Ultramicrobacteria - Penn State University

Ultramicrobacteria

Secondary article Article Contents

Ricardo Cavicchioli, University of New South Wales, Sydney, Australia Martin Ostrowski, University of New South Wales, Sydney, Australia

. Introduction . UMB: an Ecological Perspective

Ultramicrobacteria are microorganisms with a cell volume of less than 0.1 mm3 which have been cultivated from aquatic and soil environments. Nan(n)obacteria are reported to be even smaller than ultramicrobacteria and have been described in deep subsurface, meteorite and clinical samples. The size of these tiny microorganisms challenges views about the minimal size of a living cell.

. The Proliferation of Terms . Marine UMB . Clinical UMB and NB . Soil UMB . Subsurface NB . Minimum Cell Size

Introduction The size of microorganisms varies considerably. Even amongst the prokaryotes (Archaea and Bacteria) cells with volumes of 0.02–180 000 000 mm3 have been isolated. At the smallest end of this size spectrum are the ultramicrobacteria (UMB). UMB are highly abundant in most natural aquatic and terrestrial environments and for years microbial ecologists have recognized that they play important roles in the biological cycling of nutrients and in the formation of biomass. More recently, nan(n)obacteria (NB) have been reported in human kidney stones, deep subsurface samples and as fossil remains in meteorites. Accompanying these new findings has been intense discussion debating the minimum size for a living cell. It is clear that UMB and NB impact on fields of study as diverse as microbial ecology, genomics and astrobiology. This article examines the enthusiasm for research on UMB and NB. It focuses on many of the fascinating discoveries in the field, and attempts to separate fact from fiction, and demystify the jargon which has proliferated.

UMB: an Ecological Perspective The term ‘ultramicrobacteria’ was first adopted by Torella and Morita (1981) to describe extremely small bacteria (less than 0.3 mm diameter) isolated from seawater that formed ‘ultramicrocolonies’ on agar plates, retained their small cell size when growing on agar plates, and grew very slowly in the presence of high concentrations of nutrients. MacDonell and Hood (1982) modified this description to include isolates from an estuary obtained by filtration through a 0.2-mm membrane and which could form normal-sized colonies on low-nutrient agar. In their review, Schut et al. (1997) further modified the description of UMB to include microorganisms which have a cell volume of less than 0.1 mm3, and which retain this volume irrespective of growth conditions. This description, using volume as the defining criterion, is particularly useful for studies of natural communities as a range of cell shapes is often encountered, and volume provides a measurement of

size that is independent of shape. A list of criteria defining UMB is also described by Velimirov (2001). There are two distinct types of cells from the environment which have small volumes: UMB and ultramicrocells (UMC). UMC are characterized by a larger sized (greater than 0.1 mm3) reproductive form, and a miniature form that may have a volume of less than 0.1 mm3. Unlike UMB, which retain a volume of less than 0.1 mm3 when growing, the miniature form of UMC is a dormant, stress-resistant cell, similar to a spore in differentiating bacteria. The presence of distinct classes of tiny microorganisms is consistent with studies of natural aquatic and soil communities. Microscopy observations of environmental samples reveal cells with volumes of 0.02–0.12 mm3 (Schut et al., 1997). However, once the environmental samples have been cultured on agar plates, cells typically have volumes of the order of 0.34–6.4 mm3. Many of the larger cells are UMC and in practice UMC are more easily isolated than UMB. However, since the discovery of true UMB from marine and soil environments, a key area of research in contemporary microbial ecology has been to determine the proportion and physiological state of UMB and UMC, and to determine the contribution of each class in the ecology of their respective environments.

