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General Microbiology II

Microbial Evolution and Systematics

Animated Overview of Evolution

Physical Evolution of Earth

• Big Bang leads to generation of our solar system and primitive Earth
  • as the theory goes, before the big bang, all matter in the universe was concentrated as a point-source of infinite mass
  • 12.5 (+/- 3) billion years ago (Ba), this infinitely massive point-source exploded, very rapidly expanding the matter to ever-enlarging volume, spewing intense radiation and energy (recent evidence indicates that this process may be a recurrent one, with cycles of expansion followed by contraction followed by big bangs)
  • within a few minutes, when the temperature had decreased enough, protons and neutrons formed, then merged to form the nuclei of light elements like hydrogen, helium and lithium
  • these atomic nuclei then captured electrons to form atoms
  • by 300,000 years, the universe was composed mostly of clouds of hydrogen and helium atoms
  • at some point, slightly higher densities of hydrogen grew larger and clumpier until they coalesced via collisions to form stars ... and galaxies
  • these stars gave rise to all the heavy elements via fusion reactions
  • subsequently, some of these stars disintegrated via novas or supernovas, generating clouds of interstellar matter
• Patchy condensation of a cloud of interstellar matter from one of these disintegrating stars formed our solar system; most of the matter coalesced near the center to form Sol (our sun) and most of the remaining matter coalesced to form orbiting planetismals, then planetoids, then planets
• Planet Earth formed ~4.6 Ba (K40/Ar40 radiodating)
  • geology
    • inner core (0-1227km) - solid center composed of crystalline iron and nickel formed due to immense pressure of collisions during coalescence
    • outer core (1227-3486km) - liquid layer composed of molten iron, oxygen and sulfur formed during coalescence, maintained by heat energy released by radioactive decay of heavy elements in inner core
    • mantle (3486-6350km) semi-liquid layer composed of oxides of Si, Al, Mg surrounds the outer core
    • crust (6350km-surface) - solid layer composed of lighter elements (mainly silicates) plus some lighter mantle layer elements "floats" on the mantle layer formed as Earth cooled by radiating heat into space
      • oceanic crust formed from relatively smooth basalt
      • continental crust formed from irregular granite protocontinents - as cooling continued, the crust contracted and cracked, releasing molten mantle layer rock and gasses by volcanic activity, increasing crust thickness, and generating a primitive atmosphere
  • primitive atmosphere (alternative ideas)
    • reducing - if Earth coalesced slowly, generating little heat, iron exposed on the surface and would capture all molecular oxygen, so the primitive atmosphere would have consisted mostly of water, hydrogen, nitrogen, methane, and ammonia, with little carbon dioxide or monoxide
    • mildly oxidizing - if Earth coalesced rapidly, generating much heat, most iron would have melted and flowed to the core, allowing oxygen to combine with carbon, and the primitive atmosphere would have consisted mostly of water, carbon monoxide and carbon dioxide, with some nitrogen, sulfides, methane and ammonia, but very little oxygen
  • liquid water appeared on Earth's surface ~4 Ba as indicated by discovery of ~3.8 billion year-old sedimentary rocks (which require liquid water for their formation) in Greenland
    • upon its release from volcanoes, water vapor expanded and cooled, then condensed and fell back towards the surface as rain
    • because the crust was still very hot, rain evaporated before it reached the surface, resulting in continuing cycles of water expansion, cooling, condensation and rainfall, thus initiating the water cycle
    • this water cycle slowly cooled the crust to temperatures at which liquid water could form, leading to sequential water accumulation to form droplets, puddles, pools, ponds, lakes, rivers and oceans
    • because the rain was rather acidic (carbon dioxide, sulfur dioxide, etc. form acid when they dissolve in water) the flowing water (on its way to the lower elevation basaltic surface) dissolved some of the granitic material, generating salt water (the ocean is ~3% salt)

Origin of Life on Earth

Primitive organisms capable of metabolism (ability to accumulate and modify nutrients and energy) and reproduction (ability to generate more organisms like themselves) first appeared ~3.6 Ba, were most likely thermophilic anaerobes, and may have depended on RNA for both enzymatic and genetic activities

