The simplest cell , Mycoplasma pneumoniae

One Way To Think About the Complexity of the “Simplest” Life Form 1

I have always been fascinated by the question, “How simple can life get?” After all, anything that is alive has to perform certain functions such as reacting to external stimuli, taking in energy and converting that energy to its own use, reproducing, etc. Exactly how simple can a living system be if it has to perform such tasks? Many biologists have investigated this question, but there isn’t a firm answer. Typically, biologists talk about how simple a genome can be. The simplest genome belongs to a bacterium known as Carsonella ruddii. It has 159,662 base pairs in its genome, which is thought to contain 182 genes.1 However, it is not considered a real living organism, as it cannot perform all the functions of life without the help of cells found in jumping plant lice.


The bacterium known as Pelagibacter ubique has the smallest genome of any truly free-living organism. It weighs in at 1,308,759 base pairs and 1,354 genes.2 However, there is something in between these two bacteria that might qualify as a real living organism. It is the bacterium Mycoplasma genitalium. It’s genome has 582,970 base pairs and 525 genes.3 While it is a parasite, it performs all the standard functions of life on its own. It just uses other organisms (people as well as animals of the order Primates) for food and housing. Thus, while it cannot exist without other organisms, it might be the best indicator of how “simple” life can get.

If you follow science news at all, you might recognize the name. Two years ago, Dr Craig Venter and his team constructed their own version of that bacterium with the help of living versions of the bacterium, yeast cells, and bacteria of another species from the same genus. Well, now a scientist from Venter’s lab teamed up with several scientists from Stanford University to produce a computer simulation of the bacterium!

Their work, which seems truly marvelous, gives us deep insight into how complex the “simplest” living organism really is.


Let’s start with what the computer simulation actually accomplished. It modeled all the inputs and outputs of the bacterium’s 525 genes throughout a single cell cycle. In other words, it simulated how the genome produces proteins, how those proteins interact with other proteins, and how the entire system is regulated. It followed these processes through all the events leading up to and including the cell reproducing itself.4

Now that’s a lot of work! How did the authors do it? Well, they looked at over 900 different scientific papers that had been produced on the inner workings of Mycoplasma genitalium, and they identified 1,900 specific parameters that seem to govern how the cell operates. There were several discrepancies that were found among the papers involved, and as a result, there was a lot of reconciliation that had to be done. The details of this reconciliation and other matters are found in a 120-page supplement to the 12-page scientific paper.

Once the reconciliation of these studies was accomplished, the essential workings of the cell were split into 28 separate modules that each governed specific functions of the cell. For example, one module dealt with metabolism, while another dealt with the activation of proteins once they were produced. Once each module was built and tested individually, the modules were then joined by looking at what they produced every second. If the products of one module were the kinds of chemicals used by a second module, those products were then treated as inputs to the second module for the next second of computation. The computation proceeded like this (checking the inputs and outputs of each module) for about 10 hours, which is roughly the time it takes a real Mycoplasma genitalium to reproduce.

Why would a group want to undertake such a complex endeavor? Well, one obvious reason is the reconciliation that I mentioned previously. As independent papers, each of the 900 studies to which the authors referred made sense. However, when the authors started using the results of those studies in a model that tries to take all the molecular processes of a cell into account, they found that some results didn’t mesh well with others. The reconciliation that had to take place to get the simulation working will help us better understand the limits of many of the studies related to Mycoplasma genitalium and hopefully will lead to more detailed studies that will slowly wipe away such discrepancies. Also, as the authors state, these kinds of models will:

…accelerate biological discovery and bioengineering by facilitating experimental design and interpretation. Morever, [this study and others] raise the exciting possibility of using whole-cell models to enable computer-aided rational design of novel microorganisms.

So in the end, not only will such models help us better design and interpret experiments, they might one day lead us to ways that we can engineer new microorganisms.

This is fantastic work, and I do think it opens up new vistas in cell and molecular biology. However, we need to pull back for a moment and think about the direct implications of this computer simulation. It simulated, in very basic terms, the molecular interactions that occur in a cell that might be a good analog for the simplest possible life form. It skipped over a lot of details, of course, so it is not a complete simulation by any means. Nevertheless, it is a great first step towards understanding how a living system really works.

