The Origin Evolution and Classification of Microbial Life

Bacteriology at UW- Madison

The Microbial World

University of Wisconsin - Madison

The Origin, Evolution and Classification of Microbial Life

Opalescent Pool in Yellowstone National Park, Wyoming USA. Conditions for life in this environment are similar to Earth over 2 billion years ago. In these types of hot springs, the orange, yellow and brown colors are due to pigmented photosynthetic bacteria which make up the microbial mats. The mats are literally teeming with microbes. Some of these bacteria such as Synechococcus conduct oxygenic photosynthesis, while others, such as Chloroflexus, conduct anoxygenic photosynthesis. Other non-photosynthetic bacteria, as well as thermophilic and acidophilic Archaea, are also residents of the hot spring community.

THE ORIGIN OF CELLULAR LIFE

When life arose on Earth about 4 billion years ago, the first types of cells to evolve were procaryotic cells. For approximately 2 billion years, procaryotic-type cells were the only form of life on Earth. The oldest known sedimentary rocks found in Greenland are about 3.8 billion years old. The oldest known fossils are procaryotic cells, 3.5 billion years in age, found in Western Australia and South Africa. The nature of these fossils, and the chemical composition of the rocks in which they are found, indicates that these first cells made use of simple chemical reactions to produce energy for their metabolism and growth.

The primitive earth's atmospheric gases, such as ammonia (NH₃), hydrogen (H₂) and hydrogen sulfide (H₂S) could be oxidized to produce energy that allowed conversion of CO₂ to cellular (organic) material. As organic material developed, it became the substrate to support the growth and metabolism of other cells that use simple organic compounds as their source of energy. The use of inorganic chemicals as a source of energy is called chemolithotrophy; the use of organic chemicals as energy sources is called chemoheterotrophy. Thus, chemolithotrophy and chemoheterotrophy, were the first two types of metabolism to evolve. An important group of archaea that were involved in this process were the methanogens, which grow by using H₂ as an energy source and CO₂ as a carbon source, resulting in the production of the simplest of all organic molecules, methane (CH₄). Archaea and bacteria probably arose from a universal ancestor but are thought have split early during the evolution of cellular life into the two groups of procaryotes that we recognize today.

Photosynthesis (metabolism which uses of light as an energy source) developed in bacteria about 3.2 billion years ago. The first type of photosynthesis to appear is called anoxygenic photosynthesis because it does not produce O₂. Anoxygenic photosynthesis preceded oxygenic photosynthesis (plant-type photosynthesis, which produces atmospheric O₂) by half a billion years. However, oxygenic photosynthesis also arose in procaryotes, specifically in a group of bacteria called cyanobacteria, and existed for millions of years before the evolution of plants.

As molecular oxygen (O₂) began to appear in the atmosphere, organisms that could use O₂ for respiration began their evolution, and "aerobic" respiration became a prevalent form of metabolism among bacteria and some archaea. A time scale for major events in evolution of the first (procaryotic) cells is given in Table 1 below.

Table 1. Timescale for some major events in procaryotic evolution. Battistuzzi, et al. MC Evol Biol. 2004. 4: 44

Origin of life: prior to 4.1 billion years ago (Ga)
Origin of methanogenesis: 3.8 - 4.1 Ga
Origin of phototrophy: prior to 3.2 Ga
Divergence of the major groups of Archaea: 3.1 - 4.1 Ga
Origin of anaerobic methanotrophy: after 3.1 Ga
Colonization of land: 2.8 - 3.1 Ga
Divergence of the major groups of Bacteria: 2.5 - 3.2 Ga
Origin of aerobic methanotrophy: 2.5 - 2.8 Ga

Eucaryotic cells evolved into being between 1.5 and 2 billion years ago. Eucaryotic cells appear to have arisen from procaryotic cells, specifically out of the Archaea. Indeed, there are many similarities in molecular biology of contemporary archaea and eucaryotes. However, the origin of the eucaryotic organelles, specifically chloroplasts and mitochondria, is explained by evolutionary associations between primitive nucleated cells and certain respiratory and photosynthetic bacteria, which led to the development of these organelles and the associated explosion of eucaryotic diversity.