• Primordial soup (solution) theories - predict that the original organisms were thermophilic anaerobic organotrophs (most likely fermenters) that obtained both energy and carbon from organic compounds
  • chemical reactions formed fatty acids, sugars, amino acids, purines, pyrimidines, nucleotides, and polymers of all these ~4.1 Ba when primitive (reducing) atmospheric gases were exposed to lightning and intense UV light while dissolved in the salty acidic water
  • accumulation of these compounds over time, leading to oceans (puddles?) with very rich organic molecule content (primordial soup)
  • spontaneous aggregation of lipids and proteins ~3.9 Ba formed primitive membranes, inside of which were accidentally incorporated just the "right" combination of organic (and inorganic) chemical components
• Surface theories - predict that the original organisms were thermophilic anaerobic lithotrophs (obtained both energy and carbon from inorganic compounds) 
  • pyrite theory - life started as a result of a metabolic process that could occur on iron pyrite surfaces
    • iron pyrite provides positive charges for bonding of phosphates, etc., thus fostering polymerization reactions
    • polymerization of lipids formed semi-permeable membranes across which proton gradients could be generated and maintained, thus providing energy for synthetic reactions involving organic compounds generated either inside or outside of these membranes
  • clay theory - life started on montmorillonite clay surfaces, where polymerization reactions could readily occur (similar to pyrite theory, but lacks some of the metabolic possibilities)

• RNA World Hypothesis
  • discovery of ribozymes gave rise to the idea that the earliest (pre-cellular) lifeforms may have used RNA for genetic information storage and for enzymatic activity
  • evidence that clay surfaces can promote RNA evolution

Early Evolution of Organisms with Increasing Complexity

• Fossils are prevalent in rocks formed during Precambrian time ... as early as the Archaean Era 3.8-2.5 Ba and continuing through the Proterozoic Era 2.5-0.543 Ba ... and up to current time
  • Bacterial fossils
    • unicellular and filamentous
    • stromatolites and oncolites - microbial mats comprised of layers of filamentous prokaryotes containing trapped sediment
  • Archaeal fossils
• Mutations caused by high levels of UV irradiation (and other mutagens) occurred continually, and selection among them allowed adaptations that led to development of more complex microorganisms with cell walls, greater biosynthetic capabilities, more extensive membranes, cytochromes, and chlorophylls, thus giving rise to phototrophs, which derive energy from sunlight and carbon from inorganic compounds
  • Anoxygenic photosynthesizers
    • evolved ~0.2 billion years after the first organisms
    • use photosystem I exclusively - purple or green photosynthetic bacteria probably formed the original stromatolites, since they are anaerobic photosynthesizers and conditions on Earth were still anoxic
  • Oxygenic photosynthesizers
    • evolved ~1.2 billion years after the first organisms
    • use a combination of photosystem I and photosystem II

Atmosphere Evolved as Microbes Produced Oxygen ~2.5 Ba

• Oxygen content slowly increased
  • 0.1% oxygen in atmosphere after 0.1 billion years (~2.4 Ba)
  • 1% oxygen in atmosphere after 0.5 billion years (~2 Ba))
  • 10% oxygen in atmosphere after 1 billion years (~1.5 Ba))
  • 21% oxygen in atmosphere after 1.6 billion years (~0.9 Ba))
• Ozone layer formed as UV light energy was absorbed by oxygen
  • lower UV flux on surface - absorption of UV by ozone
  • lower mutation rate - due to decreasing UV flux

Atmospheric Oxygen Availability Led to Increasing Diversity

Organisms with oxygen as final electron acceptor evolved as the atmosphere changed from reducing to oxidizing - more energy available with aerobic respiration than with anaerobic metabolism

• Microbial diversity increased ~0.5 billion years after oxygen generation began
• Modern eukaryotes evolved ~1.3 Ba (1.2 billion years after oxygen generation began)
  • eukaryotic diversity increased ~0.1 billion years later (1.2 Ba)
  • metazoans appeared ~0.3 billion years later (0.9 Ba, when atmospheric oxygen reached ~21%)

Broad Evolutionary Picture

• Original organism replicated and evolved for millions of years, then gave rise to the universal ancestor (common to all later forms of life known to currently exist on earth)
• Universal ancestor was the result of considerable evolution of the original life form and gave rise to three domains of living things Bacteria, Archaea and Eukarya, via considerable evolution

Classification

• Phylogeny vs. Taxonomy - phylogeny depends upon genotypic analysis as a basis of classification, whereas taxonomy depends upon phenotypic (generally biochemical) analysis
• Binomial nomenclature - organisms are always given two names; one designates their species (similar strains), one their genus (similar species)

