Monera

Prokaryotes And The Evolution Of Metabolism

The history of the earth is divided into two time eons - the Precambrian and the Phanerozoic.

The Precambrian comprises the earliest and almost seven-eighths of the history of the Earth (4,500 to 544 million years ago).

The Precambrian is divided into three eras: the Hadean (4,500-3,800 mya) during which the earth formed and cooled; the Archaean (2800 to 2500) during which life originated and the prokaryotes evolved; and the Proterozoic (2500 to 544 mya) during which eukaryotes appeared and multicellular life evolved.

In this lecture we are concerned with the Archaean and prokaryote evolution.

Prokaryotes are composed of cells that contain:

DNA in a circular loop
Outer cell membrane surrounded by a cell wall
Cytoplasm containing ribosomes
Flagellum composed of protein subunits
The cells divide by constriction, not mitosis.
These cells lack:
membrane-bound organelles
chromosomes with histone proteins
microtubules (and therefore, no mitosis or meiosis)

Some might suppose, on seeing such structurally simple organisms, that the Archaean was a period of relatively little evolutionary change.

This is not the case because the prokaryotes are biochemically very diverse and have played a central role in shaping the past and present environments of Earth. In fact we can say that the evolution of most of the important biochemical or metabolic pathways used by life on the planet today evolved in prokaryotes during the Precambrian.

Furthermore, it is an interesting dicotomy that prokaryotes are structurally simple but metabolically diverse, whereas eukaryotes (such as ourselves) are structurally diverse but metabolically fairly uniform. In fact, if a eukaryote wants to do something that requires innovative biochemistry (such as break down cellulose to sugar) they often co-op bacteria into a symbiotic relationship to do it for them.

Evolution of the Prokaryotes

The reconstruction of the evolutionary history of prokaryotic organisms is difficult, but not impossible if we use clues from comparative molecular biology and the fossil record.

Twenty years ago, the most primitive prokaryotes were thought to be cyanobacteria (also called the blue-green algae). Why?

stromatolites

2. Cyanobacteria are photosynthetic autotrophs. That is they can produce every resource they need by converting light energy from the sun into chemical energy by photosynthesis. This is a fairly passive process and so was seen as "simplier" than engulfing food and so was thought to be more primitive.

More recently we realized:

1. Fossils of non-photosynthetic organisms found that date to 3.8 bya.

2. Photosynthesizing organisms while certainly passive in appearance are actually doing something that is biochemically very complex. How could something as complex as photosynthesis evolve quickly when the intermediate steps of photosynthesis give no advantage that could be favored by natural selection ?

Norman Horowitz proposed:
1. The primitive ocean, because of outgassing and concentration of organic compounds a rich organic soup. The first organisms to arise would be the ones that used this rich store of organic chemicals. They would have been heterotrophs.

They would not have been photosynthetic
Their respiration would have been by fermentation because no free oxygen (O2) would have been present.
They would need few metabolic (that is enzyme driven) pathways - everything would be slurped up from the environment (in other words, naturally occurring organic chemical synthesis would supply the nutrients).
The simplist way to view metabolism in living organisms is illustrated in the following diagram:

anabolism

catabolism

ATP is used to store energy and when it is broken down into ADP through the loss of phosphate ions. ATP will form by naturally occuring chemical processes, and it may have accumulated in the Earth's early oceans. The earliest forms of life would only need to take in ATP and break it down into ADP to obtain energy (only need to do catabolism).

However, as life forms multiplied, this free ATP would have been used up. At this point in time, prokaryotes that were able to create ATP through simple chemical pathways appeared and were favored by natural selection (in other words they need to also be able to do anabolism). We have such prokaryotes still living today - they are called the Archebacteria

Archebacteria

These are the only type of prokaryotes that could have survived in the world 3.8 billion years ago. Archaea are similar to other prokaryotes in most aspects of cell structure and metabolism (they have DNA in a circular loop, a cell wall (although its chemical composition is unique), ribosomes, flagella may be present, lack membrane-bound organelles).

Individual archaeans range from 0.1 to over 15 µm in diameter, and some form aggregates or filaments up to 200 µm in length. They occur in various shapes, such as spherical, rod-shaped, spiral, lobed, or rectangular.

However, their genetic transcription and translation do not show the typical bacterial features, but are extremely similar to those of eukaryotes.

For instance, archaean translation uses eukaryotic initiation and elongation factors, and their transcription involves TATA-binding proteins and TFIIB as in eukaryotes.

Several other characteristics also set the Archaea apart.