Now let’s look at this in very practical terms. In order to be able to match the speed at which the organism operates, this less-than-complete simulation required a cluster of 128 computers to get the job done. Think about that for a moment. In order to simulate most (but not all) of the processes that take place in an analog for what might be the simplest possible living organism, the authors needed the power of 128 computers running together! That should tell us something very clearly:

There is no such thing as a simple living organism.
The more we understand life, the more clear it becomes that even the “simplest” version of it has to be the result of design.

REFERENCES

1. Atsushi Nakabachi, et al., “The 160-Kilobase Genome of the Bacterial Endosymbiont Carsonella,” Science 314:267, 2006.
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2. Stephen J. Giovannoni, et al., “Genome Streamlining in a Cosmopolitan Oceanic Bacterium,” Science 309:1242-1245, 2005.
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3. According to the Comprehensive Microbial Resource Manual.
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4. Jonathan R. Karr, et al., “A Whole-Cell Computational Model Predicts Phenotype from Genotype,” Cell 150(2):389-401, 2012.
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1) http://blog.drwile.com/?p=8161

Transcriptome Complexity in a Genome-Reduced Bacterium 1

To study basic principles of transcriptome organization in bacteria, we analyzed one of the smallest self-replicating organisms, Mycoplasma pneumoniae. We combined strand-specific tiling arrays, complemented by transcriptome sequencing, with more than 252 spotted arrays. We detected 117 previously undescribed, mostly noncoding transcripts, 89 of them in antisense configuration to known genes. We identified 341 operons, of which 139 are polycistronic; almost half of the latter show decaying expression in a staircase-like manner. Under various conditions, operons could be divided into 447 smaller transcriptional units, resulting in many alternative transcripts. Frequent antisense transcripts, alternative transcripts, and multiple regulators per gene imply a highly dynamic transcriptome, more similar to that of eukaryotes than previously thought.



Our work revealed an unanticipated complexity in the transcriptome of a genome-reduced bacterium. This complexity cannot be explained by the presence of eight predicted transcription factors (26). Furthermore, the fact that the proteome organization is not explainable by the genome organization (28) indicates the existence of other regulatory processes. The surprisingly frequent expression heterogeneity within operons, the change of operon structures leading to alternative transcripts in response to environmental perturbations, and the frequency of antisense RNA, which might explain some of these expression changes, suggest that transcriptional regulation in bacteria resemble that of eukaryotes more than previously thought.

Simple' bacterium shows surprising complexity 2

The inner workings of a supposedly simple bacterial cell have turned out to be much more sophisticated than expected.

An in-depth "blueprint" of an apparently minimalist species has revealed details that challenge preconceptions about how genes operate. It also brings closer the day when it may be possible to create artificial life.

Mycoplasma pneumoniae, which causes a form of pneumonia in people, has just 689 genes, compared with 25,000 in humans and 4000 or more in most other bacteria. Now a study of its inner workings has revealed that the bacterium has uncanny flexibility and sophistication, allowing it to react fast to changes in its diet and environment.

"There were a lot of surprises," says Peer Bork, joint head of the structural and computational biology unit at the European Molecular Biology Laboratory (EMBL) in Heidelberg, Germany. "Although it's a very tiny genome, it's much more complicated than we thought."



1) http://www.sciencemag.org/content/326/5957/1268.full
2) http://www.newscientist.com/article/dn18206-simple-bacterium-shows-surprising-complexity.html#.VZlsARtVhBc

A Genome-Scale Metabolic Reconstruction of Mycoplasma genitalium, iPS189

http://journals.plos.org/ploscompbiol/article?id=10.1371/journal.pcbi.1000285

https://intelldesign.wordpress.com/tag/intelligent-design/

“We’ve learned that cells move in much more complex ways than previously believed,” said Christian Franck, assistant professor in engineering at Brown and the co-lead author of the study published online in the Proceedings of the National Academy of Sciences. “Now, we can start to really put numbers on how much cells push and pull on their environment and how much cells stick to tissues as they move around and interact.”
In the study, Franck and co-lead author Stacey Maskarinec, who both conducted the experiments while graduate students at the California Institute of Technology, placed cells on top of a 50-micron-thick water-based gel designed to mimic human tissue. They added into the gel spheres about a half-micron in diameter that lit up when jostled by the cells’ actions. By combining two techniques — laser scanning confocal microscopy and digital volume correlation — the scientists tracked the cells’ movement by quantifying exactly how the environment changed each time the cell moved. The team recorded results every 35 minutes over a 24-hour period.
What they found was cells move in intriguing ways. In one experiment, a cell is clearly shown operating in three dimensions by extending feelers into the gel, probing at depth, as if thrusting a leg downward in a pool. The Brown and Caltech scientists also found that as a cell moves, it engages in a host of push-pull actions: It redistributes its weight, it coils and elongates its body, and it varies the force with which it “grips,” or adheres, to a surface. Combined, the actions help the cell generate momentum and create a “rolling motion,” as Franck described it, that is more like walking than shuffling, as many scientists had previously characterized the movement.
“The motion itself is in three dimensions,” Franck said.

