PANEL 2

Is there a relationship between minimum cell size and environment?

Is there a continuum of size and complexity that links conventional bacteria to viruses?

What is the phylogenetic distribution of very small bacteria?

Discussion

Summarized by Kenneth Nealson, Panel Moderator

Goals of the Session

Panel 2 focused in general discussions on the issue of whether any given kind of environment appeared to favor very small microbes. Experts with experience in a wide variety of intracellular and extracellular niches, including host cells (Van Etten and Kajander), aquatic environments (Button and DeLong), hydrothermal environments (Stetter and Adams), and soils and sediments (Staley) presented their views (Table 1). Insofar as it was possible, the discussion was focused on questions relating to the size ranges of organisms found in each environment, and the question of whether some properties of the environment (nutritional, physical, or chemical) might lead to the favoring of very small, nanometer-sized cells. In essence, this discussion sought to use the natural experiences of field and laboratory microbiologists to reach consensus on questions such as the following:

    1. What are the smallest sizes of viable organisms actually seen in the environment?

    2. What are the environmental issues that impose or relieve restrictions on cell size?

    3. What strategies are used to attain and maintain small size in nature?

Organisms Encountered in Natural Environments

What are the smallest viable organisms actually encountered in the various environments? In pursuit of the answer to this question, the speakers focused on their environments of interest (see Table 1) and the sizes of organisms encountered there. Included were organisms such as obligate parasites and symbionts, as well as free-living organisms, both rapidly growing and in various types of resting stages. For the sake of completeness, mitochondria and chloroplasts were included, although no



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PANEL 2 Is there a relationship between minimum cell size and environment? Is there a continuum of size and complexity that links conventional bacteria to viruses? What is the phylogenetic distribution of very small bacteria? Discussion Summarized by Kenneth Nealson, Panel Moderator Goals of the Session Panel 2 focused in general discussions on the issue of whether any given kind of environment appeared to favor very small microbes. Experts with experience in a wide variety of intracellular and extracellular niches, including host cells (Van Etten and Kajander), aquatic environments (Button and DeLong), hydrothermal environments (Stetter and Adams), and soils and sediments (Staley) presented their views (Table 1). Insofar as it was possible, the discussion was focused on questions relating to the size ranges of organisms found in each environment, and the question of whether some properties of the environment (nutritional, physical, or chemical) might lead to the favoring of very small, nanometer-sized cells. In essence, this discussion sought to use the natural experiences of field and laboratory microbiologists to reach consensus on questions such as the following: 1. What are the smallest sizes of viable organisms actually seen in the environment? 2. What are the environmental issues that impose or relieve restrictions on cell size? 3. What strategies are used to attain and maintain small size in nature? Organisms Encountered in Natural Environments What are the smallest viable organisms actually encountered in the various environments? In pursuit of the answer to this question, the speakers focused on their environments of interest (see Table 1) and the sizes of organisms encountered there. Included were organisms such as obligate parasites and symbionts, as well as free-living organisms, both rapidly growing and in various types of resting stages. For the sake of completeness, mitochondria and chloroplasts were included, although no

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Table 1 Organisms, Environments, and Presenters Organism Environment Speaker Viruses Animal or plant cells Van Etten Nanobacteria Animal serum Kajander Attached bacteria Soils, sediments, rocks Staley Hyperthermophiles Hot springs and vents Stetter Hyperthermophiles Hot springs and vents Adams Aquatic bacteria Lakes and oceans Button; DeLong Table 2 Size Ranges of Organisms or Organelles, and Niches Where They Are Found Organism Diameter Range (nm) Life Style Virus 30 to 200 Host-dependent Nanobacteria 100 to 200 Host-dependent Marine bacteria 100 and larger Free-living Attached forms 100 and larger Free-living Hyperthermophiles 200 and larger Free-living Mitochondria 200 and larger Host-dependent Chloroplasts 200 and larger Host-dependent presentations were specifically made in these areas. 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. For example, there was considerable debate with regard to the nanobacteria, as summarized by Dr. Kajander. While such nanobacteria have been reported to be smaller than 100 nm in diameter, Dr. Kajander was of the opinion that the only organisms for which growth could be established with certainty were those of 100-nm diameter or larger. This represents an area of considerable importance in terms of being able to search for and recognize very small organisms (e.g., Are there organismal fragments that appear to have similar morphologies, but are not actually viable, growing entities?). A point of interest with regard to this area is that virtually all of the microbiologists present had encountered structures resembling cells in the size range of 100 to 200 nm, but whether or not these could be demonstrated to be viable or cultivable microbes had usually not been established. The timeworn method of filtration through a 200-nm (0.2 micrometer) pore-size filter was still very dependable in terms of delineating cultivable bacteria. Environmental Parameters and Size What are the environmental issues that may impose or relieve restrictions on the smallest sizes that can be achieved by organisms? In pursuit of this question, the speakers considered a variety of different environmental factors that might lead to organisms adopting a smaller size. These included:

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1.   Nutrient-rich environments, which allow evolution to small cells with less biosynthetic capacity, such as obligate parasites or symbionts; 2.   Nutrient-poor environments, which lead to adaptation of small, starved cells; 3.   High or low temperature; and 4.   Attachment to surfaces. Of the issues discussed, that of nutrient availability was repeatedly noted as one of potential importance. Two major issues were emphasized: (1) the effect of nutrient limitation and starvation, which leads to adaptation of normally large cells to resting stages that are considerably smaller; and (2) the effect of nutrient richness, which leads to evolution of cells that are host dependent, and often considerably smaller. In nutrient-poor environments, organisms were deemed to be small in the starved state, although the lower size limit of this starvation state appears to be on the order of 200 rim. The mechanisms for achieving such small size (or for returning to a state of larger, rapidly growing cells) are not well understood. 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. In the case of intracellular symbiosis or parasitism in nutrient-rich environments, considerable discussion occurred as to whether or not such organisms could eliminate enough functions to evolve to a very small size. Dr. Adams presented a general discussion of the theoretical limits of life, based on organisms with the same basic biochemistry as those we are familiar with. At the theoretical extreme are the viruses, which are obligate intracellular parasites and which have no need for their own transport systems, translation machinery, or transcription apparatus. These organisms can be quite small, as they consist of a protein coat surrounding the genetic material. The lower size limits are seen in some RNA viruses like the Qβ virus (which contains only three genes), and in certain animal viruses (e.g., poliovirus) that are in the range of 25 to 50 nm in diameter, while most others are in the range of 100 to 200 nm or even larger. Symbiotic organelles or bacteria are also commonly found in the 200-nm range and are sometimes smaller. These include non-cultivable bacteria from a wide variety of organisms, intra-cellular organelles (e.g., mitochondria or chloroplasts), and the enigmatic nanobacteria discussed by Dr. Kajander. It should be clear, however, that the strategies used for attaining and maintaining small size will be very different for the oligotrophic organisms, which become small as a matter of optimizing their surface-to-volume ratio under diffusion-limited growth conditions, and the eutrophic organisms, which are allowed to become small because of the richness of their environment. In the latter case, these organisms are not faced with the maintenance of the genetic or physiological capacity for either extensive biosynthesis or diverse catabolism. While it is often possible to maintain such "obligate" symbionts or parasites in a host-free growth phase using a very rich medium, discussion of their role(s) as very small bacteria may be relevant only in the context of their existence as parasites or symbionts. Perhaps the liveliest discussion in Panel 2 centered on the specification of the smallest sizes actually seen in the environment and the criteria that one accepts for a living cell. To this end, Dr. Kajander proposed that nanobacteria may fragment into non-growing entities that appear considerably smaller than the true, viable organisms, and that these fragments may come together at a later time to form a viable organism. In terms of this possibility, Dr. Van Etten pointed out that some plant viruses exhibit just such a pattern. Each particle packages separate RNA, and sometimes three separate particles are needed to establish an infection. It was also noted that many estimates of the smallest sizes for viable