The most striking chemical differences between Archaea and other living things lie in their cell membrane. Their are four fundamental differences between the archaeal membrane and those of all other cells: (1) chirality of glycerol, (2) ether linkage, (3) isoprenoid chains, and (4) branching of side chains. These may sound like complex differences, but a little explanation will make the differences understandable:

Remember: The basic unit from which cell membranes are built is the phospholipid . This is a molecule of glycerol which has a phosphate added to one end, and two side chains attached at the other end. When the cell membrane is put together, the glycerol and phosphate end of the molecules are hydophilic hang out at the surface of the membrane, with the long, hydrophobic side chains sandwiched in the middle:

Like bacteria and eukaryotes, archaea possess glycerol based phospholipids. However, three features of the archaeal lipids are unusual:

1. the archaeal lipids have an L-glycerol while bacteria and eukaryotes have a D-glycerol. This is strong evidence for a different biosynthetic pathway:
2. Most bacteria and eukaryotes have membranes composed mainly of glycerol-ester lipids, whereas archaea have membranes composed of glycerol-ether lipids:

The hydrophobic side-chains in Eukaryotes and Bacteria are fatty acids while the chains in Archaea are made up of isoprenes. These isoprenes can be branched and joined in a greater variety of ways and this gives Archaea more variability in their membranes.

Like Bacteria, Archaea have a cell wall. In bacteria, however, the cell wall is made up of a material called peptidoglycan, while it is made of a variety of other materials.

Habitats

Archaea are usually harmless to other organisms and none are known to cause disease. Many archaeans are extremophiles (that is they live in extreme environments). Some live at very high temperatures, often above 100°C, as found in geysers and black smokers. Others are found in very cold habitats or in highly saline, acidic, or alkaline water. However, other archaeans are mesophiles, and have been found in environments like marshland, sewage, and soil. Many methanogenic archaea are found in the digestive tracts of animals such as ruminants, termites, and humans.

Important to our story of evolution are one of there - the Methanogens

anaerobes - intolerant to O2.
Organisms able to generate energy from chemical reactions involving inorganic molecules that they take in from the environment. The methanogenic bacteria are examples of such bacteria. These bacteria use CO2 to split H2 release energy and produce ATP from ADP. The waste products at the end are methane and water. They are responsible for almost all biologically produced methane on this planet:

In the Precambrian, methanogens could have existed almost anywhere. Today they live only where O2 is excluded and where H and CO2 are available. -Stagnent water, sewage treatment facilities, hot springs, rumen of cattle.

The methane sometimes ignites into a blue flame called a will o'wisp or jack-o-lantern.
: The methane sometimes ignites into a blue flame called a will o'wisp or jack-o-lantern.

Most archebacteria are extreme heterotrophs and would have depleted the organic soup and caused an evolutionary crisis. We can call it the living world's first energy crisis. The precursors of the organic compounds would still be naturally occurring. Natural selection would favor those organisms which could produce their own complex organic molecules from the naturally occurring precursors. This led to the evolution of the Eubacteria (means true bacteria : as opposed to archebacteria) - Cell structure a little more complex (cell walls contain murine, mesosomes-complex membrane folds that are the site of many biochemical pathways) but more importantly, these bacteria had more complex metabolic pathways that allowed fermentation to produce energy.

Eubacteria

1. Fermenting bacteria

Chemotrophs - In the absence of oxygen, these bacteria break glucose (which has been absorbed from the environment) into two molecules of pyruvate. This releases enough energy to produce two molecules of ATP. This process is known as anaerobic glycolysis (in other words, glycolysis is a metabolic heirloom from these earliest cells):

(pyruvate is broken down to lactic actid in animals)
----
The bonanza of free glucose in the biotic soup could not last. If the earliest heterotrophs continued to consume glucose molecules at a rate greater that they could be produced by slow geochemical reactions, then eventually life would have died out.

In this early famine, organisms with the ability to produce their own glucose would have been favored by natural selection. Life on earth today depends primarily on photosynthesis as the process of glucose production - a solution invented more than 2 billion years ago.

The earliest photosynthetic organisms lived in an anaerobic atmosphere. They did not fix CO 2 or generate O 2 or even use chlorophyll. They used light to make ATP and absorb nutrients. We have seen this already. Archaeal photosynthesis in Halobacterium halobium uses the purple pigment bacteriorhodopsin in the external membrane and light energy to expel protons from the cell. The proton gradient used for nutrient absorption (by co-transport with protons) and for ATP production. This has a selective advantage.

Why was a purple pigment selectively advantageous to the Archaea? A purple pigment absorbs in the middle of the visible spectrum, where the sun emits light most copiously. It absorbs green light, but reflects red and blue so it looks purple. Without the ability to fix CO 2 Archaea cannot photosynthesise without external reducing agents and organic carbon. So, they use light to make energy, but need reduced carbon (food) on the menu.

2. Photosynthetic Bacteria
Trapping light energy and converting it to chemical energy is the process of photosynthesis. Organisms that do it use chlorophyll. Chlorophyll is a porphyrin molecule.