http://spectrummagazine.org/article/book-reviews/2009/10/06/signature-cell

According to Meyer the “simplest extant cell, Mycoplasma genitalium — a tiny bacterium that inhabits the human urinary tract — requires ‘only’ 482 proteins to perform its necessary functions….” If, for the sake of argument, we assume the existence of the 20 biologically occurring amino acids, which form the building blocks for proteins, the amino acids have to congregate in a definite specified sequence in order to make something that “works.” First of all they have to form a “peptide” bond and this seems to only happen about half the time in experiments. Thus, the probability of building a chain of 150 amino acids containing only peptide links is about one chance in 10 to the 45th power.

In addition, another requirement for living things is that the amino acids must be the “left-handed” version. But in “abiotic amino-acid production” the right- and left-handed versions are equally created. Thus, to have only left-handed, only peptide bonds between amino acids in a chain of 150 would be about one chance in 10 to the 90th. Moreover, in order to create a functioning protein the “amino acids, like letters in a meaningful sentence, must link up in functionally specified sequential arrangements.” It turns out that the probability for this is about one in 10 to the 74th. Thus, the probability of one functional protein of 150 amino acids forming by random chance is 10 to the 164th. If we assume some minimally complex cell requires 250 different proteins then the probability of this arrangement happening purely by chance is one in 10 to the 164th multiplied by itself 250 times or one in 10 to the 41,000th power.

there are about 10 to the 80th elementary particles in our observable universe. Assuming a Big Bang about 13 billion years ago, there have been about 10 to the 16th seconds of time. Finally, if we take the time required for light to travel one Plank length we will have found “the shortest time in which any physical effect can occur.” This turns out to be 10 to the minus 43rd seconds. Or turning it around we can say that the most interactions possible in a second is 10 to the 43rd. Thus, the “probabilistic resources” of the universe would be to multiply the total number of seconds by the total number of interactions per second by the total number of particles theoretically interacting. The math turns out to be 10 to the 139th.

Bacterial - Mycoplasmas (Mollicutes)

Mollicutes, informally referred to as the mycoplasmas, are extraordinary organisms! They are
the smallest known cells, typically the size of large viruses at only 200 to 300 nm in diameter (1
nanometer, 1 nm = 1 millionth of a millimetre) and they have the smallest genomes of any known
bacteria. The genome of Mycoplasma genitalium, for example, is only 580,073 bases long and
contains a mere 517 genes. Though still complex in real terms, this is a very minimilistic genome
and mycoplasmas are of especial interest to scientists working to build simple cells in the lab.

Mycoplasmas are often described as the simplest free-living cells, although most are parasitic or
commensals dependent on their host for certain functions, however, they are independent in the
sense that they have their own power sources, fermenting fuels to make ATP (unlike the tiny
chlamydial elementary bodies which are energy parasites dependent on ATP provided by the
host cells they live within). Most 'respire' anaerobically by fermentation without the use of
electron transport chains (ETCs) although rudimentary ETCs may be present in the cytoplasm
(or attached to the cell membrane in Acholeplasma). Their simplicity is in large part due to
evolutionary degeneration - they are dependant on their hosts for many metabolic functions that
they would otherwise have to carry out on their own and so they have lost these functions,
including the ETC.

As prokaryotes they lack a nuclear envelope, possessing a DNA/protein nucleoid rather than a
nucleus. As bacteria we tend to think of them as single-celled organisms, however, like most
bacteria they can form biofilms - loose associations in which the cells remain distinctly separate
but are embedded in a common slime matrix. Despite this, they do not seem to use
quorum-sensing to sense the presence of other bacteria and communicate in order to form
biofilms. Most bacteria use quorum-sensing in biofilm formation, but it is thought that
mycoplasmas form these structures without communication between the component cells.
Biofilms are not true multicellular organisms, since the cells do not communicate via cell-to-cell
junctions (pores and channels that join the cytoplasm of neighbouring cells together, as do gap
junctions in animal cells, plasmodesmata in plants and microdesmata in some cyanobacteria).
Indeed, in a typical biofilm the cells are not generally in direct physical contact with other cells.