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organisms come from filtration studies, and that bacteria with non-rigid cell walls may pass through filters of pore size smaller than their actual diameter. As a final point, one would like to have an indication of the minimum cell volume needed to sustain life. Dr. de Duve emphasized that diameter alone is not a sufficient parameter, pointing out the practical difficulty of estimating true diameter from random thin sections To this end, the discussion by Dr. Adams focused almost entirely on the intracellular volumes of variously sized and shaped organisms, and the possibility that such volumes could accommodate the machinery of life. Strategies for Attaining and Maintaining Small Cell Size Are there strategies that can allow the minimum size of an organism to be smaller than might be anticipated through studies of extant organisms? 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. While this strategy is known for some RNA viruses, there are as yet no examples among the prokaryotes. A second strategy considered was that employed by parasites and symbionts, which simply discard a sizable fraction of their genetic information and adopt a host-dependent life style. Such organisms, while achieving a very small size, sacrifice the freedom of being host-free. Other approaches that might allow attainment and maintenance of a smaller cell size are (1) reduction of the average size of proteins; (2) an RNA-world approach in which a single type of molecule accomplishes both catalytic and genetic functions; and (3) the use of overlapping genes and genes on complementary strands. In no case has a systematic analysis of any of these approaches been done. Consensus? In terms of reaching a consensus, Panel 2 members, with the exception of Dr. Kajander, who described nanobacteria in the size range of 100 nm, considered that the lower size limit of bacteria-like particles believed to be cultivable corresponded to spherical organisms with a diameter in the size range of 200 to 250 nm. The nanobacteria of Kajander are “obligate” parasites (e.g., they require very rich media to achieve host-free growth) and so may fall into the category of organisms adopting a host-dependent life style. Thus, despite a very large amount of discussion, a general consensus was reached that was in agreement with the theoretical arguments put forward during the workshop, that the lower limit of size for a free-living, DNA-based organism corresponds to a spherical organism with a diameter in the size range of 200 to 250 nm. For host-dependent organisms the size may be smaller, and the extent of the smallness will certainly depend on the extent to which genetic and physiological functions have been discarded. For an organism that used one type of molecule for both catalysis and replication, the size could be considerably smaller, as discussed by Dr. Benner and others.

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Can Large dsDNA-Containing Viruses Provide Information About the Minimal Genome Size Required to Support Life? James L. Van Etten Department of Plant Pathology University of Nebraska at Lincoln Abstract The genomes of a few viruses, such as Bacillus megaterium phage G (670 kb) and the chlorella viruses (330 to 380 kb), are larger than the predicted minimal genome size required to support life (ca. 320 kb). A comparison of the 256 proteins predicted to be required for life with the putative 376 proteins encoded by chlorella virus PBCV-1, as well as those encoded by other large viruses, indicates that viruses lack many of these "essential" genes. Consequently, it is unlikely that viruses will aid in determining the minimal number and types of genes required for life. However, viruses may provide information on the minimal genome size required for life because the average size of genes from some viruses is smaller than those from free-living organisms. This smaller gene size is the result of three characteristics of virus genes: (1) virus genes usually have little intragenic space between them or, in some cases, genes overlap; (2) some virus-encoded enzymes are smaller than their counterparts from free-living organisms; and (3) introns occur rarely, if at all, in some viruses. Introduction Two recent estimates of the minimum genome size required to support life arrived at similar values. (1) The effect of 79 random mutations on the colony-forming ability of Bacillus subtilis resulted in the conclusion that a genome of 318 kb could support life (Itaya, 1995). Assuming 1.25 kb of DNA per gene (Fraser et al., 1995), this amount of DNA would encode 254 proteins. (2) A comparison of the genes encoded by Mycoplasma genitalium and Haemophilus influenzae led Mushegian and Koonin (1996) to suggest that as few as 256 genes are necessary for life. Using the same 1.25 kb gene size, the minimum self-sufficient life-form would have a 320 kb genome. Interestingly, these estimates are smaller than the genomes of some viruses (Table 1). Bacteriophage G, which infects Bacillus megaterium, has a genome of about 670 kb (Hutson et al., 1995); phycodnaviruses that infect chlorella-like green algae have 330 to 380 kb genomes (Rohozinski et al., 1989; Yamada et al., 1991); and some insect poxviruses have genomes as large as 365 kb (Langridge and Roberts, 1977). Other large, dsDNA-containing viruses, such as herpesviruses, African swine fever virus (ASFV), coliophage T4, baculoviruses, and iridoviruses, have genomes ranging from 100 to 235 kb (see Table 1). However, except for the common property of having large dsDNA genomes, these viruses differ significantly from one another in such characteristics as particle morphology, genome structure, and the intracellular site of replication. For example, poxviruses, herpesviruses, and baculoviruses have an external lipid envelope, whereas iridoviruses and phycodnaviruses have an internal lipid component. Baculovirus genomes are circular, iridoviruses and phage T4 have linear circular permuted genomes with terminal reduncancy, and the linear genomes of herpesviruses have sequences from both termini that are repeated internally in an inverted form. The phycodnaviruses, poxviruses, and ASFV have linear genomes with covalently closed hairpin ends. Finally, herpesviruses and baculoviruses primarily replicate in the nucleus, whereas

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Table 1 Representative Large dsDNA Viruses Virusa Virus Family Host Genome Size (bp) Minimum No. of Codonsb No of Genes Average Length of Gene (Bases) Reference Phage G Myoviridae Bacillus megaterium ˜670,000 — — — Hutson et al., 1995 PBCV-1 Phycodnaviridae Chlorella NC64A 330,742 65 376c 880 Li et al., 1997 MsEPV Poxviridae Grasshopper 236,120 (222,120)d 60 267 (257)d 884 (864)d Afonso et al., 1998 MCV Poxviridae Human 190,289 (180,889)e 60 182 (180)e 1,046 (1,005)e Senkevich et al., 1996 ASFV Unclassified Swine 170,101 (166,613)f 60 151 1,127 (1,103)f Yanez et al., 1995 Coliphage T4 Myoviridae E. coli 168,800 29 288g 586 Kutter et al., 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 a G, Giant; PBCV-1, Paramecium bursaria chlorella virus 1; MsEPV, Melanoplus sanguinipes entomopoxvirus; MCV, Molluscum 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. b Minimum number of codons used by the authors to calculate an open reading frame (ORF). c Four of the genes are diploid. d MsEPV has a 7 kb inverted repeat at each terminus. This 14 kb encodes 10 small ORFs (60 to 155 codons). Removal of 14 kb and 10 ORFs from the calculations produces the smaller genome size (in parentheses). e MCV has a 4.7 kb inverted repeat at each terminus. This 9.4 kb encodes two 488 codon ORFs. Removal of 9.4 kb and 2 ORFs from the calculations produces the smaller genome size (in parentheses). f ASFV has a 2134 bp inverted repeat at each terminus. The most terminal 1744 bp at each end do not encode an ORF and thus 3488 bp were removed from the calculations, which leads to the smaller genome size (in parentheses). g This includes 161 genes known to encode proteins and 127 suspected of encoding proteins (Gisela Mosig, personal communication). h HSV-2 has 473 met-initiated ORFs of 50 codons or longer of which 74 are known to be functional genes. If some of the additional 399 ORFs prove to be protein encoding, the average length of a herpesvirus gene would decrease substantially. the entire life cycle of the poxviruses occurs in the cytoplasm. Iridoviruses and phycodnaviruses initiate replication in the nucleus, but capsids are assembled and DNA is packaged in the cytoplasm. With the exception of bacteriophage G, the genome of at least one representative of each of these dsDNA-containing viruses has been sequenced, and the number of putative genes encoded by the viruses are listed in Table 1. Because the 330,742 bp genome of the phycodnavirus PBCV-1 is the largest virus genome sequenced to date (Lu et al., 1995, 1996; Li et al., 1995, 1997; Kutish et al., 1996), it will be used to illustrate the organization and diversity of genes that can be encoded by a large dsDNA-containing virus. The PBCV-1 genome encodes 701 open reading frames (ORFs), defined as continuous stretches of DNA that translate into a polypeptide initiated by an ATG translation start

