Panel 2 (Continued)

Abstract

The smallest members of the domain bacteria known to date are found in the following phylogenetic groups: Proteobacteria, Chlamydia, gram-positive bacteria, spirochetes, and Verrucomicrobia. The spirochetes contain very thin bacteria with some species having cell diameters of about 0.1 to 0.15^mm that are at least 5 to 6^m m in length. Apart from this group, the author is not aware that any of other phylogenetic groups produce cells or buds that are less than 0.2 to 0.25^m m in diameter. Likewise, buds, baeocytes, resting, and dispersal stages such as spores and cysts are not known to be less than 0.25^m m in diameter.

Subcellular bacterial structures, such as fimbriae, gas vesicles, prosthecae, and stalks may be as small as 5 to 10 nm in diameter. Some of these are released from cells into environments and may become fossilized. However, the author is not aware that any such structures have ever been reported as fossils even though the remnants of some structures, such as the heavily encrusted stalk of Gallionella, would appear to be excellent candidates for this. The search for and verification of fossils of small, single-celled microorganisms and subcellular microbial structures is warranted.

"Metallogenium" is the name given to a structure of microbial size found in the hypolimnion of lakes. This heavily salified rosette structure has been regarded as a bacterium by some, but current evidence suggests that it is non-cellular.

Introduction

Prokaryotic cells show a tremendous range in size. The largest known bacterium is Thiomargarita, the denitrifying sulfur-oxidizer found off the west coast of southern Africa; its cells are over 500^m m in diameter. However, such large cell sizes are a rarity in the prokaryotic world.

Certain physical constraints dictate the minimum size of an organism. All cells have a cell membrane, cytoplasm, ribosomes, and nuclear material. Cell membranes are about 8 to 10 nm thick, and sufficient DNA, ribosomes, and enzymes are needed for cells to metabolize and reproduce.

The cell size of many bacterial species is variable, being influenced by growth conditions. Actively growing cells of bacteria are typically larger than senescent cells, and starving cells may be very small indeed. In fact, it is possible that starving cells may turn over so much of their cell matter that they are no longer able to reproduce, and therefore persist in the environment as nanocarcasses less than 0.2^mm in size. From a macromolecular perspective, these organisms would be expected to be depleted in RNA and protein, but rich in DNA. The finding of high concentrations of DNA in particulate materials from natural oligotrophic environments (e.g., Holm-Hansen et al., 1968) is a likely indication that many of the bacteria in such environments are either growing at very low rates or not growing at all.

Also, the effects of physical parameters may be very important in determining cell sizes. Factors such as gravity, pressure, pH, and temperature may influence cell sizes during evolution and selection.

Of course, if the question is, what is the smallest size of a living entity, then bacteria may not be our best example. It is possible that the smallest living entities are precellular. Thus, if life-forms on other planets are different from those on Earth, bacteria may not be the ideal model for comparison.

Selective Advantages of Small Size

Fossil and geochemical records indicate that microorganisms have existed successfully on Earth for more than 3.5 Ga. Indeed, they have persisted despite the evolution of morphologically complex macroorganisms. This observation suggests that there are certain selective evolutionary advantages of small size. Conceivably, small organism sizes could be selected because of (a) the exploitation of niches found in microenvironments, (b) parasitism, (c) oligotrophy, and (d) production of small reproductive cells and spores. Each of these potential selective advantages is discussed briefly below.

Exploitation of Niches Found in Microenvironments

Abundant evidence indicates that microorganisms flourish in microenvironments that are too small to be exploited by macroorganisms. For example, narrow vertical gradients of sulfide and light found in intertidal marine sediments have selected for microbial mat communities structured in millimeter-thick strata. Likewise, anaerobic sediment gradients in which alternate electron acceptors exist are dominated by various bacterial groups involved in fermentations and anaerobic respirations. The microbial loop, which consists of various microbial groups that ingest and degrade microorganisms and small detritus particles, is another example of a microenvironment. However, although these microenvironments are small, they are not of nanometer size, and there are no specific examples of microorganisms less than 0.1 to 0.25^mm in diameter that are known to occupy such a habitat.

