Size Limits of Very Small Microorganisms Proceedings of a Workshop (1999) / Chapter Skim
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From page 39... ...
Experts with experience in a wide variety of intracellular and extracellular niches, including host cells (Van Etten and Kajander) , aquatic environments (Button and DeLong)
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From page 40... ...
The size ranges shown in Table 2 represent the consensus values reached in the presentations and in ensuing discussions by the assembled group. In many cases it was hard to reach consensus on firm estimates for the smallest organisms or organelles encountered, and the reader is referred to specific arguments in the individual papers.
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From page 41... ...
However, such organisms are not regarded as true nanobacteria, because under nominal growth conditions, they are considerably larger than the diminutive forms discussed here. These larger forms are thought to represent a true evolutionary lower size limit for DNA-based life.
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From page 42... ...
With regard to this question, several strategies were considered by Panel 2 speakers. The first, discussed briefly above, was that of Kajander and Van Etten, in which organisms actually fragment so that each very small organism is incapable of growth, but the population is capable of achieving success.
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From page 43... ...
Introduction Two recent estimates of the minimum genome size required to support life arrived at similar values.
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f 1995 Coliphage Myoviridae E cold 168,800 29 288g 586 Kutter et al., T4 1994 HSV-2 Herpesviridae Human 154,746 74h 2,091 Dolan et al., 1998 AcNPV Baculoviridae Insects 133,894 50 154 890 Ayres et al., 1994 LCDV Iridoviridae Flounder 102,653 40 110 933 Tidona and Darai, 1997 aG, Giant; PBCV-1, Paramecium bursaria chlorella virus 1; MsEPV, Melanoplus sanguinipes entomopoxvirus; MCV, Mol luscum contagiosum virus; ASFV, African swine fever virus; HSV-2, Herpes simplex virus type 2; AcNPV, Autographa californica multinucleocapsid nuclear polyhedroses virus; LCDV, lymphocystis disease virus.
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From page 45... ...
-I- - -- 7 -- -- -- -- -- - -- -- -- -- - - -- - - - -r This gene diversity undoubtedly reflects the natural history of the chlorella viruses. The viruses are ubiquitous in freshwater collected worldwide, and titers as high as 4 x 104 infectious viruses per ml of native water have been obtained (Van Etten et al., 1985; Yamada et al., 1991~.
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From page 47... ...
On the other hand, viruses may provide useful information about the minimum genome size required for the genes to support life. In Table 1, we have calculated the average length of a virus gene by dividing the genome size by the number of putative genes.
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From page 48... ...
However, the finding that, on average, virus genes can be 10 to 50% smaller than those from bacteria indicate that the minimum genome size required to support life may be smaller than previously thought. Acknowledgments I thank Les Lane, Mike Nelson, Myron Brakke, and Mike Graves for their comments on this manuscript and Dan Rock and Gisela Mosig for the information on MsEPV virus and coliphage T4, respectively.
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From page 49... ...
1989. Chlorella viruses contain linear nonpermuted double-stranded DNA genomes with covalently closed hairpin ends.
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From page 50... ...
The theoretical minimum diameter of a cell, based on the size of those macromolecules now considered to be necessary for a living cell, has been calculated to be about 0.14 ~m (Himmelreich et al., 1996; Mushegian and Koonin, 1996~. Some nanobacterial cells appear smaller than that.
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From page 51... ...
So, nanobacterial cell walls do have typical gramnegative components, although their ultrastructure is unique and varies during their growth phases.
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Conventional culture methods do not support the growth of L-form microbes. L-forms can pass through sterile filters but can be easily lysed and their nucleic acids and proteins extracted (Darwish et al., 1987~.
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From page 53... ...
Small size is not directly linked to the genomic size: Myxococcus xanthus genome size 9.4 Mb (Chen et al., 1990) is among the largest, whereas mycoplasmas have the smallest genome sizes, 0.58-1.6 Mb (Barley and Borchsenius, 1991~.
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From page 54... ...
Hoescht 33258 staining indicated that nanobacteria should have DNA amounts between that of mycoplasmas and mitochondria. Can bacteria have novel nucleic acids contributing to smallness?
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From page 55... ...
1991. Continuous distribution of Mycoplasma genome sizes.
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From page 56... ...
Methods The light scatter attending single cells can be analyzed by flow cytometry, separating the bacteria from one another hydrodynamically and from debris according to the intensity of DNA-specific stains. Because the DNA content is large, from 1.5 to 8 fg/cell for most aquatic bacteria, there is little interference from debris and other organisms, and observed signals are thought to emanate almost entirely from the bacteria.
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From page 57... ...
Assuming this smallest component is either non-bacterial or at the most 2% of the biovolume, the smallest known bacteria are of the order of 0.4 rim in diameter with the possible exception of Nanobacterium sanguineum discussed in the proceedings from this workshop. Bacterial Size and Genome Size According to flow cytometry data, genome sizes of cultivated aquatic bacteria range upward from the lowest known for a conventional isolate, 1.6 Mb for Pseudomonas LBrevandimonas]
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From page 58... ...
. Moreover, while significant changes in bacterial size are often reported with depth, cell sizes by flow cytometry are relatively constant from the surface to several thousand meters where minimal grazing is expected.
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From page 59... ...