The Proliferation of Terms Marine ecologists describe a size-graded series of plankton with dimensions ranging from 0.02 mm to 200 cm (Sieburth et al., 1978). The terms femto-, pico-, nano-and microplankton are used to distinguish size classes with 10-fold increments between 0.02 mm and 200 mm, respectively (Table 1). According to these descriptions, UMB most closely correlate with femtoplankton (or femtobacterioplankton). A range of other terms have been coined to describe small microorganisms (Table 1). The prefix recently adopted in a number of fields is ‘nano’; e.g. nan(n)obacteria (NB), nanobe, nanocell and nanosize. The use of ‘nano’ in

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Table 1 Description of terms relating to very small microorganisms, or structures thought to be microorganisms or of microbial origin Ultramicrobacteria (UMB) Microorganisms with a cell volume of less than 0.1 mm3 that maintain their size with only minor changes, irrespective of growth conditions. Observed by light microscopy Ultramicrocells (UMC) Smaller forms (usually starved) of microorganisms that are larger when actively growing. Usually associated with reductive cell division during starvation. Observed by light microscopy Nan(n)obacteria (NB) Possible synonym for UMB. In the literature usually associated with structures in geological samples with sizes ranging from 0.01 to 0.1 mm. Usually associated with uncultured and unsubstantiated descriptions of microorganisms. Observed by electron microscopy Microfossils Mineral structures with morphologies and dimensions that resemble microbial cells. May be formed as a result of biomineralization of cell surfaces. Observed by electron microscopy Femtoplankton Marine microorganisms 0.02–0.2 mm Picoplankton Marine microorganisms 0.2–2.0 mm Nanoplankton Marine microorganisms 2.0–20 mm Microplankton Marine microorganisms 20–200 mm Other related terms Dwarf cells/bacteria, lilliputanian cells, femtobacterioplankton, miniature cells/bacteria, nanocells, nanosized, nanobe, nano-organisms, nannograins, nanofossils

this context is intended to refer to a size range much smaller (e.g. tens of nanometers) than in the definition of nanoplankton (0.2–2 mm). The use of the term ‘nanobacteria’ may derive from Morita (1988) where it was described as a synonym for UMB. This usage may be consistent with that of Hamilton (2000), who described NB as ‘extremely small cellular forms’, and Uwins et al. (1998) who described nanobes as having a ‘significantly different size to bacteria and archaea’. However, the term is being increasingly equated with sizes of objects that may not be large enough to support life (reviewed in Velimirov, 2001; Trevors and Psenner, 2001). Most of the recently described NB come from geological or mineral samples, and none of these have been conclusively proven to be biological life forms. Moreover, the term has been used to describe microfossils, for which proof of origin is even more difficult to establish. In view of this, Southam and Donald (1999) argue that NB should no longer be used to describe geomicrobiological formations. Irrespective of personal view and convention, it is likely that the term ‘nanobacteria’ and other associated ‘nano’ terms will continue to evolve and continue to be used. This is well illustrated by the description of the first ‘nano’ archaeal isolate which has been formerly proposed as Nanoarchaeum equitans, within the new archaeal kingdom Nanoarchaeota (Huber et al., 2002).

Marine UMB UMB appear to be most prevalent in oligotrophic environments, such as the open ocean, where the concentration of microorganisms is of the order of 105 – 106 cells mL 2 1 (Schut et al., 1997). At least one-third of the ocean is oligotrophic and the total number of 2

microorganisms in the ocean is predicted to be approximately 1030 cells (reviewed in Cavicchioli et al., 2003). This implies that UMB make an enormous contribution to microbial biomass and are likely to play a key role in biogeochemical cycling of organic and inorganic matter. According to the definition of a UMB (volume less than 0.1 mm3) only one marine strain has been isolated and extensively studied: Sphingopyxis alaskensis (formerly Sphingomonas alaskensis) (Table 2). However, a number of other marine bacteria that are only marginally larger have also been isolated, including the photosynthetic cyanobacterium Prochlorococcus (Table 2). A range of morphological, physiological and ecological characteristics of UMB and small microorganisms are summarized in Table 3. The fact that UMB are often associated with oligotrophic conditions is consistent with these small-sized cells having a high surface area to volume ratio, which enhances the opportunity for cells to uptake nutrients from the environment. By containing less mass, UMB also require less nutrients than larger cells to produce progeny. UMB may also be less subject to grazing pressure by larger predators (e.g. marine protozoa). S. alaskensis fulfils many of the criteria expected for a model oligotroph (Table 3 and reviewed in Cavicchioli et al., 2003). These properties are consistent with a UMB that is adapted to growth under nutrient limitation and not simply a dormant member of the population. These physiological properties are corroborated by ecological data demonstrating the presence of S. alaskensis as one of the most numerically abundant microorganisms from a number of ocean sites (reviewed in Cavicchioli et al., 2003). There are many examples of UMC from the marine environment. Changes in cell volume for UMC are associated with growth phase and culture conditions. For example, the cell volume of Vibrio sp. ANT 300 may vary