Molecular Approaches to Phylogeny

• Evolutionary distances (ED) between phylogenetic groups can be measured by differences in nucleic acid (amino acid) sequence, if the molecules used are:
  • Universally distributed across the group studied
  • Functionally homologous - have identical function
  • Properly aligned - so regions of homology and heterogeneity can be identified correctly
  • Rate of sequence changes - commensurate with the evolutionary distance between members
• Molecules used:
  • Initial studies used proteins with fundamental physiological function, such as cytochrome c
  • then 5S ribosomal RNA (rRNA), which is small and easy to isolate, but has little complexity
  • Some current studies use ATP synthase - functionally homologous in species where it is found, properly aligned, changes sequence at rate commensurate with ED, genes are easy to isolate
  • Most current studies use 16S rRNA (eukaryotic, 18S rRNA) isolated from the small subunit of ribosomes because it is highly conserved, meets the precepts outlined above, has the needed level of complexity, and is relatively easy to isolate and work with using current techniques
    • first, the primary structure is determined using reverse transcriptase to make a DNA copy (cDNA) of rRNA, cDNA is sequenced, then rRNA sequence is inferred from cDNA sequence
    • rRNA sequences are then aligned and their degree of homology analyzed by one of two methods:
      • (cataloging (80%) - uses signature sequences determined from secondary structure of rRNA
      • (distance matrix (20%) - ED calculated by recording number of positions at which compared molecules differ; ED matrix then analyzed by computer program that produces phylogenetic trees
  • Ef-Tu and Ef-G, protein synthesis elongation factors used in were used to "root" the Three Domain Tree