Parasitism

Parasites rely on host organisms for materials and in some cases even energy generation. Thus, parasites do not need genes that code for materials and functions provided by the host. Examples of such host-dependent, degenerate bacteria include the obligately intracellular parasites Rickettsia and Chlamydia. Chlamydia species produce special elementary reproductive bodies in cells that can be as small as 0.2^mm in diameter, somewhat smaller than cells of Rickettsia spp.

Another small parasitic bacterium is Bdellovibrio, which has a typical gram-negative cell wall. This Proteobacterium is about 0.25^mm in diameter and about 0.5^mm in length. It is a parasite of other gram-negative bacteria.

The mycoplasmas comprise yet another group of small parasitic bacteria. These organisms lack cell walls and may be as small as about 0.2 to 0.3^mm in diameter. It is noteworthy that the mycoplasmas are all host-dependent parasites and pathogens, so they would typically be found associated with larger host organisms. It is much more likely that the host would leave a fossil record than these cell wall-less bacteria.

Oligotrophy

Many natural aquatic and soil environments, such as the pelagic marine water column, have very low concentrations of nutrients. Living in these environments are oligotrophic bacteria that select for organisms with high surface area to volume ratios (SA/V) to enhance nutrient uptake. Because the environment is nutrient limited, oligotrophic bacteria do not need to grow rapidly and therefore do not need to produce large numbers of ribosomes and enzymes. Thus, small organisms that have a high SA/V and few ribosomes and enzymes have a selective advantage in such environments.

Production of Small Reproductive Cells and Spores

Most bacteria divide by binary transverse fission. In this process two cells of comparable size and mirror-image symmetry are produced. The daughter cells receive about half of the material and energy of the parent cell, and the cell diameter remains unchanged throughout the division cycle. One possible strategy for reproduction would be to produce a small reproductive cell that would have the minimal requirements for independent growth. The mother cell in this instance would not commit so much of its resources to reproduction as would be required if the daughter cell were the same size as the parent cell. Two examples of cell division processes, budding and baeocyte production, are known in bacteria that result in the production of cells that are smaller than the parent. In addition, some bacteria produce special hardy cells referred to as endospores, cysts, or exospores that may be smaller than the parent cell.

Buds and Baeocytes. Many bacteria produce buds. Examples of budding bacteria are reported in the phylogenetic groups Proteobacteria (e.g., Hyphomicrobium, Prosthecomicrobium, Ancalomicrobium, Gemmiger, etc.) and Planctomycetes (Pirellula, Planctomyces, Gemmata, and Isosphaera). However, in all groups reported above, the cell size of the mother cells is quite large, so although the bud diameters are smaller, they are still greater than 0.2^mm in diameter when they separate from their mother cells (Bergey's Manual, 1989).

Some Pleurocapsaen cyanobacteria undergo multiple fission to produce small cells referred to as baeocytes. However, those that have been reported are more than 1.0^mm in diameter (Waterbury and Stanier, 1978).

Endospores, Cysts, and Exospores. Endospores are special survival cells produced by some gram-positive bacteria, particularly those that live in sediments, soil, and rock environments. The classical genera Bacillus and Clostridium are best known for endospore production, but others such as Sporo Bacillus also are known. The endospore contains DNA, ribosomes, and several layers of wall material referred to as a coat. The mature endospore is dehydrated and contains high concentrations of calcium and dipicolinic acid. Usually the endospore is somewhat smaller in diameter than its vegetative mother cell, but in some cases, such as Clostridium tetani (which causes tetanus), it is actually larger. However, none of the endospores reported is less than 0.25 ^mm in diameter (Bergey's Manual, 1986).

Cysts are produced as resting stages by some gram-negative bacteria found in soils. Azotobacter species are one example. The myxobacteria also produce cysts termed microcysts or microspores. Cysts of both of these Proteobacterial groups are relatively large, ultimately larger than 0.25 ^mm, because they are formed from a vegetative cell by the addition of extra layers outside the cell wall.

Exospores or conidiospores are produced by many of high mol% G + C gram-positive bacteria such as Streptomyces spp. These specialized cells are produced in the aerial mycelium as a resistant dispersal reproductive cell. They are about the same diameter as the filament diameter, greater than 0.5 ^mm (Bergey's Manual, 1989).

Other Small Free-living Organisms

A recently discovered small bacterium is a member of the division Verrucomicrobia, one of the major, more recently described phylogenetic groups of the Bacteria (Hedlund et al., 1996). This anaerobic free-living bacterium is about 0.35 ^mm in diameter and 0.5 vm in length (Janssen et al., 1997). Thermoplasma is an example of a small (0.2 ^mm diameter), free-living, cell-wall-less archaeon that is found in natural environments.