Endosymbiosis

Endosymbiosis is the name given to processes wherein one cell lives inside of another cell in a mutualistic fashion. There are many examples of endosymbiosis in the microbial world, usually involving a smaller procaryotic cell living within the cytoplasm of a eucaryotic cell (see Endosymbiosis.html). Endosymbiotic events between eucaryotic and procaryotic cells has been taking place since the origin of the eucaryotic cell. It is an endosymbiosis between early eucaryotes and bacterial cells that has given rise to eucaryotic chloroplasts and mitochondria as stated above. In fact, possibly all eucaryotic membranous structures may have arisen from procaryotic cells through independent processes of endosymbiosis.

About 1.5 -2 billion years ago, oxygenic photosynthesis and aerobic respiration were predominant types of metabolism in the bacteria. Cyanobacteria produced all of the earth's atmospheric O₂, and respiratory bacteria had developed sophisticated membrane systems allowing them to reduce O₂ and generate relatively large amounts of energy. If these procaryotes invaded or were captured by primitive eucaryotic cells, which had only sluggish modes of chemoheterotrophic metabolism, they could provide new ways to produce energy from light or during aerobic respiration. In return, the eucaryotic cell could provide nutrients and a protected habitat for its invader or prey. Hence, the two organisms were able to enter into a mutually beneficial and stable relationship, and thus, microbiologists believe that the origin of eucaryotic chloroplasts (organelles for photosynthesis) and mitochondria (organelles for aerobic respiration) are in cyanobacteria and respiratory bacteria that entered into a partnership with eucaryotic cells in the evolutionary past.

Figure 1. The probable events of endosymbiosis that gave rise to the chloroplasts and mitochondria of eucaryotic cells.

If mitochondria and chloroplasts are evolutionary remnants of bacteria, there ought to be some similarities between contemporary eucaryotic organelles and the bacteria from which they arose, and indeed, there are. Mitochondria and chloroplasts are membrane-enclosed structures, the size of procaryotes, that arise from pre-existing structures. They contain their own genome (DNA) and ribosomes, both of which have a bacterial configuration and function. They synthesize their own proteins in the same way as bacteria. Chloroplasts have the same type of chlorophyll, enzymes, and metabolism as cyanobacteria, and mitochondria have the same type of metabolism as respiratory bacteria such as Pseudomonas. and other "proteobacteria". Interestingly, on the basis of RNA analysis, the closest relatives of mitochondria are the rickettsia bacteria, which are modern-day intracellular parasites of eucaryotic cells!

For more about endosymbiosis see Kimball's biology page Endosymbiosis

Structure of eucaryotic and procaryotic cells

It is appropriate to review the structure of eucaryotic and procaryotic cells now that we have an idea of how and when they evolved as distinct types of cells. Then we will look at the classification schemes that have attempted to organize the microbial forms of life in ways that demonstrate their origin and apparent evolutionary relationships.

Procaryotic organisms (archaea and bacteria) and eucaryotic organisms (both unicellular and multicellular forms) have evolved as two distinct types of cells, differing fundamentally in their cell structure. Eucaryotes always contain a membrane-enclosed nucleus, multiple chromosomes, and various other membranous organelles, such as mitochondria, chloroplasts, the golgi apparatus, vacuoles, etc. Procaryotic cells are typically much smaller in size and never contain a nuclear membrane around their genetic material. The fundamental differences between eucaryotic and procaryotic cells, as well as the similarities and differences between eucaryotes, bacteria and archaea, are evidenced by their nuclear organization, their cell wall, cell membrane and ribosome structure, and their modes of protein synthesis, as shown in Figure 2 and Table 2 below.

Figure 2. (above) The structure of a typical procaryotic cell, in this case, a Gram-negative bacterium, compared with (below) a typical eucaryotic cell (plant cell). The procaryote is about 1 micrometer in diameter and about the size of the eucaryotic chloroplast or mitochondrion. Drawings by Vaike Haas, University of Wisconsin-Madison.

Table 2. Phenotypic properties of Bacteria and Archaea compared with Eucarya.