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by more than an order of magnitude, from 0.048 to 5.9 mm3. The small cells are typically produced by the process of reductive cell division during periods of nutrient deprivation. In contrast to UMC, growth and media conditions have a minimal effect ( 2-fold) on the cell volume of the UMB S. alaskensis (reviewed in Cavicchioli et al., 2003). An important hurdle in this field is the isolation of UMB from the environment. Insight into overcoming this problem can be gained from the isolation attempts which yielded S. alaskensis. Strains from waters near Alaska, the North Sea and Japan were obtained using an extinction dilution method. This relies on diluting samples in low nutrient media until the final dilution tube contains the most abundant members of the population which are able to grow in the media. The next phase in the isolation is storage of cells at low temperature in low-nutrient liquid medium for up to 12 months, with monthly culturing on to solid medium. A consistent finding for S. alaskensis was initial growth of cells only in liquid medium, with the eventual formation of colonies on solid media. It is important to establish if these methods can be successfully used to isolate UMB other than Sphingomonads from these environments. Filtration has also been employed, although unsuccessfully, to isolate UMB from the ocean (reviewed in Cavicchioli et al., 2003; Velimirov, 2001). Isolates obtained from the filtrates of 0.2-mm filters have been larger than UMB and are likely to be UMC. This method, however, may be useful if the filtrates are initially grown in low-nutrient liquid medium, and then processed in a similar way to that described for the extinction dilution cultures. Unique to the oligotrophic waters that have yielded S. alaskensis, are the remarkable hydrothermal vent systems. Within the vent fluid a diverse range of thermally adapted (hyperthermophilic) microorganisms have been isolated. Most of these are archaea and many of them have unusual

cell shapes and develop a large variation of cell sizes (Workshop, 1999). Species such as Thermodiscus produce flat disks with a thickness of only 0.1–0.2 mm, however their diameter is 0.2–3.0 mm. A variable cell size appears to be characteristic of these archaea and growth conditions producing batches with homogeneous cell sizes have not been established. In effect these archaea may be equated to extreme UMC. In contrast to the size-variable hyperthermophiles, the recently described archaeal symbiont Nanoarchaeum equitans appears to consistently form coccoidshaped cells with a diameter of 0.4 mm (Huber et al., 2002). The cells require an actively growing archaeal host (a species of Ignicoccus) and grow at 70–988C. During late exponential growth phase, the N. equitans cells detach from Ignicoccus. In this form they contain a small genome of approximately 0.5 Mb. While this UMB is not free-living, it provides a unique opportunity to examine the growth and survival strategies of an archaeal UMB.

Clinical UMB and NB Clinically important and obligately parasitic UMB and UMC have been isolated that belong to a few distinct taxonomic groups. Most of the intracellular parasites and vertebrate pathogens are members of the Bdellovibrio, Brucella, Mycoplasma, Rickettsia and Chlamydia genera. The latter four groups are parasitic or pathogenic in humans and animals while the bdellovibrios are parasites of Gram-negative bacteria. The pathogenic UMB are fastidious and have a strict host dependency. They have incomplete cell structures and metabolic pathways, and possess extremely small genomes (as small as 0.5 Mb). It will be interesting to compare the minimal genomes of these parasites with that of the archaeal symbiont N. equitans. Genes in common will represent those that are


