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National Research Council (US) Steering Group for the Workshop on Size Limits of Very Small Microorganisms. Size Limits of Very Small Microorganisms: Proceedings of a Workshop. Washington (DC): National Academies Press (US); 1999.
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Size Limits of Very Small Microorganisms: Proceedings of a Workshop.
Show detailsMonica Riley
Woods Hole Marine Biological Laboratory
Abstract
The size of Earth-bound bacteria is dictated by a number of factors, the most important of which is the growth rate. If we postulate that ancient cells had very slow growth rates and therefore required few ribosomes, and if they lived in nutritionally rich surroundings, thus requiring few biosynthetic enzymes, they could conceivably be as small as a 200-nanometer-diameter sphere. More drastic scenarios, such as supposing a one-polymer information system or a sharing system for part-time genes, could reduce the size requirement further.
Introduction
We are asking ourselves what, from the point of view of traditional microbiology and biochemistry, are the factors that dictate a lower limit to the size of microorganisms of the kind that are present in our life system? The question has different answers depending on the composition of the environment of the microorganism, but it is possible to consider in turn major factors that influence the estimate.
The Size and Contents of an Average Gram-Negative Organism
Escherichia coli is a typical gram-negative rod bacterium. Its dimensions are those of a cylinder 1.0-2.0 micrometers long, with radius about 0.5 micrometers. Another gram-negative rod, less metabolically independent than E. coli, is Hemophilus influenzae, which has half the length and diameter. Bacteria such as mycoplasma, which have a more modest metabolic capability, are even smaller.
The Effect of Growth Conditions on Cell Constituents
If the environment of the culture of microorganisms is rich, the growth rate will be fast relative to the growth rate in minimal medium. The distribution of contents of E. coli changes with growth rate (1-4) as shown in the Table 1.
Table 1
Cell Contents at Fast and Slow Growth Rates (Percent by Weight).
The cytoplasm, which contains the soluble proteins and the protein synthesis apparatus, is the dominant space-filling aspect of the microbial cell. Ribosomes dominate the cytoplasm and are major space-occupying cellular elements. Compared to the cytoplasmic proteins and the ribosomes, cellular ingredients that do not occupy much volume are the DNA, the cell wall, and the membranes with their component transport systems. Nucleoids are composed of compacted DNA neutralized with either positive ions or basic proteins. The DNA is so compacted that within the nucleoid the concentration of DNA reaches 10-20% and constitutes only about 10% of cell volume.
Distribution of Genetic Determinants of E. coli K-12
The genetic determinants of E. coli K-12 distribute as follows:
Enzymes | 1,300 |
Transporters | 500 |
Regulators | 500 |
RNAs | 115 |
Structure | 200 |
Factors | 25 |
Unknown | 1,800 |
The numbers and kinds of genes expressed at any one time are determined by the composition of the environment. Enzymes are repressed unless needed, induced when needed. Numbers of ribosomes are determined by environmental conditions (1,3).
Essentials of Conceptually Pared Down Average Cell Like E. Coli
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.
Summary
Can we think, then, about a very early cell with an enclosing membrane that has fewer transporters than at present, only partially controls transport in and out, and contains only a few ribosomes? It would contain only about 300 proteins including enzymes, a small number of generalized regulators, and perhaps no DNA, only RNA performing tasks of coding, processing, and replication. Such a cell would need to have a volume of at least 200 nanometers#03>3. To imagine smaller cells than this requires a higher level of speculation, such as having only part-time genes that come and go, or having a one-polymer system where RNA is possessed of adequate catalytic powers to be able to substitute for proteins (7), or where proteins can replicate themselves without a polynucleotide template.
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- Correlates of Smallest Sizes for Microorganisms - Size Limits of Very Small Micr...Correlates of Smallest Sizes for Microorganisms - Size Limits of Very Small Microorganisms
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