Property	Biological Domain
	Eucarya	Bacteria	Archaea
Cell configuration	eucaryotic	procaryotic	procaryotic
Nuclear membrane	present	absent	absent
Number of chromosomes	>1	1	1
Chromosome topology	linear	circular	circular
Murein in cell wall	-	+	-
Cell membrane lipids	ester-linked glycerides; unbranched; polyunsaturated	ester-linked glycerides; unbranched; saturated or monounsaturated	ether-linked branched; saturated
Cell membrane sterols	present	absent	absent
Organelles (mitochondria and chloroplasts)	present	absent	absent
Ribosome size	80S (cytoplasmic)	70S	70S
Cytoplasmic streaming	+	-	-
Meiosis and mitosis	present	absent	absent
Transcription and translation coupled	-	+	+
Amino acid initiating protein synthesis	methionine	N-formyl methionine	methionine
Protein synthesis inhibited by streptomycin and chloramphenicol	-	+	-
Protein synthesis inhibited by diphtheria toxin	+	-	+

TAXONOMY AND CLASSIFICATION OF MICROBES

Three Kingdom System (1866)

Haeckel (1866), a Swiss naturalist, was the first to create a natural kingdom for the microbes, which had been discovered nearly two centuries before by Antony van Leeuwenhoek. Haeckel placed all unicellular (microscopic) organisms in a new kingdom, "Protista", on the level with the existing kingdoms for plants (Plantae) and animals (Animalia), which are multicellular (macroscopic) organisms.

Figure 3. Haeckel's 3-Kingdom system for the classification of life. Haeckel separated life into three kingdoms and rooted them as a "tree of life". What seemed the most primitive forms of life were closest to the main trunk of the tree, and what seemed the most advanced forms are at the tips of the branches. Also, in keeping with the Darwinian ideas of the day, Haeckel supposed that simpler forms of life lead to more advanced or complicated forms of life. All of the microscopic forms of life are landed in the Protista, and low among the first branches of the Protista are microbial groups that are still recognized today, including bacteria, algae and protozoa.

Four Kingdom System (circa 1950)

The development of the electron microscope in the 1950's revealed a fundamental dichotomy among Haeckel's "Protista": some cells contained a membrane-enclosed nucleus, and some cells lacked this intracellular compartment. The latter were temporarily shifted to a fourth kingdom, Monera (or Moneres), the procaryotes (also called Procaryotae). Protista remained as a kingdom of unicellular eucaryotic microorganisms.

Five Kingdom System (1967)

Whittaker, a botanist at the University of California, refined the system into five kingdoms in 1967, by identifying the Fungi as a separate multicellular eucaryotic kingdom of organisms, distinguished by their absorptive mode of nutrition.

Figure 4. Whittaker's phylogenetic Tree of 1967. The 5-Kingdom system is based on three levels of organization- procaryotic (Kingdom Monera), eucaryotic unicellular (Kingdom Protista), and eucaryotic multicellular (Kingdoms Plantae, Fungi and Animalia). At he microbial levels there is divergence in relation to principal modes of nutrition - photosynthetic, absorptive and ingestive. Ingestive nutrition is lacking in Monera, but the three modes are continuous along numerous evolutionary lines in the Protista giving rise to the three higher Kingdoms of Plantae, Fungi and Animalia. Note that the tree is rooted in the Procaryotes (Monera) and that the more distant an organism is removed from the root, the more highly (and recently) evolved is the organism.

Carl Woese's Three Domain System (1988)

In the late 1970s, Carl Woese, at the University of Illinois, began phylogenetic analysis of all forms of cellular life based on comparison of nucleotide sequences of the small subunit ribosomal RNA (ssrRNA) that is contained in all organisms. Woese considered other conserved molecules in cells including certain proteins, and conserved genes (DNA), but settled for the ssrRNA for a number of reasons.

1. rRNA is found in all cells.

2. rRNA is present in thousands of copies and is easy to isolate from cells

3. rRNA can be analyzed to determine the exact sequence of nucleotide bases in its makeup.

4. The sequence of bases in RNA is a complementary COPY of the sequence of bases in the gene (DNA) that encodes for RNA.

5. Base sequences in different rRNA molecules can be compared by computer analyses and statistical methods to reveal precise similarities and differences in cellular genomes.

Woese's analysis of RNA molecules from different types of cells revealed a new dichotomy, this time among the procaryotes. There exist two types of procaryotes, as fundamentally unrelated to one another as they are to eucaryotes. Thus, Woese defined three cellular domains of life as they are displayed in Figure 5 (below): Eukaryotes, Eubacteria and Archaebacteria. Whittaker's Plant, Animal and Fungi kingdoms (all of the multicellular eucaryotes) are at branch tips of the Eukaryote Domain, while other eukaryote branches lead to protists (unicellular algae and protozoa).