Mycoplasmas also lack protective cell walls - they have no peptidoglycan like most bacteria and
no rigid layer at all in their cell envelopes, which instead consists of single cell membranes
coated in carbohydrates (forming the glycocalyx or slime coat) rather like animal cells and
protozoa. Like mammalian cells they are osmotically sensitive. Having no wall to maintain their
shape they will swell and burst in distilled water. Again, this may be a feature that their ancestors
had which was lost as their host maintains an osmotically stable environment which is also
generally free of mechanical trauma. Many do incorporate sterols, manufactured by their hosts,
however, which strengthen their membranes by making them more rigid. Acholeplasma is able to
grow without sterols, but will incorporate them if they are available and may manufacture
carotenoids to strengthen its membrane.

Life as parasites

Most mycoplasmas are parasites or commensals, living inside other organisms. Thermoplasma
acidophilum is unusual in being found in acidic coal refuse piles where internal temperatures
reach 55 degrees C. Spiroplasma infects plants and the arthropods which carry it from plant to
plant, infecting the haemolymph, gut and salivary glands of insects. Some may cause repiratory
infections in humans, for example Mycoplasma pneumoniae can cause a type of pneumonia
(though it is by no means the only cause of this disease). Mycoplasma may also infect the
synovial membranes of the joints of vertebrates, causing a form of arthritis.

Mycoplasmas require complex nutritional requirments if grown in the lab, since they depend on
their hosts for complex growth requirements, such as fatty acids, vitamins, purines, pyramidines,
and also the sterols for their membranes. When grown on agar they form characteristic small
circular colonies with a nipple-like or fried egg-like appearance.

Ureaplasma lives in the mouth, respiratory and genital tracts of mammals and humans and has a
novel way of obtaining energy. Whereas most mollicutes generate ATP by anaerobic
fermentation, Ureaplasma exhibits an unusual form of respiration in which urea (a waste product
of mammalian metabolism) is hydrolysed by an enzyme called a urease to form ammonium
(NH4+) which acts as a source of protons (H+) to power the ATPase by generating a proton
gradient across the cell membrane (essentially generating positive electric charge which flows
through the ATPase which acts as an electric motor whose rotation energy is sued to make ATP).

Many mycoplasmas have been implicated in causing plant disease, though since these are often
poorly characterised they are often referred to tentatively as mycoplasma-like organisms (MLOs).

Cell shape and cell motility - novel mechanisms of locomotion

Mycoplasmas are often described as pleomorphic: having variable shape, especially types like
Mycoplasma, although this is more true when growth conditions are sub-optimal (it is dificult to
grow parasites out of their hosts!) and in optimal conditions their form is more consistent.

The simplest cells?

Mycoplasma

Above: Mycoplasma grown under optimal conditions tends to have a more regular form. These organisms
glide by means of the apical protrusion protruding from their front end.
For a long time it was though that bacteria lacked an internal cytoplasmic skeleton (cytoskeleton)
as found in eukaryotic cells. This is because bacterial cells are much smaller and have a cell wall
to support them (for a while it was thought that plant cells might not have a cytoskeleton because
of their cell walls, but they do). However, it is now known that bacteria do have a cytoskeleton,
although one that is much less developed than in plant and animal cells. Indeed, in normal
electron microscopy no cytoskeleton may be evident at all in bacteria, except perhaps the
occasional tubular structure. It is now realised, however, that cytoskeletal structures form during
cell division and also during cell growth, at least in some forms, where cytoskeletal filaments
direct the deposition of new peptiodoglycan fibres in the cell wall (see growth in bacteria for more
details). In some cases cytoplasmic filaments are also seen to anchor flagella motors.
Mycoplasmas, although tiny, lack supporting cell walls and so their cytoskeletons have more work
to do and are better developed. (Animal cells and protozoa, being large and wall-less have
especially well developed cytoskeletons).

The cytoskeletons of mollicutes are also involved in locomotion. These bacteria lack flagella,
possibly because they have no rigid cell walls in which to anchor the rotary motors. Spiroplasma,
being helical in shape moves by flexing, creeping and swimming by rotating in cork-screw
fashion, rather like spirochaetes, except that spirochaetes use endoflagella to produce these
movements whereas Spiroplasma has no endoflagella. Instead Sprioplasma has a unique helical
protein fibres (3.6 nm diameter) in the cytoplasm, grouped into bundles or ribbons and a second
helical structure made of an actin-like protein MreB. Cryo-electron tomography, a new technique
for visualising cell structure at a molecular scale (less than 5 nm resolution) whilst preserving the
structure of living cells almost intact has revealed two ribbons of thicker filaments with a band of
thinner filaments in-between. At least one of these structures is thought to be contractile.