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Table 3 Characteristics of selected UMB and small microorganisms Marine isolates

Characteristics

Sphingopyxis alaskensis

Isolated as an abundant species from ocean waters near Alaska and in the North Sea and the oligotrophic North Pacific Ocean High-affinity, broad-specificity nutrient-uptake systems predicted to enable successful competition in oligotrophic waters 3.2 Mb genome Single copy of rRNA operon, maximum 2000 ribosomes cell 2 1, minimum 200 ribosomes cell 2 1 High level of regulation of proteome and rates of ribosome, protein and RNA synthesis Intrinsically resistant (no starvation cross-protection) to oxidative stress, UV, ethanol, high temperature and antibiotics. Even higher levels of resistance to hydrogen peroxide during low, nutrient-limited growth Most abundant phototrophic organism Global distribution between 408N and 408S and 0–200 m depth Smallest known phototroph 1.8 Mb genome Accounts for more than 50% of chlorophyll and contributes 30–80% of the total photosynthesis in the oligotrophic oceans Isolated from same location as S. alaskensis from Resurrection Bay, Alaska Dilute cytoplasm 3 Mb genome Utilizes only a few aromatic hydrocarbons and acetate as growth substrates Kinetic constants for uptake compatible with growth on ambient concentrations of nutrients in seawater Hyperthermophilic archaeon Isolated from a hydrothermal vent Symbiosis with archaeal host, Ignicoccus sp. Represents a previously unknown phylum of Archaea Harbours the smallest archaeal genome of 0.5 Mb

Prochlorococcus marinus

Cycloclasticus oligotrophus

Nanoarchaeum equitans

Soil isolates Verrucomicrobiales lineage Pseudomonas sp. & Xanthomonas sp. Clinical isolates Mycoplasma genitalium

Chlamydia

Anaerobic heterotroph isolated from anoxic rice paddy soil Abundant isolate Aerobic copiotrophic heterotrophs isolated from polluted soil in Japan

Pathogen of animals Fastidious and host dependent for growth Anaerobic or facultatively anaerobic Devoid of cell walls and surrounded by only a plasma membrane Smallest genome (0.58 Mb) completely sequenced 468 predicted protein encoding genes Obligate intracellular parasite of animals and insects 1.0–1.2 Mb genome

important for a microorganism with a minimal genome, irrespective of the cell’s environment. Mycoplasma and Chlamydia form ‘elementary bodies’ as small as 0.13 mm (Morowitz, 1967). These forms may survive out of their host and remain infectious, however they do not represent the main growing forms of the cells. For example, actively growing Chlamydia are typically 4

0.6–1.5 mm in diameter. This changeable cell size conforms to the description of UMC. NB have been reported from human and cow blood and commercial cell culture serum (Kajander and Ciftcioglu, 1998). The isolated NB are reported to produce biogenic apatite on their cell envelope and be responsible for pathogenic calcification observed in a number of human


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diseases, including kidney stone formation. The organisms causing this are reported to be coccoid with a cell size of 0.2–0.5 mm (0.1–0.7 mm3) when growing. However, smaller forms (0.05–0.2 mm diameter) have also been described and these are believed to account for the ability of the NB to pass through 0.1-mm membrane filters and establish growing cultures. There is no evidence provided to substantiate that the smaller forms are complete, live cells. It is possible that the smaller forms are staining artefacts, or are UMC that re-establish growth after filtration, similar to those observed for the marine strains. Alternative explanations for the nanobacterial-induced biomineralization have been provided by Cisar et al. (2000). They isolated mineralized particles from fetal blood serum, human saliva and dental plaque. The particles possessed all of the properties of the NB, including the ability to initiate biomineralization. However, the authors could find no evidence that the particles were living entities and suggested that the biomineralization was dependent on the nucleating activities of nonliving biological macromolecules. In addition they provided evidence that Nanobacterium sanguineum, which was implicated as the aetiological agent of kidney stone formation, was caused by a problem of experimental design (i.e. polymerase chain reaction (PCR) artefact or a contaminant organism).

