most cofactor precursors (i.e., vitamins) are provided by the environment. Our proposed minimal cell performs only the steps for the syntheses of the strictly necessary coenzymes tetrahydrofolate, NAD+, flavin aderine dinucleotide, thiamine diphosphate, pyridoxal phosphate, and CoA.
Several attempts have been made to try to define the characteristics of a minimal cell. A computer model of a such minimal cell was performed a few years ago and was called the E-CELL Project (81
). The proposed virtual self-surviving cell (SSC) contained only 105 protein-coding genes that allowed the cell to maintain protein and membrane structure. The only functions considered in this virtual cell were glycolysis (using exogenous glucose to obtain ATP), phospholipid biosynthesis (from exogenous fatty acids and glycerol), transcription, and translation. Therefore, this virtual minimal cell is able to maintain metabolic homeostasis but not to reproduce and evolve. Among the 105 genes proposed for SSC, 97 are included in our proposed minimal set. The only differences involve the fmt
gene (methionyl-tRNA formyltransferase) and the genes proposed in SSC for phospholipid biosynthesis, since we have included a different pathway for this purpose. Another computational approach obtained by comparative analysis of 21 complete genomes of bacteria, archaea, and eukaryotes (48
) suggested that a bare-bones set of about 150 genes would be sufficient to maintain basal systems for transcription, translation, and replication, a reduced repair machinery, a small set of molecular chaperones, an intermediate metabolism reduced to glycolysis, a primitive transport system, and no cell wall. Our proposed minimal cell has a similar number of genes devoted to such functions. However, we also included in our minimal set the genes involved in a nonoxidative pentose phosphate pathway, the maintenance of the membrane proton motive force, and nucleotide and coenzyme syntheses, although some of these biosynthetic pathways are not present in some bacteria with reduced genomes. The lack of such pathways reflects that these bacteria are very dependent on their hosts due to an irreversible degenerative process of their metabolic abilities. Therefore, we think they should be present in a hypothetical minimal cell able to perform all the necessary reactions to maintain a minimal and coherent metabolic functionality, although it remains questionable whether such a minimal cell could survive under any realistic conditions.
At any rate, we should accept that there is no conceptual or experimental support for the existence of one particular form of minimal cell, at least from a metabolic point of view. In this sense, our conclusions must be regarded as provisional. Different approaches, ours among others, should converge in several solutions (35
). Finally, one must keep in mind that this kind of research has little relevance for the study of the origin of life, since it is impossible to identify any of the above-mentioned diverse solutions with the one adopted by the more primitive cells (63
). This is especially true in the cases where a bacterium-centered approach is followed, as described in this paper. Any attempt to universalize the conclusions would necessarily include the comparison with archaeal genomes, more specifically the smallest ones (84
). We are sure that future studies will highlight a diversity of minimal ecologically dependent metabolic charts supporting a universal genetic machinery. In a more technological vein, the development of more sophisticated techniques for genomic engineering, together with the continued efforts in defining the minimal gene set, could help to achieve the exciting goal of experimentally constructing a minimal living cell (73