The cost of additional permeases in cytoplasmic enzymes is less certain since less degradative enzymes may be required, and the excess requirement could be offset by the need for fewer anabolic enzymes. The trend toward smaller dry mass with more limited substrate can be seen from a few examples.
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From page 60... ...
. On the other hand, about half are autoradiographypositive for single amino acids, and small cells sorted by flow cytometry, following incubation with radiolabeled amino acids alone, were as radioactive as the larger fraction, indicating that the small cells are not slowly losing biomass and activity.
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From page 61... ...
(1998) , Determination of the biomasses of small bacteria at low concentration in a mixture of species with forward light scatter measurements by flow cytometry.
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From page 62... ...
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.
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From page 63... ...
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 rim in diameter.
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From page 64... ...
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.
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From page 65... ...
, 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.
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From page 66... ...
1968. Measurement of deoxyribonucleic acid in the ocean and its ecological significance.
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From page 67... ...
1994. Gas vesicles.
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From page 68... ...
The volume of cells in the same culture may vary by more than four orders of magnitude. The smallest cell sizes observed in hyperthermophilic archaea are rods 0.17 rim in diameter in Thermoftlum, spheres 0.3 rim in diameter protruding from rod-shaped cells of Thermoproteus and Pyrobaculum, and disks 0.2 to 0.3 rim in diameter and 0.08 to 0.1 rim wide in Thermodiscus and Pyrodictium.
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From page 69... ...
A great many exhibit a chemolithoautotrophic mode of nutrition: inorganic redox reactions serve as energy sources, and CO2 is the only carbon source required to build up organic cell material (Table 2~. Depending on the organisms, hyperthermophiles are able to use H2, ferrous iron, and reduced sulfur compounds as electron donors.
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From page 70... ...
An unanticipated variation of cell sizes can be observed within pure cultures of members of the Thermococcales, Desulfurococcales, and Thermoproteales, which represent the deepest and shortest phylogenetic branches among the hyperthermophiles (Stetter, 1996~: Cultures of Thermococcus and Pyrococcus usually show duplex-shaped irregular spheres, about 0.5 to 2 ~m in diameter. However, during the early logarithmic growth phase, very tiny frog-egg-shaped cells about 0.2 1lm in diameter arranged in clusters up to about 20 individuals may be observed.
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From page 72... ...
The cannulae penetrate into the periplasmic space of the cells and connect those to each other, building up a huge network and greatly extending the range of a single cell. The flakes of Pyrodictium may be seen
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From page 73... ...
The enormous variation in cell size and volume appears to be a rather primitive feature and is in line with the 16S rRNA phylogeny of the corresponding hyperthermophiles. The smallest cell sizes observed are in the 200 to 300 nanometer range and the ability of hyperthermophilic archaea to form those may be of great advantage to pass narrow pores of soils and rocks in order to colonize hot subterranean environments.
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From page 74... ...
More likely, the absolute minimum size is closer to 250 rim where the cell has sufficient DNA to enable it to grow on simple compounds commonly found in various natural environments including, possibly, extraterrestrial ones. Introduction The question of minimum microbial size was recently brought to the fore by the report of McKay and coworkers (1)
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From page 75... ...
Obviously, these are organisms that require nothing more than the simplest of chemicals, such as CO2, O2, H2 and NH3. These so-called autotrophic organisms can synthesize all amino acids, cofactors, nucleotides, etc., with CO2 as the sole carbon source, using the oxidation of H2 as an energy source, and with ammonia (or even N2 gas)
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From page 76... ...
At the other extreme are nutrient-rich environments, such as those experienced by parasitic bacteria, and here lifeforms can have a minimum biosynthetic capacity. So, how many types of proteins (enzymes)
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From page 77... ...
The genome sizes and number of proposed protein-encoding genes in these two organisms are 1.67 and 1.55 Mb, and 1,738 and 1,512, respectively. It should be noted that the pathway of CO2 assimilation and the biochemistry of energy conservation in the methanogen are very different from those in A
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From page 78... ...
Of course, such a cell would have minimal biosynthetic capacity. A cell growing on CO2 as its carbon source would need at least 750 genes which, if they occupied 50% of the total volume, would require a cell of 156 rim in diameter.
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From page 79... ...
To grow, such a cell must be supplied with (and must assimilated all amino acids, fatty acids, nucleotides, cofactors, etc., because it would contain the minimum number of genes (250) and have a minimal biosynthetic capacity.
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(1996) Genome size in bacteria.
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From page 81... ...
Cell dimensions and biovolumes are now more frequently estimated via fluorescent nucleic acid staining and epifluorescence microscopy, or flow cytometry. Fluorescent DNA stains can also sometimes be misleading, because the visualized nuclear material may not accurately reflect the actual cytoplasmic volume (7~.
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From page 82... ...
A significant number of bacteria have been observed to undergo a transition from a large, actively growing state, to a dormant state of much smaller cell size (14-16~. Some of these physiologically induced small cells reduce to cell volumes as low as 0.03 - 3.
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From page 83... ...
in a relatively nutrient-rich environment, yet maintain their small cell dimensions (13~. Symbiotic and parasitic bacteria are known that have reduced physiological capacities and genome sizes (20~.
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From page 84... ...
! The very smallest bacterial cells and the very largest viral particles fall into about the same size category, raising some questions about the accuracy of currently used methods for quantifying naturally occurring virus and prokaryotes.
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