Soil UMB Cells with volumes conforming to the definition of UMB have routinely been observed in soil samples. However, similar to the situation for UMB from aquatic environments, it has been difficult to establish whether the small cells represent UMB or UMC. In 1997, Janssen et al. reported the isolation of three UMB from rice paddy soil. The isolates belong to the order Verrucomicrobiales, are anaerobic heterotrophs and retain their ultramicro-size (0.03–0.04 mm3) even when grown in rich media. The isolation of these strains demonstrates that UMB and not just UMC are present in the soil. Furthermore, the isolates were obtained from a high dilution culture series, indicating that they were abundant in the original soil sample. Iizuka et al. (1998) isolated a number of aerobic, heterotrophic bacteria with cell volumes ranging from 0.07 to 0.22 mm3, from polluted soil in Japan. Three isolates with volumes of 0.07–0.08 mm3 and one isolate with a volume of 0.12 mm3 were reported to be closely related to the species Pseudomonas lemoignei. Two additional isolates with volumes of 0.11 mm3 were closely related to Xanthomonas campestris. The bacteria were obtained by plating soil suspensions directly on to nutrient-rich media (tryptic soy agar). As a result the strains were described as copiotrophic (as opposed to oligotrophic). The strains similar to X. campestris grew rapidly (0.67 h 2 1) in tryptic soy broth, whereas two of the strains similar to P. lemoignei

grew slowly (less than 0.10 h 2 1) in the rich media but more rapidly in a defined medium (0.17 h 2 1). The strains isolated by Janssen et al. (1997) and Iizuka et al. (1998) provide excellent resources for probing the physiology of soil UMB.

Subsurface NB Preliminary evidence has been found for the existence of NB or nanobes as small as 0.01 mm in diameter in a variety of geological samples from the deep subsurface. In one publication by Uwins et al. (1998), filamentous forms 0.020–1.0 mm in diameter were identified from sandstone samples taken from a depth of 3400–5100 m below the sea bed where the pressure is 2000 atm and the temperature 115–1708C. They subsequently identified, on freshly fractured rocks, similar structures that appeared to grow spontaneously by forming colonies after 2–3 weeks in the air at 228C. Similar formations also appeared on copper, polystyrene and glass surfaces. The structures in the newly formed colonies were filaments with diameters of 0.05– 0.1 mm. The nanobes were described as exhibiting structures similar to fungi with membranes and a cell wall. They were also reported to be composed mainly of carbon, nitrogen and oxygen and to contain DNA. Similar deep subsurface NB have been reported by Hamilton (2000), however no independent publications have appeared to verify the existence of the NB from either of these groups. Folk (1999) reports the existence of NB in a broad variety of locations from diverse geological to exotic sites including birdbaths and groundwater pipes. While the work by Folk and colleagues does not include a search for biological markers (other than morphology) they provide hypotheses for how the formations may occur. These include carbonate precipitations as a result of heterotrophic growth, or bacteria acting as nucleating centres. The reporting of NB in easily accessible and available samples (e.g. water pipes) provides the opportunity to substantiate the existence of NB by growth and other studies, but to date this has not occurred. Contributing to the controversy surrounding the existence of NB as living entities is the fact that the term has been used to describe microfossils that are thought to be the remains of NB. Deducing a biological origin for microfossils is clearly a difficult task as the causative agent may never be cultured. As a consequence, a range of circumstantial evidence must be used to derive a hypothesis. This evidence must include more than just structural morphology as it has been clearly shown that the inorganic processes may produce structures that look indistinguishable from biological cells (Southam and Donald, 1999; Ruiz et al., 2002). Recently, Raman spectroscopy, chemical composition and morphology was combined to determine whether 3.5-billion-year-old microfossils were of biological origin. Despite the use of these new


