Not So Much DNA

A minimal free-living cell need not have as much DNA as E. coli does because E. coil is able to live in many different circumstances and carries genes for many more than the minimum number of enzymes required at any one time. If one is thinking about organisms living in a biochemically rich soup or organisms like parasitic bacteria that live inside cells and draw on the processes and contents of inhabited cells, the organism could be smaller than if it were a free-living cell that makes all its own substance from scratch. Presently known smallest bacterial genomes are around 600 Kb of DNA.

Ribosomes

A major element determining the size of bacteria is their ribosomes. Under different growing conditions, E. coli has 10,000-60,000 ribosomes. Each ribosome contains two RNA molecules and a very large number, 52, of different proteins: 21 in the 30S subunit, 31 in the 50S subunit. The protein complex called L7/12 is represented 4 times in each ribosome, but probably the rest of the ribosomal proteins are present only once per ribosome (5). Other proteins are involved in directing the correct assembly of the components of the ribosomes.

Thus, ribosomes are a prominent feature determining minimum cell size in any life system using ribosomes as an obligatory element. However, we can imagine circumstances not requiring as many ribosomes as E. coil has. The number of ribosomes is steeply dependent on growth rate; namely, the slower the growth rate, the fewer ribosomes needed (1,2,4). If we postulate that growth rates of very early, very small cells could have been very slow, ribosomal content could have been correspondingly lower than today's organisms. If we postulate extremely slow growth, can we postulate as few as two ribosomes per cell? This question reaches outside our known world and cannot be answered knowledgeably today.

Proteins

Although some proteins of E. coli are the ribosomal proteins, most are soluble enzymes. Other proteins are integral membrane proteins allowing for cellular localization and carrying out transport functions. Membrane and wall components and also cellular appendages such as flagellae and pili account for a very small part of the weight of a bacterium, so we will leave them aside. Soluble cytoplasmic proteins are chiefly enzymes of metabolism and regulators of all cell processes. The numbers of necessary metabolic enzymes and their regulators required for life depends entirely on the genetic capacities and the metabolic capabilities of the cell.

Metabolism

Quasi-redundancy. E. coli is profligate in its metabolic enzymes. It squanders its information content on multiple enzymes. There are over eighty examples of small-molecule metabolic reactions that are carded out by more than one separately encoded isozyme (6). In addition, there are multiple large-molecule enzymes such as polymerases, helicases, repair enzymes. Whereas E. coil is a versatile organism able to draw on isozymes of slightly different characteristics to guarantee life under different circumstances, our theoretical very small, very early organism may not have been versatile. A minimal organism might have only one enzyme per reaction, and the number of reactions catalyzed may have been small.

"Totipotency." E. coli possesses flexibility and metabolic alternatives as a consequence of containing the enzymes for many metabolic capabilities, only some of which are working under any environmental condition or at any point in time. A minimal organism could make do with minimal metabolic capacity.

Degradation and Energy. All major degradative pathways are in E. coli: glycolysis, pentose shunt, Entner-Douderoff pathway, TCA cycle, and glyoxalate shunt. E. coli has the ability to live anaerobically as well as aerobically, with many other electron acceptors besides oxygen (among them nitrate, sulfate). E. coli can derive energy and make ATP from many oxidizable substances by using either respiration with electron carriers, both aerobic and anaerobic, or fermentation involving only reduced and oxidized organic compounds. E. coli contains the complex formate dehydrogenase system and hydrogenase, enabling it to derive energy from such simple molecules as hydrogen or formate, the simplest organic acid. E. coli can use as carbon and energy sources a wide variety of substrates (although it does not fix CO₂). Thus, the degradative and energy metabolism of E. coli is much more complex than it would be for a minimal organism.

Simple ways a minimal organism might use to gain energy are by fermentation, using one organic molecule as electron donor and another molecule as acceptor (as a chemoorganotroph) or by oxidizing inorganic substances such as H₂S (as a chemoautotroph).

Anabolism. The synthetic capacities of E. coli are also extensive. It can make all of its small-molecule building blocks such as amino acids, purines, pyrimidines, and cofactors, and for some pathways there are alternative mutes using other enzymes. If the necessary building blocks for life were in the environment of a minimal cell, the synthetic metabolic capacity would not be needed. However, one would need transport mechanisms to bring the building blocks in and not leak out essential metabolites.

If, on the other hand, primitive environments did not supply preformed metabolites, then the machinery for CO₂ fixation, perhaps also for N₂ fixation and full anabolic capability would be needed. The complexity and need for more proteins could then rise back up to E. coli levels.

Counting Up

E. coli contains over 1,000 known soluble enzymes, and a high proportion of the 1,800 unknown ORFs [open reading frames (genes likely to code for proteins)] are expected to be enzymes. In a minimal cell with minimal small-molecule metabolism and minimal macromolecule mechanisms, maybe the enzyme count could be reduced to as little as 300.

Reducing the number of proteins also reduces the number of ribosomes needed to synthesize the proteins. Primitive cells with slow growth characteristics could have had far fewer ribosomal RNA molecules and could have used many fewer individual proteins to build primitive ribosomes. Because with slow growth conditions protein (the sum of enzymes and ribosomal protein) is the major component of a cell, reduction in number of unique proteins seems to be a major factor in determining the complexity and size of the cell.

Macromolecule processes such as protein synthesis and DNA replication could have been even simpler in a very small and very ancient cell. What are the minimal needs for these processes in an "RNA world"(7)? Can the machinery be reduced to a much simpler version, eliminating altogether DNA and its replication? Do RNA and protein constitute a two-polymer system? Can we imagine a one-polymer system in which RNA is possessed of adequate catalytic powers to be able to substitute for proteins, or where proteins can replicate themselves without a polynucleotide template?

Envelope

Another factor to think about is the increased surface volume of small cells as compared to large. Although one expects the structures to be much simpler than the envelope of gram-negative bacteria, still a minimal cell would have a higher proportion of its substance in its membrane and wall fractions.