Porphyrins are ring-shaped molecules that combine with metal ions and can be formed from chemicals present on the early earth. Porphyrins include hemoglobin (which contains iron), chlorophyll (which contains magnesium), and vitamin B12 (which contains cobalt). These molecules are able to act as catalysts and cause electrons to be exchanges. Chlorophyll can capture light energy and store it by raising the energy within the porphyrin ring. This energy then causes electrons to move through the photosynthesis reaction resulting in formation of glucose.

Chlorophyll probably evolved in the ancestors of the green sulfur bacteria. It occurs in the bacterial cell membrane and transfers electrons photochemically onto NAD, and the NADH so produced is used to run glycolysis backwards. How did this evolve? The respiration in early sulfur bacteria generated NADH. This must be converted back to NAD, but could not be oxidised by oxygen in an anaerobic atmosphere. Instead the electrons were passed via ferredoxin to hydrogenase. Hydrogenase combines the electrons from ferredoxin and with protons inside the cell to make hydrogen gas. This removes protons from within the cell and generates a gradient that can be used to make ATP. If this system could be run backwards , the bacteria could generate sugars by gluconeogenesis, using hydrogen as an electron donor.

All the bacteria needed was a way of generating PGA, and they could run glycolysis backwards, turning a waste-route into a way of making sugar for 'free'. The green sulfur bacteria evolved an enzyme called ATP citrate lyase, which cleaves citrate into acetyl-CoA and oxaloacetate (this is called the reductive Krebs cycle). This allowed these bacteria to run the Krebs cycle backwards and generate PGA. The enzyme was required because the Krebs cycle is essentially irreversible, so some of the 'backwards' steps needed new enzymes, in much the same way that fructose bisphosphatase acts as a work-around in gluconeogenesis for the irreversibility of the phosphofructokinase reaction in glycolysis. [Note that a number of other enzymes were required to bypass these irreversible steps, not just the lyase].

The purple sulfur bacteria chanced upon another way. All cells already contained enzymes to convert GAP to pentose phosphates (the pentose phosphate pathway) because this is needed to make nucleic acid. RuBP became the substrate for a new enzyme (Rubisco) which made it combine with CO 2 to generate PGA. Running glycolysis backwards uses hydrogen gas as a reducing agent, and this destroys the proton gradient. Modern sulfur bacteria solve this by dehydrogenating H 2 S using sulfide dehydrogenase, to produce solid sulfur, protons and electrons outside the cell. This generates the proton gradient for ATP synthesis.

Two types:

green sulfur bacteria

In the presence of sunlight and chlorophyll:

They removed electrons from H2S (hydrogen sulfide)-

to create NADPH which can transform CO2 into glucose and water

(b) Cyanobacteria (blue-green algae)

A more abundant source of electrons than hydrogen sulfide, is water. But splitting water to remove the necessary electrons requires abour 10 times more energy that it does to split them from H2S.

Cyanobacteria accomplished this by evolving enzymes that lowered the energy threshold and a more complex photosystem: Splitting water in cyanobacterial photosynthesis generates oxygen as a waste product. Cyanobacteria created huge mats (called stromatolites) and produced a great deal of oxygen. These mats can be seen commonly in Archaean fossil rocks and, in a few places where they are protected from animal predators, they still form today (for example in saline lagoons in Australia:

About 2300 million years ago (2.3 bya) oxygen gas began accumulating in the atmosphere. This created an enormous problem for existing life forms:

Oxidation

Oxygen (while extremely important to our metabolism) is extremely reactive and can disrupt the delicate balance in a living cell. Even after millions of years with oxygen, living organisms can still be poisoned by high concentrations of O2: just breathing rapidly (hyperventilating) makes people dizzy.

ii. Block UV light - only biotic production of organic molecules can now occur in natural environments

To anaerobic microorganisms, the arrival of oxygen was an unmitigated disaster. Some (probably many) went extinct, some retreated into habitats without oxygen (oblgate anaerobes) - that is the only place we find many archebacteria today.

This leads to a third type of eubacteria:

3. Facultative anaerobic bacteria

These anaerobic bacteria were the first to have evolved the ability to produce special molecules that bind oxygen and neutralize it. These molecules, primarily porphyrins, are still used today by microorganisms and their descendants to bind oxygen.

we find porphyrins in hemoglobin--

Even bioluminescense (used in communication today by many organisms) may have originally evolved in bacteria because it generally involves reducing oxygen to a non-reactive form.

Finally, the ability to use the reactive properties of oxygen to generate ATP would have appeared:

4. Aerobic Bacteria
In respiration, pyruvate is converted into Acetyl CoA. Acetyl CoA enters a series of reactions (known as the krebs cycle) where it is broken down to give 30 molecules of ATP, carbon dioxide, and water.

Summary (in the form of a cladogram):

References
Woese, C. R. 1994. There must be a prokaryote somewhere: microbiologiy's search for itself. Microbiological Reviews 58: 1-9.