Bacteria, Archaea and Eukarya

• Bacterial Phylogeny - highly diverse (e.g., purple and green bacteria are less related than plants and animals) ... more info on bacterial phylogeny
  • Proteobacteria (Phylum 1) - although these five groups of Gram-negative bacteria are quite diverse, it appears that they evolved from a common phototrophic ancestor
    • alpha group contains phototrophs, lithotrophs and organotrophs) - Rhodobacter, purple anoxygenic phototrophs; Rhodopseudomonas, budding photoorganotrophs; Rhizobium, plant endosymbiotic organotrophs; Agrobacterium, plant pathogens; Nitrobacter, budding nitrogen oxidizing lithotrophs; Aquaspirilum, microaerophilic organotrophs; Hyphomicrobium, motile, stalked, budding organotroph; Acetobacter, aerobic organotroph; Paracoccus, hydrogen-oxidizing lithotroph; Methylcoccus, methanotroph; some Pseudomonas, respiratory
    • beta group contains phototrophs, lithotrophs and organotrophs - Rhodocyclus, phototrophs; Nitrosomonas, Spirillum, Bordetella, Neisseria, non-fluorescent Pseudomonas Sphaerotilus, sheathed metal oxidizers; Thiobacillus, sulfur- and iron-oxidizing lithotrophs; Alcaligenes, hydrogen-oxidizing lithotrophs; Neisseria, oxidative organotrophs; some Pseudomonas, respiratory organotrophs
    • gamma group contains phototrophs, lithotrophs and organotrophs - Chromatium and Thiospirillum, purple sulfide-oxidizing phototrophs; Beggiatoa, lithotroph that oxidizes sulfide; Legionella, intracellular pathogen; Azotobacter, free-living nitrogen fixer; fluorescent Pseudomonas, ; enterics (Escherichia, Shigella, Erwinia, Salmonella, Serratia, Proteus, Providencia, Vibrio, Yersinia), frequently associated with large intestine of humans and other animals; Leucothrix, sulfur-oxidizing lithotroph
    • delta group contains organotrophs (only) - Desulfovibrio, anaerobic sulfate-reducer; Bdellovibrio, parasitizes bacteria; Myxobacteria, motile swarming myxospore-forming organotrophs
    • epsilon group contains lithotrophs and organotrophs - Thiovulum, sulfur-oxidizing lithotrophs; Wolinella, hydrogen-utilizing fumarate-based fermenter; Campylobacter and Helicobacter, parasitic organotrophs
  • Gram-Positive Bacteria (Phylum 2) - motile (via flagella) or non-motile rods (Bacillus, Clostridium, Corynebacterium, Lactobacillus) and cocci (Staphylococcus, Streptococcus) with thick peptidoglycan cell walls lacking outer membrane; also wall-less forms (Mycoplasma); many spore-forming species (Actinomyces, Bacillus, Clostridium, Sporolactobacillus); anaerobic (Clostridium, Desulfotomaculum, Veillonella), facultatively aerobic (Bacillus, Streptococcus) or aerobic (Sporosarcina); exoenzymes common
  • Cyanobacteria, Prochlorophytes and Chloroplasts (Phylum 3) - Gram-negative phototrophs with gliding motility; share last ancestor with proteobacteria; unicellular forms (Anacystis, Synechococcus, Pleurocapsa) or filamentous forms (Anabena, Nostoc, Oscillatoria); heterocystous cyanobacteria such as Anabena and Nostoc are nitrogen-fixers
  • Chlamydia (Phylum 4) - Gram-negative obligate intracellular parasites whose cell walls lack peptidoglycan
  • Planctomyces/Pirella (Phylum 5) - Gram-negative budding, obligate aerobes, lacking peptidoglycan in their cell walls (proteinaceous instead); organotrophic, but require very dilute culture media for growth (oligotrophic); many have holdfasts; only two genera, Planctomyces and Pirella
  • Verrucomicrobia (Phylum 6) - Gram-negative prosthecate aerobic to facultatively aerobic sugar fermenters found in freshwater/marine waters and forest/agricultural soils
  • Bacteroides/Flavobacterium (Phylum 7) - Gram-negative; some are obligate anaerobes (Bacteroides, Fusobacterium); others are rod-shaped or filamentous respiratory aerobes with gliding motility(Flavobacteria)
  • Cytophaga (Phylum 8) - Gram-negative obligately aerobic organotrophs with gliding motility; Cytophaga, Sporocytophaga are long, slender rods with pointed ends that digest cellulose and are found on surfaces of the cellulose-containing material (or fish gills); Flexibacter is a filamentous soil and freshwater saprophyte with carotenoids that does not digest cellulose
  • Green Sulfur Bacteria (Phylum 9) - Gram-negative anaerobic photolithotrophs (Chlorobium, Prosthecocloris)
  • Spirochetes (Phylum 10) - Gram-negative, spiral (helical) in shape, with axial filaments; organotrophic; many pathogens (Borrelia, Treponema, Leptospira)
  • Deinococci (Phylum 11) - Deinococcus, Gram-positive radiation-resistant organotroph; Thermus, Gram-negative thermophilic organotroph whose cell wall contains ornithine instead of diaminopimelic acid
  • Green Nonsulfur Bacteria (Phylum 12) - Gram-negative, mostly thermophilic photoorganotrophs (Chloroflexus), but some are chemotrophs (Thermomicrobium, which has unusual membrane lipids that contain 1,2-dialcohols (rather than glycerol) with a long hydrocarbon chain (no ester or ether linkages, because these are)
  • Thermotogae (Phylum 13)
    • Thermotoga Gram-negative thermophiles from benthic hydrothermal vents and continental hot springs; these anaerobic fermenters have a sheath-like envelope (hence the term "toga" in the name)
    • Thermodesulfobacterium - Gram-negative anaerobic thermophilic sulfate-reducing organotroph with an optimum growth temperature of 70C, and ether-linked lipids (very Archaea-like!)
  • Aquifex, Thermocrinus and Relatives (Phylum 14) - Aquifex is a submarine volcanic hot spring bacterium that is hyperthermophilic (optimum temperature for growth is 80C, but can grow up to 90C) lithotroph (reverse TCA cycle) that oxidizes hydrogen, sulfur or thiosulfate and uses oxygen (microaerophilic growth) or nitrate (anaerobic growth) as terminal electron acceptors; most ancient branch of the bacterial phylogenetic tree
  • Nitrospira (Phylum 15) - Gram-negative, aerobic oxidizers (Nitrospira, Leptospirillum, Thermodesulfovibrio)
  • Deferribacter group (Phylum 16) - Gram-negative anaerobes (Defferibacter, Geovibrio, Flexistipes)