These fibrils are positioned just beneath the cell membrane and are thought to be involved in
maintaining helical shape as well as in motility and daughter-chromosome separation during cell
division. Indeed, these fibrils bear some resemblance to those seen in walled bacteria, for
example an MreB-like protein, Mbl (an MRb homologue) in the rod-shaped Bacillus subtilis form
helical filaments that direct the deposition of helical fibres of peptidoglycan in the cell wall during
cell growth (cell elongation) whilst MreB controls cell width. However, the other component of
Spiroplasma's helical fibres is apparently unique to Spiroplasma.

Spiroplasma rotates as it swims, corkscrewing its way along, which is an advantage when
swimming in highly viscous (sticky) fluids and, perhaps not surprisingly, Spiroplasma is
viscotactic, moving towards regions of high fluid viscosity and also exhibits chemotaxis. They
change helicity, from anticlockwise to clockwise at intervals, and often transitional cells are seen
with mixed helicity, caught in the act of changing. They can also change the pitch (steepness of
turns) of the helix with waves of change in helicity traveling down the length of the cell as pairs of
tiny kinks travelling from anterior to posterior. The kinks in each pair are on average 0.26
seconds apart, with the second kink appearing as the first nears the posterior end of the cell.
The kinks move at about 10.5 micrometres per second and the cell swims in the opposite
direction at about 3 micrometres per second, faster (up to about 5 micrometres per second) if
the medium is thickened (made more viscous) by the addition of 0.5% methylcellulose - like
spirochaetes they swim faster in high viscosity fluids. These observations suggest that the cells
rotate and swim due to flexing of the helical filaments.

Gliding motility in Mycoplasma

Many mollicutes move by gliding across a solid surface. Of these, the fastest is Mycoplasma
mobile which can glide at 2.0 to 4.5 micrometres per second on a glass surface. This is
astonishingly fast for a gliding organism. Like many mollicutes, Mycoplasma mobile moves by
means of an anterior nose-like anterior projection which generates traction against the substrate
apparently by means of about 400 minute (less than about 50 nm long) 'leg-like' proteins
(possibly the protein Gli349) in the cell membrane which are thought to alternately adhere to,
detach, advance and reattach to the surface, in a stepping manner, pulling the cell along.
Alternatively, other electron microscope evidence shows a much smaller number of relatively
long spikes (less than about 50 nm long) which may be the leg-like structures or separate
adhesion organelles. The cells can only glide forwards and can not reverse. ATP hydrolysis
provides the energy for this movement. Inside the apical protrusion are prominent cytoskeletal
structures - a solid nose-cap or hexagonal-lattice of a specific protein in the very tip of the
protrusion, forming an oval or hemispherical cap 235 nm wide and 155 nm long attached to
dozens of flexible protein 'tentacles' (inside the cytoplasm). The tentacles have 20 nm particles
(proteins?) attached to them at 30 nm intervals and it is thought that the leg-like proteins in the
cell membrane attach to these tentacles, transmitting the force of traction they generate to the
rest of the cell.
Suggested Bibliography

Below are some of the key references used in writing this article:

   Spiroplasma fibrils - http://www.sciencedirect.com/science/article/pii/S0014482704004525


   Makoto Miyata, and Jennifer D. Petersen, 2004. Spike Structure at the Interface between
   Gliding Mycoplasma mobile Cells and Glass Surfaces Visualized by Rapid-Freeze-and-
   Fracture Electron Microscopy. J. Bacteriology 186: 4382–4386.


   Mayer, F. 2003. Cytoskeletons in prokaryotes. Cell Biology International 27: 429–438.


   Makoto Miyata and Hiroshi Ogaki, 2006. Cytoskeleton of Mollicutes. J Mol Microbiol
   Biotechnol 11: 256–264.


   Daisuke Nakane and Makoto Miyata, 2007. Cytoskeletal ‘‘jellyfish’’ structure of
   Mycoplasma mobile. PNAS 104: 19518–19523.