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codon, and extending 65 or more codons. The 701 ORFs have been divided into 376, mostly non-overlapping, ORFs (major ORFs), which are predicted to encode proteins, and 325 short ORFs, which are probably non-protein encoding. Four PBCV-1 ORFs reside in the 2.2 kb inverted terminal repeat region of the PBCV-1 genome and consequently are present twice in the PBCV-1 genome (Strasser et al., 1991; Lu et al., 1995). The 376 PBCV-1 ORFs are evenly distributed along the genome and, with one exception, there is little intergenic space between them. The exception is a 1788-bp non-protein coding sequence near the center of the genome. This region, which has numerous stop codons in all reading frames, does code for ten tRNA genes. The middle 900 bp of this intergenic region also has some characteristics of a "CpG island" (Antequera and Bird, 1993). To put the coding capacity of the PBCV-I genome in perspective, the 580-kb genome of the smallest self-replicating organism, Mycoplasma genitalium encodes about 470 genes (Fraser et al., 1995). Computer analyses of the predicted products of the 376 PBCV-1 major ORFs indicate that about 40% of the ORFs resemble proteins in the databases, including many interesting and unexpected proteins. Some PBCV-1 encoded proteins resemble those of bacteria and phages, such as DNA restriction endonucleases and methyltransferases. However, other PBCV-1 encoded proteins resemble those of eukaryotic organisms and their viruses, such as translocation elongation factor-3, RNA guanyltransferase, and two proliferating cell nuclear antigens. The PBCV-1 genome is thus a mosaic of prokaryotic- and eukaryotic-like genes, suggesting considerable gene exchange in nature during the evolution of these viruses. 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 × 104 infectious viruses per ml of native water have been obtained (Van Etten et al., 1985; Yamada et al., 1991). The only known hosts for these viruses are chlorella-like green algae, which normally live as hereditary endosymbionts in some isolates of the ciliate, Paramecium bursaria. In the symbiotic unit, algae are enclosed individually in perialgal vacuoles and are surrounded by a host-derived membrane (Reisser, 1992). The endosymbiotic chlorella are resistant to virus infection and are only infected when they are outside the paramecium (Van Etten et al., 1991). Because of the large size of the PBCV-1 genome, it is not surprising that many of the predicted 376 PBCV-1 genes have not been found in other viral genomes. Box 1 lists some of the PBCV-1 encoded ORFs that match proteins in the databases and, in a few cases, indicate if a gene is transcribed early (E) or late (L) during virus replication. The functionality of some PBCV-1 encoded proteins has been established by either complementation of mutants and/or assaying recombinant protein for enzyme activity. (These proteins are indicated with an asterisk in Box 1.) Twenty-nine of the PBCV-1 ORFs resemble one or more other PBCV-1 ORFs suggesting that they might be either gene families or gene duplications. Sixteen families have 2 members, 8 families have 3 members, 3 families have 6 members, and 2 families have 8 members. Even if some of the suspected 376 PBCV-1 protein-encoding genes turn out to be non-coding, it is clear that PBCV-1 encodes more genes than the minimum number predicted to be necessary to support life. Comparing the genes that Mushegian and Koonin (1996) proposed were essential to support life with the PBCV-1 encoded genes indicates that the virus lacks many of these genes, including a RNA polymerase, a complete protein synthesizing system, and an energy-generating system. Consequently, PBCV-1 depends on the algal host to fulfill these essential functions. A comparison of the genes encoded by the other large dsDNA-containing viruses listed in Table 1 with those encoded by PBCV-1 indicates that a few genes are present in all of the viruses, e.g., each of the viruses encodes a DNA polymerase gene. However, there are more differences in the genes encoded by these viruses than similarities, which reflects the different life-styles of the viruses. Like PBCV-1,

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Box 1 Putative ORFs Encoded by Chlorella Virus PBCV-1a DNA Replication & Repair DNA Restriction/Modification (E) A185R DNA polymerase   A251R* Adenine DNA methylase (M.CviAII) (E) A544R* DNA ligase   A252R* Restriction endonuclease (R.CviAII) (E) A583L* DNA topoisomerse II   A252R* Restriction endonuclease (R.CviAII)   A193L PCNA (E) A517L* Cytosine DNA methylase (M.CviAIII)   A574L PCNA (L) A530R* Cytosine DNA methylase (M.CviAIV)   A153R Helicase (E) A581R* Adenine DNA methylase (M.CviAI)   A241R Helicase (E) A579L* Restriction endonuclease (R.CviAI)   A548L Helicase   A683L Cytosine DNA methylase (M.CviAV) (E) A50L* T4 endonuclease V       A39L CyclinA/cdk associated protein Sugar and Lipid Manipulation   A638R Endonuclease (L) A64R Galactosyl transferase     (E) A98R* Hyaluronan synthase Nucleotide Metabolism (E) A100R* Glucosamine synthase (E) A169R* Aspartate transcarbamylase   A114R Fucosyltransferase   A476R Ribo. reductase (small subunit) (E) A118R GDP-D-mannose dehydratase   A629R Ribo. reductase (large subunit)   A222R Cellulose synthase   A427L Thioredoxin   A295L Fucose synthase   A438L Glutaredoxin (E) A473L Cellulose synthase   A551L dUTP pyrophosphatase (E) A609L* UDP-glucose dehydrogenase   A596R dCMP deaminase   A49L Glycerophosphoryl diesterase   A416R dG/dA kinase   A53R 2-hydroxyacid dehydrogenase   A363R Phosphohydrolase   A271L Lysophospholipase   A392R ATPase       A674R Dicty Thy protein Phosphorylation/dephosphorylation       A34R Protein kinase Transcription (L) A248R* Phosphorylase B kinase   A107L RNA transcription factor TFIIB   A277L Ser/Thr protein kinase   A125L RNA transcription factor TFIIS   A278L Ser/Thr protein kinase   A166R Exonuclease   A282L Ser/Thr protein kinase   A422R Endonuclease   A289L Ser/Thr protein kinase (E) A103R* RNA guanyitransferase   A305L Tyr phosphatase   A464R RNase III   A614L Protein kinase       A617R Tyr-protein kinase Translation     (E,L) A666L Translation elongation factor-3 Miscellaneous   A85R Prolyl 4-hydroxylase alpha-subunit   A207R* Omithine decarboxylase   A105L Ubiquitin C-terminal hydrolase   A217L Monoamine oxidase   A448L Protein disulphide isomerase (L) A237R* Homospermidine synthase   A623L Ubiquitin-like fusion protein 10 tRNAs   A78R b-alanine synthase       A245R Cu/Zn-superoxide dismutase       A284L* Amidase Cell Wall Degrading   A465R Yeast ERVI protein (E) A181R* Chitinase   A598L Histidine decarboxylase (L) A260R* Endochitinase   A250R K+ ion channel protein (L) A292L* Chitosanase   A625R Transposase   A94L β1,3 glucanase     a E and L refer to early and late genes, respectively. An asterisk means that the gene encodes a functional enzyme as determined either by complementation or by enzyme activity of a recombinant protein.

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each of these viruses rely on their host cells for such basic functions as energy generation, protein synthesis, and amino acid biosynthesis. The net result is that it seems unlikely that examining virus genes will aid in determining the minimal number and types of genes required to support life. 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. Except for herpesvirus HSV 2, the size of the average virus gene varied from 586 nucleotides for coliphage T4 to 1,127 nucleotides for ASFV, with the average gene size for five of the viruses being less than 1 kb. The sizes are even smaller if one removes the non- or sparsely-coding regions in the virus genomes before making the calculations. For example, the two poxviruses MsEPV and MCV, as well as ASFV, have inverted terminal repeat regions that either are non-coding or only encode a few genes. Eliminating these non-coding regions from the calculations reduces the size of the average MsEPV gene from 884 nucleotides to 864 nucleotides, the MCV gene from 1,046 nucleotides to 1,005 nucleotides and ASFV from 1,127 nucleotides to 1,103 nucleotides (Table 1). Similar calculations made on nine Eubacteria and three Archaea indicate that the average length of Eubacteria protein-encoding genes ranges from 1,023 nucleotides for Aquifex aeolicus to 1,234 nucleotides for Mycoplasma genitalium (Doolittle, 1998). The predicted average length of the three archaea is slightly smaller—895, 943, and 961 nucleotides for Archaeoglobus fulgidus, Methanococcus thermo-autotrophicurn , and M. jannaschii, respectively. Thus, depending on the virus and bacterium being compared, the average functional virus gene is 10 to 50% smaller than the average bacterial gene. This conclusion depends on the assumption that at least the majority of the predicted virus genes, in fact, encode proteins. The apparent smaller size of genes from large dsDNA viruses can be attributed to three factors. (1) Typically, virus genomes have little intergenic space and, in some cases, genes overlap. This tight packaging of genes does not prevent gene regulation, however, as virus genes are typically expressed early or late in the replication cycle. The 376 major ORFs in chlorella virus PBCV-1 are evenly distributed along the genome, and 85% are separated by less than 200 nucleotides. Likewise, 85% of the 151 putative genes in ASFV are also separated by less than 200 nucleotides (Yanez et al., 1995). The genes in phage T4 are even more tightly packed (Kutter et al., 1994). Consequently, transcription start and stop signals plus the regulatory regions for at least some virus genes are extremely short, or they are located in the coding region of adjacent genes. (2) Some virus-encoded proteins are smaller than those from free-living organisms and may approach the minimum size required for enzyme activity. Examples include the PBCV-1 encoded 298 amino acid residue ATP-dependent DNA ligase, the 1,061 amino acid residue type II DNA topoisomerase, and the 372 amino acid residue ornithine decarboxylase. Each of these virus-encoded proteins has the expected enzyme activity. ATP-dependent DNA ligases range in size from the 268 amino acid residue enzyme from Haemophilus influenzae (Cheng and Shuman, 1997) to the 1,070 amino acid residue enzyme from Xenopus laevis (Lepetit et al., 1996). The PBCV-1 enzyme is the second smallest ATP-dependent ligase in the databases. The PBCV-1 encoded type II DNA topoisomerase is about 130 amino acids smaller than the next smallest type H topoisomerase in the databases, which is encoded by virus ASFV (Garcia-Beato et al., 1992). The PBCV-1 encoded ornithine decarboxylase is about. 90 amino acids smaller than the next smallest ornithine decarboxylase in the databases. Likewise, the large subunit of ribonucleotide reductase from the baculovirus Orgyia pseudotsugata multinucleocapsid nuclear polyhedrosis virus (OpMNpV) is 150 to 200 amino acids smaller than its counterpart from most organisms (Ahrens et al., 1997). (3) Even though introns were first discovered in adenoviruses (Berget et al., 1977; Chow et al.,