Many bacteria form very thin filaments. The spirochetes are one group that contains species whose cell diameters may be 0.1 ^mm. However, the cells are much longer, in excess of 5 ^mm (Bergey's Manual, 1984), so the minimum cell volume is comparable to that of cocci and rods.

Small Subcellular Structures

Small structures have the potential of producing small fossils, although this author is not aware that any of them have been reported as fossils. Candidate structures from contemporary bacteria include prosthecae and stalks that are extensions of the cell and that are smaller than the diameter of the cell. In addition, gas vesicles are very small proteinaceous structures formed by some Bacteria and Archaea. These structures are normally associated with the much larger organism that produces them. However, it is possible that, under some environmental conditions, they could be released from the parent cell and therefore become fossilized in its absence.

Prosthecae

Certain bacteria produce cellular appendages. Those of Caulobacter and Asticcacaulis may be quite narrow, approximately 0.1 ^mm in diameter. Furthermore, under some conditions, these structures can be separated from the cells giving rise to very thin membrane-bound structures that might be mistaken for cells. However, these structures would not be viable and would be expected to occur only rarely in natural environments. The prosthecae of Hyphomicrobium, Pedomicrobium, Ancalomicrobium, and Rhodomicrobium are about 0.2 ^mm in diameter and are less likely to become detached from the cell (Perry and Staley, 1997).

Stalks

Stalks are non-cellular appendages found on some bacteria such as Gallionella and Planctomyces spp. These structures may become encrusted with iron and manganese oxides. Planctomyces stalks are fibrillar consisting of several pilus-sized fibers several m in length that are held together in a fascicle. They are often so fine, less than 0.l ^mm in diameter, that they cannot be observed by light microscopy. However, Gallionella stalks may be much larger and because of encrustation may produce readily observable fossils in excess of 1.0 ^mm in diameter and up to several microns in length.

Gas Vesicles

Gas vesicles are proteinaceous membranes that are produced by many Bacteria and some Archaea. These structures are elongated cylinders with conical tips. They range in diameter from 45 to 200 nm and in length from 100 to more than 800 nm (Walsby, 1994). They are most abundantly produced by cyanobacteria during summer blooms in lakes, but are also produced by some heterotrophic bacteria and halophilic and methanogenic Archaea. Cyanobacterial cells may lyse at the end of a bloom releasing vesicles into the environment where they could become fossilized.

"Metallogenium"

One of the major findings in microbiology in the 20th century was the discovery of microbial fossils. The research of micropaleontologists, Barghoorn and Tyler (1965), revolutionized our thinking. The filamentous fossilized microstructures they found were so compellingly reminiscent of modern day cyanobacteria that their discovery convinced a whole generation of skeptical microbiologists about the existence of microbial fossils.

One of the major difficulties in studying ancient microbial fossils on Earth is that their predicted simple structure makes them difficult to identify. Therefore, we would predict that the first microorganisms would have been unicellular and may have lacked the typical cell wall structure of modern-day Bacteria and Archaea. Fossils of single unicellular bacteria might be very difficult to identify as biological structures. However, fossilized pairs (as cells formed during binary transverse fission) might be more readily recognized as being biological. In any event, fossil hunting in early sedimentary rocks on Earth poses special problems owing not only to the great age of the material, but also to the expected simplicity of the earliest organisms.

Most of the readily recognizable microbial fossils date from 1.0 to about 2.5 Ga bp. Convincing fossils of more ancient microorganisms are not so readily found. One of the more common precambrian fossils closely resembles modern microbial structures that have been named "Metallogenium" (Crerar et al. 1980). However, critical studies that have analyzed the modern-day counterpart that is found in the hypolimnion of lakes have cast doubt on its bacterial nature and/or viability (Klaveness, 1977; Gregory et al.,1980). Nonetheless, the possibility exists that the structure may be formed by microbial activities even though it is not a microorganism itself (Maki et al., 1987). This is an important point to verify in continuing research because, if this is true, its presence in fossilized material would be a signature of microbial life.

Acknowledgments

I appreciate the support of the National Science Foundation and the helpful comments of Brian Hedlund.

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