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methods, an editorial in the journal Nature (2002, 417: 782– 784) described reports from two independent groups which reached opposite conclusions about the same microfossils. A similar level of controversy exists for the structures observed in the Martian meteorite ALH84001 (McKay et al., 1996), where, for example, the structure of magnetite crystals was used to infer a biological origin (Thomas-Keprta et al., 2001) or contradict a biological origin (Barber and Scott, 2002). It is important to establish whether the NB in geological samples include live biological forms as this will substantiate the novelty of claims and may guide studies of microfossils. Moreover, it will clarify the targets for searches of life on Earth and in extraterrestrial environments (Cavicchioli, 2002).

Minimum Cell Size Estimates for minimum cell sizes have been proposed based on observations of the smallest sizes of cells in environmental and clinical samples, estimations of the volume occupied by the minimum number of macromolecules required for a living cell, or based primarily on estimations of the minimum size of a genome. Using these types of approaches, most reports estimate the minimum size of a cell to be in the range 0.1–0.3 mm in diameter. In 1967, Morowitz examined the physical and biochemical factors that may limit the ability of a biological entity to replicate. He deduced a physical limit for the size of a cell by calculating the number of atoms present within a defined size. For example, a cell with a diameter of 0.2 mm could contain about 4 108 atoms. Underlying principles for a living cell were then examined and ten generalizations were derived (Table 4). These included the requirement for a membrane, and a minimum set of organic molecules. The average size of essential components (e.g. a molecular weight of 40 000 for an enzyme) were combined to derive a formula for the minimum radius of a cell. The formula also incorporated a factor (n) for biochemical complexity, based on the number of enzymes required to fulfil cellular

function. Where n is 45, Morowitz calculated the minimum size to be a sphere with a diameter of about 0.1 mm. When considering 45 as a minimum level of complexity, Morowitz noted that additional complexity should be incorporated to account for the fidelity of replication and adaptation to environmental changes. ‘Complexity’ described by Morowitz (1967) may be equated to the number of protein-encoding genes in a genome. Mycoplasma genitalium has one of the smallest genomes known and is predicted to encode 468 proteinencoding genes. Using M. genitalium as a model, the minimum number of genes for a viable genome has been calculated as 250–350 (Mushegian and Koonin, 1996). Using this level of complexity and the formula derived by Morowitz (1967) the minimum cell diameter calculated is 0.16 mm or 0.17 mm, respectively. Genomic approaches have helped to clarify the minimum number of genes within known functional classes (e.g. energy generation, translation; reviewed in Trevors and Psenner, 2001). Interestingly, genomics has also led to the identification of a large number of uncategorized genes which appear to be essential for cell viability. Until the role of these hypothetical yet essential genes are determined in a wide variety of cell types it will limit the accuracy with which predictions may be made about the requirements of a cell with a minimum genome. The announcement of ovoid features resembling NB in the Martian meteorite ALH84001 (McKay et al., 1996) has invigorated debate about the minimum size of cells (Maniloff et al., 1997). This has also led to a broader debate in astrobiology, including discussions on the abiotic conditions tolerated by biological life forms; in particular, by extremophiles (Cavicchioli, 2002). In October 1998 the US National Academy of Sciences (NAS) held a workshop to discuss the size limits of very small microorganisms (Workshop, 1999). Conclusions from the workshop included a minimum viable diameter of 0.25–0.3 mm, and minimum genome size of 250–450 genes. An important factor highlighted for determining cell size was the number of ribosomes. This is due to the significant size of a

Table 4 General principles of conventional biological self-replicating systems (derived from Morowitz, 1967) 1. Biological information is structural 2. Functioning biological systems are cellular in nature 3. There is a universal type of membrane structure utilized in all biological systems 4. All populations of replicating biological systems give rise to mutant phenotypes 5. There is a ubiquitous and restricted set of small organic molecules which constitute a very large fraction of the total mass of all cellular systems 6. Biological energy utilization is accompanied by the hydrolysis of phosphate bonds, usually those of ATP 7. All replicating cells have a genome made of DNA which stores the genetic information of the cell that may be read out in sequences of RNA and translated into polypeptides 8. All growing cells have ribosomes which are the site of protein synthesis 9. The translation of information from nucleotide language to amino acid language takes place through specific activating enzymes and tRNA 10. The major component of all cellular systems is water