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1977), the genes of many large DNA-containing viruses either lack introns, e.g., poxviruses, baculoviruses, iridoviruses, and ASFV, or only have a few short introns, e.g., phycodnaviruses. An absence of introns obviously contributes to the smaller size of virus genes. To summarize, it is unlikely that studying viruses will reveal useful information about the minimum number and types of genes required to support life. 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. References Afonso, C.L., E.R. Tulman, Z. Lu, E. Oma, G.F. Kutish, and D.L. Rock. 1998. The genome of Melanoplus sanguinipes entomopoxvirus. J. Virol. (in press). Ahrens, C.H., R.L.Q. Russell, CJ. Funk, J.T. Evans, S.H. Harwood, and G.F. Rohrmann. 1997; The sequence of the Orgyia pseudotsugata multinucleocapsid nuclear polyhedrosis virus genome. Virology 229:381-399. Antequera, F., and A. Bird. 1993. CpG Islands. Pp. 169-185 in DNA Methylation: Molecular Biology and Biological Significance, P.J. Jost and P.H. Saluz (eds.), Basel, Switzerland: Birkhauser Verlag. Ayres, M.D., S.C. Howard, J. Kuzio, M. Lopez-Ferber, and R.D. Possee. 1994. The complete DNA sequence of Autographa californica nuclear polyhedrosis virus. Virology 202:586-605. Berget, S.M., C. Moore, and P.A. Sharp. 1977. Spliced segments at the 5'terminus of adenovirus 2 late mRNA. Proc. Natl. Acad. Sci. USA 74:3171-3175. Cheng, C., and S. Shuman. 1997. Characterization of an ATP-dependent DNA ligase encoded by Haemophilus influenzae. Nucleic Acids Res. 25:1369-1374. Chow, L., R. Gilinas, T. Broker, and R. Roberts. 1977. An amazing sequence arrangement at the 5'ends of adenovirus 2 messenger RNA. Cell 12:1-8. Dolan, A., F.E. Jamieson, C. Cunningham, B.C. Barnett, and D.J. McGeoch. 1998. The genome sequence of herpes simplex virus type 2. J. Virol . 72:2010-2021. Doolittle, R.F. 1998. Microbial genomes opened up. Nature 392:339-342. Fraser, C.M., J.D. Gocayne, O. White, M.D. Adams, R.A. Clayton, R.D. Fleischmann, C.J. Bult, A.R. Kerlavage, G. Sutton, J.M. Kelley, J.L. Fritchman, J.F. Weidman, K.V. Small, M. Sandusky, J. Fuhrmann, D. Nguyen, T.R. Utterback, D.M. Saudek, C.A. Phillips, J.M. Merrick, J.F. Tomb, B.A. Dougherty, K.F. Bott, P.C. Hu, T.S. Lucier, S.N. Peterson, H.O. Smith, C.A. Hutchison, and J.C. Venter. 1995. The minimal gene-complement of Mycoplasma genitalium. Science 270:397-403. Garcia-Beato, R., J.M.P. Freije, C. Lopez-Otin, R. Blasco, E. Vinuela, and M.L. Salas. 1992. A gene homologous to topoisomerase II in African swine fever virus. Virology 188:938-947. Hutson, M.S., G. Holzwarth, T. Duke, and J.L. Viovy. 1995. Two-dimensional motion of DNA bands during 120° pulsed-field gel electrophoresis. I. Effect of molecular weight. Biopolymers 35:297-306. Itaya, M. 1995. An estimation of minimal genome size required for life. FEBS Lett. 362:257-260. Kutish, G.F., Y. Li, Z. Lu, M. Furuta, D.L. Rock, and J.L. Van Etten. 1996. Analysis of 76 kb of the chlorella virus PBCV-1 330-kb genome: Map positions 182 to 258. Virology 223:303-317. Kutter, E., T. Stidham, B. Guttman, E. Kutter, D. Batts, S. Peterson, T. Djavakhishvili, F. Arisaka, V. Mesyanzhinov, W. Ruger, and G. Mosig. 1994. Genomic map of bacteriophage T4. Pp. 491-519 in Molecular Biology of Bacteriophage T4, J.D. Karam (ed). Washington DC: American Society for Microbiology. Langridge, W.H.R., and D.W. Roberts. 1977. Molecular weight of DNA from four entompoxviruses determined by electron microscopy. J. Virol . 21:301-308.

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Lepetit, D., P. Thiebaud, S. Aoufouchi, C. Prigent, R. Guesne, and N. Theze. 1996. The cloning and characterization of a cDNA encoding Xenopusa levis DNA ligase I. Gene 172:273-277. Li, Y., Z. Lu, D.E. Burbank, G.F. Kutish, D.L. Rock, and J.L. Van Etten. 1995. Analysis of 43 kb of the chlorella virus PBCV-1 330-kb genome: Map position 45 to 88. Virology 212:134-150. Li, Y., Z. Lu, L. Sun, S. Ropp, G.F. Kutish, D.L. Rock, and J.L. Van Etten. 1997. Analysis of 74 kb of DNA located at the right end of the chlorella virus PBCV-1 330-kb genome. Virology 237:360-377. Lu, Z., Y. Li, Q. Que, G.F. Kutish, D.L. Rock, and J.L. Van Etten. 1996. Analysis of 94 kb of the chlorella virus PBCV-1 330-kb genome: Map positions 88 to 182. Virology 216:102-123. Lu, Z., Y. Li, Y. Zhang, G.F. Kutish, D.L. Rock, and J.L. Van Etten. 1995. Analysis of 45 kb of DNA located at the left end of the chlorella virus PBCV-1 genome. Virology 206:339-352. Mushegian, A.R., and E.V. Koonin. 1996. A minimal gene set for cellular life derived by comparison of complete bacterial genomes. Proc. Natl. Acad. Sci. USA 93:10268-10273. Reisser, W. (ed). 1992. Algae and Symbioses. Bristol, UK: Biopress. Rohozinski, J, L. Girton, and J.L. Van Etten. 1989. Chlorella viruses contain linear nonpermuted double-stranded DNA genomes with covalently closed hairpin ends. Virology 168:363-369. Senkevich, T.G., J.J. Bugert, J.R. Sisler, E.V. Koonin, G. Darai, and B. Moss. 1996. Genome sequence of a human tumorigenic poxvirus: Prediction of specific host response evasion genes. Science 273:813-816. Strasser, P., Y. Zhang, J. Rohozinski, and J.L. Van Etten. 1991. The termini of the chlorella virus PBCV-1 genome are identical 2.2-kbp inverted repeats. Virology 180:763-769. Tidona, C.A., and G. Darai. 1997. The complete DNA sequence of lymphocystis disease virus. Virology 230:207-216. Van Etten, J.L., D.E. Burbank, A.M. Schuster, and R.H. Meints. 1985. Lyric viruses infecting a chlorella-like alga. Virology 140:135-143. Van Etten, J.L., L.C. Lane, and R.H. Meints. 1991. Viruses and viruslike particles of eukaryotic algae. Microbiol. Rev. 55:586-620. Yamada, T., T. Higashiyama, and T. Fukuda. 1991. Screening of natural waters for viruses which infect chlorella cells. Appl. Environ. Microbiol . 57:3433-3437. Yanez, R.J., J.M. Rodriguez, M.L. Nogal, L. Yuste, C. Enriquez, J.F. Rodriguez, and E. Vinuela. 1995. Analysis of the complete nucleotide sequence of African swine fever virus . Virology 208:249-278.