Models of Mycoplasma pneumoniae show a different arrangement of cytoskeletal structures in
the apical protrusion. There is still a cap-like structure right inside the tip and attached to this and
extending down the central axis of the protrusion is a rod-like structure, attached to the
surrounding cell membrane by radial spokes along its length and anchored at the base of the
protrusion in a disc-like structure, also connected to the cell membrane by radial filaments. Other
cytoskeletal structures have also been observed in this species, including a helical mesh just
beneath the cell membrane and fibres extending into other regions of the cell. A different mode
of locomotion has been suggested for Mycoplasma pneumoniae in which the apical protrusion
extends, then membrane proteins in the apical protrusion attach to the substrate followed by
contraction pulling the rest of the cell forwards before the cycle repeats.
Mycoplasma mobile
Above: a model of Mycoplasma mobile crawling across a surface, looking down from above,
showing the cap structure (orange) of the apical protrusion and the trailing 'tentacles' (purple)
and the membranous protein 'legs' (cyan). The 'legs' or 'feet' move forwards and bind to the
surface and then bend or rotate backwards in some manner, pulling the cell forwards (to the right
in this diagram) and then detach and swing forwards to repeat the cycle.
Mycoplasma pneumoniae model
Above: a model of the cytoskeleton of Mycoplasma pneumoniae, showing some of the key
protein structures involved in gliding motility. There is an apical cap or plug (cyan) with a central
core of tubules connected to it which span the length of the apical protuberance (yellow). Radial
spokes (orange) connect this tubule core to the cell membrane. At the base of the apical
protuberance, the core connects to a 'wheel-like' or 'bowl-like' structure (green and orange)
connected to the cell membrane by radiating filaments (green rods) and to cytoskeletal filaments
which cross the cell body (magenta). Some of the adhesion proteins which coat the outside of the
membrane of the apical protuberance are shown (red balls and rods). One model has the apical
protuberance extending and then adhering to the surface via the adhesion proteins whilst the
core of tubules contracts, transmitting force to the rest of the cytoskeleton via the wheel-like
structure, pulling the cell body forward.

Minimalist cells?

As the simplest living cells so far discovered on earth, the mollicutes have attracted much recent
research, not just because they are agents of disease, but because these fascinating organisms
may give some insight into how cells first evolved. (Viruses are simpler, but these are acellular).
However, with their 600 genes or so they manage a considerable degree of complexity as can be
seen by the (highly simplified) account we have given of their cytoskeleton and motility
mechanisms. Mollicutes are hard to study, because they are so small, and only recently has real
progress been made on understanding their cellular machinery, which is complex! Much remains
uncertain and controversial, which is one reason why we avoided details - it should be most
interesting to see the progress in these fascinating areas of research over the coming years!

It would appear that, despite their relative simplicity, the mollicutes are highly evolved: as
parasites and commensals with some degree of degeneration of their ancestral systems, in
addition to evolution of novel systems. Nevertheless they remain the smallest and simplest cells
that are known to be able to survive as free-living organisms (albeit under carefully controlled
laboratory conditions or in specialist habitats such as coal piles) and understanding how they
function is of major importance. This still leaves the mystery as to how the first cells evolved.
There would appear to be no surviving simpler cells, and also none appear to be currently
evolving. What special conditions enabled cells to evolve but prevent them from doing so today?
In the current absence of simpler cells this question is difficult to answer. However, what mollictes
show us is how much can be done with relatively few genes and proteins.

Simpler cells?

There is one line of thought that hypothesises that simpler cells do currently exist on earth, but
have so far evaded detection. Indeed, the smallest cellular life-forms, such as mollicutes and
Chlamydia are still poorly understood and many more similar organisms no doubt await detection.
(Such as the MLOs, also called phytoplasms, which cause over 600 known plant diseases). In
recent years controversial claims that minute bacteria, called nanobacteria, of the order of 100
nm across or less, have been discovered remain to be verified. Often found inside rocks and
other 'primordial' habitats, these 'organisms' may simply be some form of mineral growth. Indeed,
as the controversy over the Martian meteorite, ALH84001, and its possible nanofossils has
shown - proving the existence of the smallest cells, past or present, will likely be extremely difficult.

Almost certainly, the vast majority of bacteria remain undiscovered. Most do not grow on
standard agars and only recently has the vast diversity of bacteria and archaebacteria in the
Earth's oceans been realised. Bacteria are also being discovered in unlikely places, from
Antarctic deserts to rocks deep beneath the Earth's surface. No doubt there are many surprises
yet to be revealed by the ancient and complex world of prokaryotes!