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ribosome which has a diameter of approximately 0.02 mm and is predicted to be 0.057 mm in diameter when surrounded by a membrane and cell wall. The NAS meeting also identified two environments that may harbour cells with minimum cell sizes. Consistent with earlier views of Morowitz (1967), nutrient-rich (eutrophic) environments with low levels of abiotic fluctuations were thought to allow the evolution of cell types with reduced genetic and physiological capacity; examples are parasitic and symbiotic archaea and bacteria (Table 2). Alternatively, oligotrophic environments may enrich for microorganisms that maximize nutrient uptake ability by increasing their surface to volume ratio; examples include the marine UMB. The most comprehensive understanding of the physiology of a free-living UMB is the marine bacterium S. alaskensis from oligotrophic marine environments (Table 3). Some of the studies describing this UMB may shed light on the discussion of minimum cell size. S. alaskensis maintains a maximum of 2000 ribosomes per cell (reviewed in Cavicchioli et al., 2003). However, with just 200 ribosomes per cell it is able to respond to the addition of nutrients and reach maximum rates of growth without any growth lag. In fact, the ribosome content appears to be excessive at certain stages of growth and ribosomes may perform functions in addition to protein synthesis. To date, S. alaskensis sets an upper-limit for the minimum size of a cell (0.024 mm3) and the minimum number of ribosomes (200). The genome size for S. alaskensis is 3.2 Mb (reviewed in Cavicchioli et al., 2003) which may be larger than expected for a ‘minimalist’ bacterium. The fact that it maintains a genome of this size implies that it provides a competitive advantage enabling it to remain numerically abundant in the world’s oceans. Its genomic capacity may relate to its ability to mount significant changes in gene expression, maintain intrinsically high levels of stress resistance, immediately respond to nutrient availability, and scavenge and utilize oligotrophic levels of nutrients. It is also noteworthy that in the context of the theoretical predictions of minimum cell size (Morowitz, 1967), a complexity level of 3200 results in the calculation of a diameter of 0.34 mm and volume of 0.021 mm3. This predicted volume is equivalent to the minimum size observed for these rod-shaped cells (Table 2; reviewed in Cavicchioli et al., 2003).

Acknowledgements Research conducted in the lab of RC which is presented in this chapter was supported by the Australian Research Council.