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The Influence of Environment and Metabolic Capacity on The Size of a Microorganism Michael W.W. Adams Departments of Biochemistry and Molecular Biology University of Georgia Abstract Simple calculations show that there are two critical factors in considering minimum cell size: the amount of DNA that is required to support cell growth and the volume of the cell devoted to accommodate that DNA. The amount of DNA a cell contains is related to how much that cell depends upon its environment to supply nutrients. At one extreme, the environment provides only gases and minerals, and the life-forms that occupy such an environment have a high biosynthetic capacity and synthesize all cellular carbon from CO2. This requires at most 1,500 (an actual value) and perhaps as few as 750 genes. At the other extreme are nutrient-rich environments, such as those experienced by parasitic bacteria, and here life-forms have a minimum biosynthetic capacity requiring between 250 (a calculated value) and 500 genes (an actual value). For spherical cell with minimal biosynthetic capacity (250 genes), the minimum size is 172 nm diameter. This assumes that the cell consists Coy volume) of 10% DNA, 10% ribosomes, 20% protein, and 50% water. Such a cell could contain 65 ribosomes and an average of 65 proteins per gene. On the other hand, a cell that synthesizes all of its cellular components from CO2 must be at least 248 nm in diameter, assuming that its minimal DNA content (750 genes) is 10% of the cell volume. It is concluded that microorganisms cannot have diameters less than 172 nm if they have the same basic biochemical requirements for growth as all other extant life-forms. Even then, such a cell is biosynthetically challenged and would require a very specialized environment to supply it with a range of complex biological compounds. More likely, the absolute minimum size is closer to 250 nm 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) in which objects with upper dimensions of 20 by 100 nm were postulated to be of cellular origin. Subsequently, so-called ultramicrobacteria were isolated from marine environments that can pass through a 200 nm filter and have cell volumes of 0.03 to 0.08 µm3 (2). In addition, entities known as "nanobacteria" that have been cultured from blood apparently have diameters as low as 80 nm (3). In light of these studies, it is important to estimate the theoretical limit for minimum cell size. Can cells with a diameter of less than, say, 50 nm contain sufficient biological material to remain free-living? This begs the question of what is meant by "sufficient biological material"? One measure is genome size or more specifically the number of different types of proteins (enzymes) that an organism has at its disposal to support growth. Before considering just how many genes this may be, we must also define what is meant by “free-living." How dependent is this minimally sized cell upon its environment? In the following it is assumed that such cells have the same basic biochemical requirements as any other life-form that we know of, and must satisfy them with the same enzymatic reactions.

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The Influence of Environment on the Complexity of Life-forms Obviously, no life-form survives in isolation from its surroundings, but organisms vary considerably in their dependence upon their environment. Thus, humans require at a minimum ten or so amino acids, various minerals, an array of biological cofactors (vitamins), and a continual supply of O2 gas. Perhaps surprisingly, these same materials are also required by many microorganisms, although they typically differ from us in their ability to synthesize most, if not all, of the twenty amino acids as well as many, if not all, of what we term "vitamins." Like us, the vast majority of microorganisms require a fixed carbon source, which is usually a carbohydrate of some sort, although in some cases, lipids, nucleotides, or various simple organic compounds are utilized. In contrast, some microorganisms are intensely dependent upon their environments. For example, some microbial parasites do not synthesize any amino acid or lipid, and only a few enzyme cofactors and nucleotides; rather, they obtain all of these compounds from their host. Indeed, at one level, such a parasitic life-form is not too far removed from the simplest virus. This consists of a protein coat that surrounds a defined amount of nucleic acid (DNA or RNA). The latter encodes proteins that inside the host cell are synthesized and that direct vital replication. Hence a virus can be thought of as a life-form that has an extreme dependence upon its host. Not only does the host donate all of the necessary biological compounds, but it also provides transcriptional and translational machinery. What then is a plasmid? Can this be considered an extremely parasitic life-form? A plasmid obviously encodes the information to reproduce itself, i.e., to make a copy, but it is totally dependent upon the host to carry this out. Hence, we can consider plasmids, viruses, and parasitic bacteria as life-forms that vary in their dependence upon their environments. So what forms of life are at the other extreme? What life-form requires the least from its environment? 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) as the nitrogen source. Interestingly, this definition also includes green plants—with the exception that they obtain energy from visible light rather than from H2 oxidation. Of course, many autotrophic microorganisms gain energy from the oxidation with O2 of simple substances other than H2, such as CO, CH4, NH3, or H2S. Similarly, anaerobic autotrophs growing on H2 and CO2 also conserve energy during the reduction of CO2, either by the production of methane or acetate as accomplished by methanogens and acetogens, respectively. Clearly then, the variety of known life-forms can be classified by the extent to which they depend upon their environment for growth. Simple gases and salts are sufficient for many types of microorganism, both under aerobic and anaerobic conditions, whereas other microbes are intensely dependent upon their environment for a range of complex biological molecules. So how do we define "free-living" life? In simple terms, life can be thought of as an entity that has the ability to undergo self-directed reproduction when supplied with the appropriate environment and the necessary free energy. The question is, can this environment be another life-form? If this is the case, then the argument becomes how small can a virus be, and a possible answer is a plasmid. However, an important difference between viruses (plasmids) and parasitic bacteria is that the former, but not the latter, replicate by the transfer of nucleic acid into their environment (host). With the bacteria, the host environment provides "only" an array of nutrients, and the bacterium's genetic material does not contact the host (the environment). Theoretically and often practically, the parasite can thrive if such nutrients are provided to it directly in a liquid medium. Hence, a major distinction can be made between the mechanisms by which parasitic life-forms and viruses interact with their "living” environments. Moreover, we can use this logic to define the

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environment that will support our smallest possible life-form. That is, we will assume that if its environment is another life-form, then nutrients (or non-life-forms) can replace that life-form. In other words, we will not consider viruses or analogous life-forms in trying to define minimum size. Life-forms therefore occupy environments that fall between two extremes. One provides only gases and minerals, and the life-forms that occupy it must have a high biosynthetic capacity. At the other extreme are nutrient-rich environments, such as those experienced by parasitic bacteria, and here life-forms can have a minimum biosynthetic capacity. So, how many types of proteins (enzymes) are required to support cellular growth within these two types of environment? The recent availability of genome sequences for a variety of microorganisms (4) enables quantitative estimations to be made. Organisms with Low Biosynthetic Capacity Those organisms that are most dependent upon their environment are the parasitic bacteria, the prototypical example of which are the mycoplasma. Interestingly, the complete genome of one species, Mycoplasma genitalium, was one of the first genomes to be sequenced (5). At 0.58 Mb, this represents the smallest known genome of any free-living organism. The genome contains 470 predicted protein coding regions, and these include those required for DNA replication, transcription and translation, DNA repair, cellular transport, and energy metabolism. However, comparisons with the genome (1.83 Mb, encoding 1,703 putative proteins) of another parasite, Haemophilus influenzae (6), led to the conclusion that the "minimal gene set that is necessary and sufficient to sustain the existence of a modern-type cell" is (only) 256 genes, or about half of the genome of M. genitalium (7). It should be noted, however, that while both of these parasitic organisms grow in the absence of their hosts, to do so they require an extremely "rich medium" containing a range of nutrients. These organisms maintain a minimal biosynthetic capacity, a capacity that is apparently satisfied by approximately 250 different proteins. The Most Slowly Evolving Microorganisms In determining the "minimum" set of genes that a minimal-size microbe might contain, we must also consider what is meant by the term "modern-type cell" quoted above (7). Are present-day organisms highly sophisticated cells with a range of metabolic capabilities, only some of which are utilized and then under very specialized conditions? For example, E. coli could be regarded as highly evolved because it exhibits a range of metabolic modes, including growth under aerobic and anaerobic conditions, the utilization of a wide variety of different carbon sources, etc. Indeed, such a large metabolic capacity might be reflected in its genetic content of 4.64 Mb encoding 4,288 genes (8). Similarly, metabolically diverse species such as Bacillus subtilus and Pseudomonas putida have genomes of comparable size ( Mb). Indeed, a recent survey of gram-negative bacteria gave a mean genome size of 3.8 Mb (9). In other words, it is not unusual for microorganisms, or at least those that have been well characterized, to contain 4,000 or more genes. Hence, are there more-slowly-evolving organisms, and do they contain less genetic material and have fewer metabolic choices? By phylogenetic analyses based on 16S rRNA sequence comparisons, the most-slowly-evolving microorganisms are the deepest branches, the first to have diverged within the two major lineages corresponding to the Bacteria and the Archaea (10). Remarkably, in both domains these are the hyperthermophiles, organisms that grow optimally at temperatures of 80°C and above. Within the bacteria domain this includes two genera, Thermotoga and Aquifex, while there are almost twenty genera of hyperthermophilic archaea (11). In fact, one of the two major branches within the archaeal