Figure 4. Carl Woese's "universal" phylogenetic tree of 1988 determined from ribosomal RNA sequence comparisons. Note the three major domains of living organisms: The Eubacteria (Bacteria), the Archaebacteria (Archaea) and the Eukaryotes (Eucarya). The evolutionary distance between two groups of organisms is proportional to the cumulative distance between the end of the branch and the node that joins the two groups. Compare with the Pace Tree, Figure 5 below.

Although the definitive difference between Woese's Archaea and Bacteria is based on fundamental differences in the nucleotide base sequence in the ssrRNA, there are many biochemical and phenotypic differences between the two groups of procaryotes as shown in Table 2 above. The phylogenetic tree indicates that Archaea are more closely related to Eucarya than are Bacteria. This relatedness seems most evident in the similarities between transcription and translation in the Archaea and the Eucarya. However, it is also evident that the Bacteria have evolved into chloroplasts and mitochondria, so that these eucaryotic organelles derive their lineage from this group of procaryotes. Perhaps the biological success of eucaryotic cells springs from the evolutionary merger of the two procaryotic life forms.

The Universal Tree of Life

On the basis of small subunit ribosomal RNA (ssrRNA) analysis, the Woesean tree of life gives rise to three cellular domains of life: Archaea, Bacteria, and Eucarya (Figure 6). Bacteria (formerly known as eubacteria) and Archaea (formerly called archaebacteria) share the procaryotic type of cellular configuration, but otherwise, they are not related to one another any more closely than they are to the eucaryotic domain, Eucarya. Between the two procaryotes, Archaea are apparently more closely related to Eucarya than are the Bacteria. Eucarya consists of all eucaryotic cell-types, including protista, fungi, plants and animals.

Figure 6. The Universal Tree of Life as derived from sequencing of ssrRNA. N. Pace. Note the three major domains of living organisms: Archaea, Bacteria and Eucarya. The "evolutionary distance" between two organisms is proportional to the measurable distance between the end of a branch to a node to the end of a comparative branch. For example, in Eucarya, humans (Homo) are more closely related to corn (Zea) than to slime molds (Dictyostelium); or in Bacteria, E. coli is more closely related to Agrobacterium than to Thermus.

Notes on the Tree

It is interesting to note several features of phylogeny and evolution that are revealed in the Unrooted Tree.

--Archaea are the least evolved type of cell (they remain closest to the common point of origin). This helps explain why contemporary Archaea are inhabitants of environments that are something like the earth 3.86 billion years ago (hot, salty, acidic, anaerobic, low in organic material, etc.).

--Eucaryotes (Eucarya) are the most evolved type of cell (they move farthest from the common point of origin). However, the eucaryotes do not begin to diversify (branch) until relatively late in evolution, at a time when the Bacteria diversify into oxygenic photosynthesis (Synechococcus) and aerobic respiration (Agrobacterium).

--Mitochondria and the respiratory bacterium, Agrobacterium, are derived from a common ancestor; likewise, chloroplast and the cyanobacterium, Synechococcus, arise from a common origin. This is good evidence for the idea of evolutionary endosymbiosis, i.e., that the origin of eucaryotic mitochondria and chloroplasts is in procaryotic cells that were either captured by, or which invaded, eucaryotic cells and subsequently entered into a symbiotic association with one cell living inside of the other.

--Diversification in Eucarya is mainly within the Protista (unicellular protozoa, algae). The only multicellular eucaryotes on the Tree are Zea (plants), Homo (animals) and some fungi. Since the protists, along with the archaea and bacteria, constitute the microbial ("microorganismal") community of the planet, this helps to substantiate the claim that microorganisms are the predominant and most diverse form of life on Earth.

--Humans (Homo) are more closely related to yeast (Saccharomyces) than the are to corn (Zea). There are more genetic differences between E. coli and Bacillus than there are between humans and a paramecium. The protozoan Trichomonas is more closely related to the archaea than it is to fellow protozoan, Trypoanosoma. When the tree branches are amplified there many other surprising relationships to biologists.

--Most biology and anthropology students have been presented with fossil and other structural evidence that humans (Homo) emerged a very short time ago on the evolutionary clock. The Tree confirms this evidence on the basis of comparative molecular genetic analysis.