References Barber DJ and Scott ERD (2002) Origin of supposedly biogenic magnetite in the Martian meteorite Allan Hills 84001. Proceedings of the National Academy of Sciences of the USA 99: 6556–6561. Cavicchioli R (2002) Extremophiles and the search for extra-terrestrial life. Astrobiology 2: 281–292. Cavicchioli R, Ostrowski M, Fegatella F, et al. (2003) Life under nutrient limitation in oligotrophic marine environments: an eco/physiological perspective of Sphingopyxis alaskensis (formerly Sphingomonas alaskensis). Microbial Ecology (in press). Cisar JO, Xu DQ, Thompson J, et al. (2000) An alternative interpretation of nanobacteria-induced biomineralization. Proceedings of the National Academy of Sciences of the USA 97: 11511–11515. Folk RL (1999) Nannobacteria and the precipitation of carbonate in unusual environments. Sedimentary Geology 126: 47–55. Hamilton A (2000) Nanobacteria: gold mine or minefield of intellectual enquiry? Microbiology Today 27: 182–184. Huber H, Hohn MJ, Rachel R, et al. (2002) A new phylum of Archaea represented by a nanosized hyperthermophilic symbiont. Nature 417: 63–67. Iizuka T, Yamanaka S, Nishiyama T and Hirashi A (1998) Isolation and phylogenetic analysis of aerobic copiotrophic ultramicrobacteria from urban soil. Journal of General and Applied Microbiology 44: 75–84. Janssen PH, Schuhmann A, Morschel E and Rainey FA (1997) Novel anaerobic ultramicrobacteria belonging to the Verrucomicrobiales lineage of bacterial descent isolated by dilution culture from anoxic rice paddy soil. Applied and Environmental Microbiology 63: 1382– 1388. Kajander EO and Ciftcioglu N (1998) Nanobacteria – an alternative mechanism for pathogenic intra-and extracellular calcification and stone formation. Proceedings of the National Academy of Sciences of the USA 95: 8274–8279. MacDonell MT and Hood MA (1982) Isolation and characterization of ultramicrobacteria from a Gulf Coast estuary. Applied and Environmental Microbiology 43: 566–571. Maniloff JN, Nealson KH, Psenner R, Loferer M and Folk R (1997) Nannobacteria – size limits and evidence. Science 276: 1776–1777. McKay DS, Gibson EK, Thomas-Keprta KL, et al. (1996) Search for past life on Mars: possible relic of biogenic activity in Martian meteorite ALH84001. Science 273: 924–930. Morita RY (1988) Bioavailability of energy and its relationship to growth and starvation survival in nature. Canadian Journal of Microbiology 34: 436–441. Morowitz HJ (1967) Biological self-replicating systems. Progress in Theoretical Biology 1: 35–58. Mushegian AR and Koonin EV (1996) A minimal gene set for cellular life derived by comparison of complete bacterial genomes. Proceedings of the National Academy of Sciences of the USA 93: 10268–10273. Ruiz JMG, Carnerup A, Christy AG, et al. (2002) Morphology: an ambiguous indicator of biogenicity. Astrobiology 2: 353–369. Schut F, Prins RA and Gottschal JC (1997) Oligotrophy and pelagic marine bacteria: facts and fiction. Aquatic Microbial Ecology 12: 177– 202. Sieburth JMN, Smetacek V and Lenz J (1978) Pelagic ecosystem structure: heterotrophic compartments of the plankton and their relationship to plankton size fractions. Limnology and Oceanography 23: 1256–1263. Southam G and Donald R (1999) A structural comparison of bacterial microfossils vs nanobacteria and nanofossils. Earth-Science Reviews 48: 251–264. Thomas-Keprta KL, Clemett SJ, Bazylinski DA, et al. (2001) Truncated hexa-octahedral magnetite crystals in ALH84001: presumptive


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biosignatures. Proceedings of the National Academy of Sciences of the USA 98: 2164–2169. Torrella F and Morita RY (1981) Microcultural study of bacterial size changes and microcolony and ultramicrocolony formation by heterotrophic bacteria in seawater. Applied and Environmental Microbiology 41: 518–527. Trevors JT and Psenner R (2001) From self-assembly of life to presentday bacteria: a possible role for nanocells. FEMS Microbiology Reviews 25: 573–582. Uwins PJR, Webb RI and Taylor AP (1998) Novel nano-organisms from Australian sandstones. American Mineralogist 83: 1541–1550. Velimirov B (2001) Nanobacteria, ultramicrobacteria and starvation forms: a search for the smallest metabolizing bacterium. Microbes and Environments 16: 67–77.

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Workshop (1999) Size Limits of Very Small Microorganisms: Proceedings of a Workshop. Washington, DC: National Academy Press.

Further Reading Folk RL and Lynch LF (2001) Organic matter, putative nannobacteria and the formation of ooids and hardgrounds. Sedimentology 48: 215– 229. Hutchison CA, Peterson SN, Steven GR, et al. (1999) Global transposon mutagenesis and a minimal Mycoplasma genome. Science 286: 2165– 2169. Peterson SN and Fraser CM (2001) The complexity of simplicity. Genome Biology 2: 2002.1–2002.8.