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domain consists almost entirely of hyperthermophiles, while in the other branch the hyperthermophiles are the most slowly evolving of the known genera. A great deal is known about the genome contents of these hyperthermophilic organisms as several have been or are being sequenced. These include the genomes of the archaea, Methanococcus jannaschii, Pyrobaculum aerophilum, Pyrococcus horikoshii, P. furiosus, P. abyssi, and Archaeoglobus fulgidus, and of the bacterium Thermotoga maritima (4). Interestingly, all of these organisms have genomes only about half the size of that found in E. coli, with those of Archaeoglobus fulgidus and Aquifex aeolicus being the largest (2.18 Mb) and smallest (1.55 Mb) of this group, respectively. Thus, the most slowly evolving organisms known (at least as determined by 16S rRNA analyses) do indeed have relatively small genomes, although they are still highly complex life-forms. Organisms with High Biosynthetic Capacity So, how many different proteins are required to support growth of organisms on nothing more than gases and a few minerals, and is there a hyperthermophilic example of such an organism? To date, the genomes of two hyperthermophilic autotrophs have been sequenced. One is the archaeon, Methanococcus jannaschii (12), which is a methanogen that grows up to 90°C using H2 and CO2 as energy and carbon sources and generates methane as an end product. The other is a bacterium, Aquifex aeolicus (13), which grows up to 95°C on H2 and CO2, but it is not an anaerobe like the methanogen, as it requires low concentrations of O2. 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. aeolicus, yet approximately the same number of genes are required. On the other hand, these genomes are much larger than the genomes of the two parasites discussed above. Presumably, A. aeolicus and M. jannaschii require many more genes because they must synthesize all of their cellular components from CO2. Hence they contain about three times the genetic information of M. genitalium This seems appropriate considering that the latter organism is supplied with all of its amino acids, nucleotides, fatty acids, "vitamins," and with an energy source (glucose). From this direct comparison we might conclude that about two-thirds of the DNA in A. aeolicus and M. jannaschii, or approximately 1,000 genes, encodes proteins that function to carry out these biosynthetic tasks and produce all of these compounds from CO2. The Smallest Cell From the above discussion it can be concluded that a cell with minimal biosynthetic capacity that is growing in a nutrient-rich medium requires between 250 (a calculated value) and 500 genes (the approximate number in M. genitalium) to grow. At the other extreme is a cell that synthesizes all of its cellular material from CO2, and this requires at most 1,500 genes (the approximate number in A. aeolicus) and probably closer to 750 genes (half of the actual value). With these values in mind, let us consider how much biological material can be contained within a cell of, say, 50 nm diameter. For example, if one allows 5 nm in thickness for a lipid bilayer, a spherical cell of 50 nm diameter would have an internal volume of 33,500 nm3. For comparison, an E. coli cell, with dimensions of about 1.3 by 4.0 µm, has an internal volume of about 5 ×109 nm3, or almost 2 million times the volume of the 50 nm diameter cell. The question is, What quantifies of the various biochemical structures found in a typical prokaryotic cell can be accommodated within a volume of 33,500 nm3? A ribosome has a diameter of about 20 nm, and ribosomes are typically 25% of the mass (dry weight) of a bacterial cell

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(although this varies considerably depending on the growth rate). Assuming a similar percentage of the volume of a 50 nm diameter cell is devoted to them, such a cell could contain only two ribosomes (4,200 nm3 each). Whether only two would limit cell growth to any extent is unknown; nevertheless, the cell is certainly large enough to contain ribosomes, albeit only two. On the other hand, proteins usually constitute about half of the dry weight of bacterial cells. Let us assume that they also occupy approximately 50% of the volume of the 50 nm diameter cell, and that, in general, proteins have an average molecular weight of 30 kDa, which corresponds to a diameter of about 4 nm per protein. If a cell of 50 nm diameter were 50% protein by volume, then this would correspond to about 520 such molecules (average 30 kDa) per cell. Are two ribosomes and 520 "average-sized" protein molecules sufficient to support the growth of a cell? Note this would correspond to, on average, two copies of each protein for a cell with minimal biosynthetic capacity (calculated to contain 250 genes). But can we neglect DNA? As this is typically only about 3% of the total mass (dry weight) of a bacterial cell, at first glance it would seem unlikely that the volume of genetic material, especially in an organism with a minimum gene content, would affect cell size. For example, with a diameter of 2 nm and length of 0.34 nm/bp, the 4.64 Mb of E. coli has a volume of 4.9 × 106 nm3, which is less than 1% of the cell volume. Surprisingly, however, simple calculations show that DNA is a determining factor in much smaller cells. Thus, the hypothetical 50 nm diameter cell, 75% of which Coy volume) is occupied by proteins and ribosomes, could contain, even if the remaining 25% of the cell were devoted to DNA, only 8 genes (of 1000 bp each)! DNA Content Determines Cell Size If a 50 nm cell can only reasonably accommodate 8 genes, the question is, What is the minimum cell size that could reasonably accommodate 250 genes (or 250 kb of DNA)? Remarkably, even if the cell were 50% DNA, such a cell would have a diameter of at least 110 nm. Assuming that half of the remaining volume (25%) is protein and half of that (12.5%) is occupied by ribosomes, the 110 nm cell could contain up to 4,000 protein molecules (average 30 kDa) or an average of 8 proteins per gene, together with 15 ribosomes. 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 nm in diameter. Such a cell could also contain 12,400 protein molecules (25% by volume, or 16 copies for each gene) and 48 ribosomes. Although such calculations still leave 12.5% of the cell volume for other cellular components, such as lipids, cofactors, metabolites, and inorganic compounds, the most abundant component of a typical cell, namely water, is not included. Water typically occupies about 70% of a microbial cell, so let us assume 50% for the hypothetical cell. The volume of a cell containing 250 genes then increases to 136 nm, while that with 750 genes is now 194 nm. From these calculations it is obvious that DNA content is the main factor in determining cell size. A critical parameter is, therefore, the maximum amount of a cell that can be devoted to DNA. It seems extremely unlikely that DNA could represent 25% of the cellular volume (where water is 50%) if one considers just the volume occupied by the DNA molecule, with no allowance for neutralization of the negative charges, the bending of the DNA molecule, the unwinding of the double helix during replication and transcription, etc. It is hard to imagine that DNA could occupy more than 10% of the volume of a cell and still function. Thus, a cell that contains 250 genes that occupy 10% of its volume would be 172 run in diameter, while one containing 750 genes occupying the same relative volume would be 248 nm in diameter. Assuming such cells contain 20% by volume protein and 10% by volume ribosomes,

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the 172 nm cell could accommodate 64 ribosomes and over 16,000 proteins, or 65 per gene, and the 248 nm cell could contain three times as much. It may be concluded that the minimum theoretical size for a cell is 172 nm diameter. To grow, such a cell must be supplied with (and must assimilate) all amino acids, fatty acids, nucleotides, cofactors, etc., because it would contain the minimum number of genes (250) and have a minimal biosynthetic capacity. The cell would have a 5 nm membrane but no cell wall. It would consist, by volume, of 10% DNA, 10% ribosomes, 20% protein, and 50% water, and would contain approximately 65 proteins per gene as well as 65 ribosomes. In comparison, a cell with a much higher biosynthetic capacity, such that it could synthesize all cellular components from CO2, would be 248 nm in diameter, assuming that its DNA is also 10% of the cell volume. Note that these calculations assume a theoretical minimum gene content, which is about half of that present in known life-forms. The amount of DNA in a known autotrophic organism (approximately 1,500 genes in A. aeolicus) would require a cell of at least 314 nm diameter, assuming that it occupied 10% of the cell by volume. Hence, depending on the biosynthetic capacity of a cell, and the extent to which the calculated minimum gene content (7) is realistic, its minimum diameter is between 172 and 314 nm. Overall, one can conclude that microorganisms cannot have diameters significantly less than 200 nm if they have the same basic biochemical requirements for growth as all other extant life-forms, but even then they would require very specialized environments. More likely, the absolute minimum size is closer to 250 nm where the cell is able to grow on simple compounds commonly found in various natural environments. Acknowledgments I thank Juergen Wiegel, Kesen Ma, and Jim Holden for helpful discussions. References 1. McKay D.S., Gibson E.K., Thomas-Keprta K.L., Vali H., Romanek C.S., Clemett S.J., Chillier X.D.F., Maechling C.R., Zare R.N. (1996) Search for past life on Mars—possible relic biogenic activity in Martian meteroite ALH84001. Science 273, 924-930. 2. Eguchi M., Nishikawa T., Macdonald K., Cavicchioli R., Gottscha J.C., Kjelleberg S. (1996) Responses to stress and nutrient availability by the marine ultramicrobacterium Sphingomonas sp. strain RB2256. Appl. Environ. Microbiol. 62, 1287-1294. 3. Kajander E.O., Kuronen I., Akerman* K., Pelttaari A., and (Çiftçioglu N. (1997) Nanobacteria from blood, the smallest culturable autonomously replicating agent on Earth. SPIE 3111, 420-428. 4. Doolittle R.F. (1998) Microbial genomes opened up. Nature 392, 339-342. 5. Fraser C.M., Gocayne J.D., White O., Adams M.D., Clayton R.A., Fleischmann R.D., Bult C.J., Kerlavage A.R., Sutton G., Kelley J.M., Fritchman J.L., Weiman J.F., Small K.V., Sandusky M., Fuhrmann J., Nguyen D., Utterback T.R., Saudek D.M., Phillips C.A., Merrick J.M., Tomb J.F., Dougherty B.A., Bott K.F., Hu P.C., Lucier T.S., Peterson S.N., Smith H.O., Hutchison C.A., Venter J.C. (1995) The minimal gene complement of Mycoplasma genitalium. Science 270, 397-403. 6. Fleischmann R.D., Adams M.D., White O., Clayton R.A., Kirkness E.F., Kerlavage A.R., Bult C.J., Tomb J.F., Dougherty B.A., Merrick J.M., McKenney K., Sutton G., Fitzhugh W., Fields C., Gocayne J.D., Scott J., Shirley R., Liu L.I., Glodek A., Kelley J.M., Weidman J.F., Phillips C.A., Spriggs T., Hedblom E., Cotton M.D., Utterback T.R., Hanna M.C., Nguyen D.T., Saudek D.M., Brandon R.C., Fine L.D., Fritchman J.L., Fuhrmann J.L., Geoghagen N.S.M., Gnehm C.L., McDonald L.A., Small K.V., Fraser C.M., Smith H.O., Venter J.C. (1995) Whole genome random sequencing and assembly of Haemophilus influenzae RD . Science 269, 496-512. 7. Mushegian A.R., Koonin E.V. (1996) A minimal gene set for cellular life derived by comparision of complete bacterial genomes. Proc. Natl. Acad. Sci. USA 93, 10268-10273.

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8. Blattner F.R., Plunkett G., Bloch C.A., Perna N.T., Burland V., Riley M., ColladoVides J., Glasner J.D., Rode C.K., Mayhew G.F., Gregor J., Davis N.W., Kirkpatrick H.A., Goeden M.A., Rose D.J., Mau B., Shao Y. (1997) The complete genome sequence of Escherichia coli K-12. Science 277, 1453-1462. 9. Trevors J.T. (1996) Genome size in bacteria. Antonie van Leeuwenhoek 69, 293-303. 10. Woese C.R., Kandler O., and Wheelis M.L. (1990) Towards a natural system of organisms: proposal for the domains of Archaea, Bacteria and Eucarya. Proc. Natl. Acad. Sci. USA 87, 4576-4579. 11. Stetter, K.O. (1996) Hyperthermophilic prokaryotes. FEMS Microbiol. Rev. 18, 149-158. 12. Bult C.J., White O., Olsen G.J., Zhou L.X., Fleischmann R.D., Sutton G.G., Blake J.A., Fitzgerald L.M., Clayton R.A., Gocayne J.D., Kerlavage A.R., Dougherty B.A., Tomb J.F., Adams M.D., Reich C.I., Overbeek R., Kirkness E.F., Weinstock K.G., Merrick J.M., Glodek A., Scott J.L., Geoghagen N.S.M., Weidman J.F., Fuhrmann J.L., Nguyen D., Utterback T.R., Kelley J.M., Peterson J.D., Sadow P.W., Hanna M.C., Cotton M.D., Roberts K.M., Hurst M.A., Kaine B.P., Borodovsky M. , Klenk H.P., Fraser C.M., Smith H.O., Woese C.R., Venter J.C. (1996) Complete genome sequence of the methanogenic archaeon Mechanococcus jannaschii. Science 273, 1058-1073. 13. Deckert G., Warren P.V., Gaasterland T., Young W.G., Lenox A.L., Graham D.E., Overbeek R., Snead M.A., Keller M., Aujay M., Huber R., Feldma R.A., Short J.M., Olsen G.J., Swanson R.V. (1998) The complete genome of the hyper-thermophilic bacterium Aquifex aeolicus. Nature 392, 353-358.

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Diminutive Cells in the Oceans—Unanswered Questions Edward F. DeLong Monterey Bay Aquarium Research Institute Abstract The marine environment harbors enormous numbers of viruses and prokaryotes, existing in complex communities that span a wide spectrum of biotopes, lifestyles, and size ranges. Many naturally occurring marine bacterioplankton are extremely small, some measuring < 0.3 µm in their largest dimension, having estimated biovolumes as low as 0.027 µm3. Available data suggest that the majority of naturally occurring bacterioplankton resist cultivation, and have not been phylogenetically identified at the single cell level. Phylogenetic evidence for the evolution of major lineages that are characteristically small have not been reported (but they may exist). Because a large fraction of naturally occurring microorganisms have not been cultivated, their specific physiological traits are largely unknown. Consequently, the fraction of very small marine microbes that transiently and reversibly exist as "dwarf cells" is also unknown. Finally, although extremely small (< 0.1 µm) DNA-containing particles are very abundant in seawater and are thought to be viruses, the fraction of these particles that may actually represent cellular organisms is uncertain. Introduction Small microorganisms are ubiquitous in ocean waters, averaging about 5 × 105 cells/ml in the upper 200 m, and 5 × 104 cells/ml below 200 m depth. The total number of prokaryotic cells in ocean waters is about 1 × 1029(1). Assuming a biomass of approximately 20 fg carbon per cell, this represents 2.2 × 1015 g of prokaryotic carbon in the world's oceans. This biomass represents an enormous pool of genetic variability, a large fraction of which is represented by very small cells (2,3). Extremely small cells (< 0.5 µm) may result from a genetically fixed phenotype maintained throughout the cell cycle. Alternatively, very small cells may reflect physiological changes associated with starvation, or other aspects of the cell cycle. Both explanations likely hold for different members of complex mixed populations of small cells found in the ocean. Extremely small (<0.1 µm) DNA-containing particles are also very abundant in seawater, reaching concentrations of about 1 × 107 particles/ml in surface waters (4-6). These small particles are thought to consist largely, although not necessarily entirely, of viruses. Cell dimensions of cultured or naturally occurring bacteria can be derived from several sorts of data, each with inherent limitations. A number of uncertainties can be associated with cell size estimates. Historically, the existence of very small bacteria and viruses was first documented by observations of infectious filterable agents. Indirect cell size estimates have more recently been derived from filter fractionation experiments using membrane filters of uniform pore size. These sorts of size estimates can be compromised by filter trapping effects, as well as differential retention of cells with varying shapes or cell wall compositions. 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). Most estimates by light microscopy, electron microscopy, and flow cytometry also involve the use of fixatives that may cause cell shrinkage or other artifacts (3). Nevertheless, it is apparent that the majority of naturally occurring prokaryotes in marine plankton are

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about 1.0 µm or less in their largest dimension, and a good number of these are 0.5 mm or less in diameter (2,3). Critical Assessment of the Issue 1. What is the phylogenetic distribution of small bacteria? This question can be broken down into several components: A. What is the phylogenetic distribution of cultivated prokaryotes with a stable, very small cell size? The ongoing efforts of microbiologists to cultivate new microbial groups are currently providing new perspectives and answers to this question. It is still an open-ended question, because new microbial groups continue to yield to cultivation efforts. Recently isolated bacteria having stable, maximal dimensions of around 0.5 µm, fall into the alpha Proteobacterial lineage, as well as the Bacterial order Verrucomicrobiales. Very small bacteria in the order Verrucomicrobiales have been recently isolated. New strains isolated from an anoxic rice paddy displayed a stable cell size of about 0.5 µm in length and 0.35 µm in diameter yielding a cell volume of about 0.03 µm3 (8). These bacteria were oxygen-tolerant heterotrophs, exhibiting strictly fermentative growth on sugars. Other cultivated relatives, including Verrucomicrobium spinosum, are generally larger than 1 µm and possess prosthecae (9). Small cell size is therefore not an inherent property of members of this order. A very small marine isolate with cell volume ranging from 0.03 to 0.07 µm3 was isolated using the dilution culture technique of Button and Schut (10). This isolate was found to be associated with the alpha Proteobacterial genus Sphingomonas (11). Sphingomonas sp. strain RB2256 is heterotrophic, contains about 1.5 fg DNA/cell, and grows at a maximal rate of about 0.16 hr-1. This marine Sphingomonas isolate showed very little variation in growth rate or cell size in response to 1,000-fold variation in nutrient supply, indicating the stability of the small cell phenotype (12). Other Sphingomonas species have larger, more typical cell sizes, so diminutive size is not a specific characteristic of the genus. Nanobacteria species have been reportedly found in association with human and cow blood (13). They have been cultured in serum-free media, and have cell diameters, estimated from electron microscopy, of 0.2 to 0.5 µm (13). They have been reported to pass through 0.1 mm falters, apparently due to pleomorphic forms even smaller, about 0.05-0.2 µm (13). Ribosomal RNA sequences originating from these microorganisms are associated with the alpha subdivision of the Proteobacteria, and are most closely related to Phyllobacterium rubiacearum. B. What is the phylogenetic distribution of cultured prokaryotes that undergo an induced cell cycle transition from a "typical" to very small cell size? 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 µm3. Different bacterial genera have been reported to undergo a starvation-induced response resulting in cell miniaturization, including the gamma Proteobacteria genera Vibrio, Pseudomonas, Alcaligenes, Aeromonas, and Listonella (14). This reduction in cell size may be a common phenomenon for heterotrophic microorganisms adapted for growth at relatively high nutrient concentrations. In many of these microorganisms, the transition from large to

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dwarf cells is fully reversible upon nutrient upshift. This physiological strategy appears to be common, but its actual distribution among diverse bacterial phyla is poorly characterized. It is unknown what fraction of naturally occurring "ultramicrobacteria" represent typically larger cells that have experienced nutrient downshift and undergone cellular miniaturization. It is also not clear what fraction of these readily reverse to a large actively growing state (15), or alternatively have entered a hypothetical "viable but nonculturable" state (16). C. What are the phylogenetic identities of (uncultivated) very small cells frequently observed in natural environmental samples? This remains an open question. It has been estimated that only about 0.1-1% of naturally occurring prokaryotes have been cultivated from many specific habitats (17,18). Culture independent surveys have indicated the presence of many new, yet uncultivated, and previously unrecognized prokaryotic groups (19). Most of these have not yet been specifically identified at the single cell level. It will be interesting to determine whether a significant fraction of recently discovered, uncultivated prokaryotic groups represent some of the more diminutive cell forms. Are there inherent properties of very small cell lineage that render them recalcitrant to cultivation? 2. Is there a relationship between minimum size and environment? In low-nutrient habitats in marine plankton, cells typically appear smaller in size than those of comparable higher nutrient habitats. To the extent that some cells undergo a starvation response that involves reduction in cell size, there may be a loose relationship between cell size and ambient nutrient concentration. However, it is still unknown what fraction of naturally occurring small cells represent physiologically induced forms, versus stable, diminutive phenotypes. Smaller cells have a greater surface area to volume ratio, postulated to be adaptive for low-nutrient environments (11). However, small cell size does not necessarily imply adaptation to an oligotrophic (low-nutrient) lifestyle. For instance, new Verrucomicorbiales isolates (8) grow well and maintain small cell size under relatively high nutrient growth conditions (e.g., 4 mM glucose, or 0.1% starch). Nanobacteria dwell (and are cultivated) 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). It is possible that symbionts in environments rich with host-supplied growth factors may actually have reduced genetic and physiological demands, thereby facilitating cell size reduction. It is possible that small cell size is adaptive for free-living cells in low nutrient environments, but symbiotic species may tend toward small cell size in a nutrient-replete environment provided by the host. 3. Is there a continuum (or quanta?) of size and complexity that links conventional bacteria and viruses? Direct examination of concentrated seawater samples by electron microscopy have revealed the presence of large numbers of vital-like particles (VLPs) in the world's oceans (4,5). Ranging in numbers from about 2 × 105 to 5 × 108 particles/ml, VLP numbers typically exceed bacterial cell numbers in aquatic samples by 10-fold. Most quantitative studies to date have employed ultracentrifugation or ultrafiltration coupled with electron microscopy, or filtration, fluorescent DNA staining, and epifluorescence microscopy. A few studies have succeeded in enumerating naturally occurring viable

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infectious particles (especially in marine Synechococcus sp.) to determine the host range, in situ titers, and ecological variability of naturally occurring cyanophages (21). In the marine environment there is certainly a continuum of size in both bacterioplankton and virioplankton. Bacterioplankton can range from large filaments > 10 µm, to small coccoid cells with diameters approaching 0.3 µm (2). Marine virus isolates range in length from about 40 nm to as large as 120 nm (5). Electron micrographs of naturally occurring infected cells suggest that some bacterial hosts are considerably less than 10-fold larger than their vital parasites, having a burst size of about 7 (6)! 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. Commonly used epifluorescence techniques are convenient and reproducible, but the identity of the fluorescently stained particles is certainly subject to some uncertainty. What fraction of VLPs are actually viruses? What fraction of VLPs are viable viruses? What fraction of DNA-containing particles < 0.1 µm are actually cells, and not viruses? If some of the < 0.1 µm DNA-containing particles are cells, are they viable? These remain open-ended questions. With regard to the complexity of these populations, the issue of cultivability is a serious one. It still appears from available data that a large fraction of naturally occurring microbes have resisted cultivation attempts. The specific physiological traits and life histories of these microorganisms remain unknown, as does that of their vital parasites. A major challenge to contemporary microbiology is to devise and implement approaches to better characterize this large and uncharacterized biota. References 1. Whitman, W.B., Coleman, D.C., Wiebe, W.J. (1998), Proc. Natl. Acad. Sci. USA 95:6578-6583. 2. Watson, S.W., Novitsky, T.J., Quiaby, H.L., Valois, F.W. (1977), Appl. Environ. Microbiol. 33:940-946. 3. Fuhrman, J.A. (1981), Mar. Ecol. Prog. Ser. 5:103-106. 4. Bergh, O., Borsheim, K.Y., Bratbak, G., Heidal, M. (1989), Nature (London) 340:467-468. 5. Borsheim, K.Y. (1993), FEMS Microb. Ecol. 102:141-159. 6. Steward, G.F., Smith, D.C., Azam, F. (1996), Mar. Ecol. Prog. Ser. 131:287-300. 7. Suzuki, M.T., Sherr, E.B., Sherr, B.F. (1993), Limnol. Oceanogr . 38:1566-1570. 8. Janssen, P.H., Schuhmann, A., Morschel, E., Rainey, F.A. (1997), Appl. Environ. Microbiol. 63:1382-1388. 9. Hedlund, B.P., Gosnik: J.J., Staley, J.T. (1996), Appl. Environ. Microbiol. 46:960-966. 10. Schut, F., DeVries, E.J., Gottschal, J.C., Robertson, B.R., Harder, W., Prins, R.A., Button, D.K. (1993), Appl. Environ. Microbiol. 59 :2150-2160. 11. Schut, F., Prins, R.A., Gottschal, J.C. (1997), Aquat. Microb. Ecol. 12:177-202. 12. Eguchi, M., Nishikawa, T., MacDonald, K., Cavicchioli, R., Gottschal, J., Kjelleberg, S. (1996), Appl. Environ. Microbiol. 62:1287-1294. 13. Kajander, E.O., Çiftçioglu, N. (1998), Proc. Natl. Acad. Sci. USA 95:8274-8279. 14. MacDonnell, M.T., Hood, M.A. (1982), Appl. Environ. Microbiol. 43:566-571. 15. Kjelleberg, S., Hermansson, M., Marden, P. (1987), Ann Rev. Microbiol . 41:25-49. 16. Rozak, D.B., Colwell, R.R. (1987), Microbiol. Rev. 51:365-379. 17. Staley, J.T., Konopka, A. (1985), Ann. Rev. Microbiol. 39:321-346. 18. Amann, R.I., Ludwig, W., Schleifer, K.H. (1995), Microbiol. Rev . 59:143-169. 19. Pace, N.R. (1997), Science 276:734-740. 20. Fraser et al. (1995), Science 270:397-403. 21. Waterbury, J.B., Valois, F.W. (1993), Appl. Environ. Microbiol . 59:3393-3399.