• Archaeal Phylogeny - Archaea appear to have evolved more slowly than other organisms, perhaps due to the extreme environments they inhabit; they are prokaryotic, nonsporulating, lack peptidoglycan (use polysaccharide, glycoprotein, protein, or pseudopeptidoglycan instead) and have ether (rather than ester) linkages in membrane lipids; reproduce by binary fission ... more on archaeal phylogeny
  • Nanoarchaeota - very small hyperthermotrophic cells that replicate only when attached to cells of Ignicoccus
  • Korarchaeota - newly discovered extreme thermophiles; these are the most primitive life forms discovered to date
  • Crenarchaeota - most are extreme thermophiles with growth optima at temperatures greater than 80C and many of these are anaerobic lithotrophs (Pyrodictium), organolithotrophs or organotrophs (Thermococcus, Thermoproteus) that require elemental sulfur for optimal growth (Desulfurococcus)
  • Euryarchaeota includes:
    • extreme halophiles includes Halobacterium, Halococcus, Haloferax, Halorubrum, Haloarcula, Natronobacterium, Natronococcus; require high salt (all require at least 1.5M salt, and most require 3-4M salt for growth; obligate aerobes; most are organotrophic (via respiration), others (some Halobacterium) use photophosphorylation via bacteriorhodopsin
    • methanogens are Gram-positive or Gram-negative, strictly anaerobic organolithotrophs that can generate methane from carbon dioxide and hydrogen or from formate, acetate or organic methyl groups; includes Methanopyrus, Methanococcus, Methanothermus, Methanobacterium, Methanospirillum, Methanosarcina and some methanogenic halophiles; Methanobacterium and Methanococcus are also halophilic
    • Thermoplasmatales includes Thermoplasma and Picrophilus
    • Hyperthermophilic euryarchaeota includes Thermococcales and Methanopyrus
    • sulfur utilizers Archaeoglobus is a sulfate-reducing, methanogenic, thermophilic anaerobe; many others also utilize sulfur
    • extreme thermophiles such as Thermoplasma, an acidophilic thermophilic aerobic organotrophs that lacks a cell wall 
• Eukaryal Phylogeny - the nuclear line that led to Eukarya appears to be just as old as the prokaryotic lines that led to Bacteria and Archaea; original "nucleus" was very simple with little DNA; as the DNA content and nuclear complexity increased, DNA was compartmentalized into chromosomes, nuclear membranes evolved to segregate them from the cytoplasm, and specialized microtubules evolved as mitotic spindles; eukaryotes evolved rapidly after atmospheric oxygen and ozone levels developed; evolution of organelles from endosymbiotic bacteria (mitochondria, related to proteobacteria; chloroplasts, ancestors of Cyanobacteria); although very successful, Eukarya did not compete Bacteria and Archaea out of existence because the Eukarya could not survive in habitats that allow Bacteria and Archaea to thrive
  • Groups of Eukaria
    • Protozoa - eukaryotic; unicellular; no cell wall; chemoheterotrophic aerobes (some anaerobes) with absorptive or ingestive nutrient uptake; motility via pseudopodia or undilipodia; some form (dormant) cysts; asexual reproduction via binary fission or budding; sexual reproduction via fusion of haploid gametes
    • Fungi - eukaryotic; generally larger than bacteria; uni- or multicellular; dimorphic (yeast or mold); chitinous cell walls; aerobic heterotrophs, absorptive nutrient uptake; asexual reproduction via spores, binary fission, budding, fragmentation; sexual reproduction via fusion of haploid nuclei leading to formation of spores
    • Plants - eukaryotic; uni- or multicellular; cellulosic cell walls; chloroplasts with chlorophyll a; motility (in unicellular forms) via undilipodia; photosynthetic aerobes (some are facultatively organotrophic); asexual reproduction via binary fission, fragmentation or sporulation; sexual reproduction via fertilization of eggs by sperm, leading to zygospores which form zygote that matures into adult form
    • Animals - eukaryotic; multicellular; lack cell walls; possess organs and organ systems; sexual (only) reproduction via fertilized eggs that mature into offspring
  • Endosymbiotic theory of eukaryotic organelle origin and evolution
    • endosymbiosis has been proposed as the mechanism underlying the origin and evolution of mitochondria and chloroplasts in Eukarya based on this evidence and the fact that a number of primitive protozoa clearly exist as consortia of symbiotic microorganisms
    • endosymbiotic theory proposes that ...
      • prokaryotes and primitive eukaryotes (members of an ancient cell line that possessed a nucleus, but lacked organelles) formed symbiotic associations (probably parasitic at first) that eventually developed into today's nuclear cells with mutually-interdependent remnants of those prokaryotic endosymbionts that we now call organelles
      • through the millenia during which these associations were established and refined the genes that were originally present in the endosymbiotes were gradually moved to the nucleus (although it is much larger in plant cells than in animal cells, organelle DNA is rather small compared with that of the typical prokaryote)
    • evidence supporting the endosymbiotic theory includes the facts that mitochondria and chloroplasts:
      • are enclosed in double lipid bilayer membranes
      • replicate themselves independently of the cell nucleus
      • contain a single, circular, double-stranded DNA chromosome that replicates itself independently of the nucleus of the cell
      • contain 70S ribosomes, and use them for synthesis of proteins not coded for by nuclear genes
        • the 16S rRNA of mitochondria shows a high degree of homology with proteobacteria
        • 16S rRNA of chloroplasts shows a high degree of homology with cyanobacteria
    • analysis of rRNA sequences of a large number of mitochondria and chloroplasts has shown that:
      • endosymbiotic events leading to generation of mitochondria appear to have been quite restricted (only one, most likely) and to have occurred earlier than those leading to generation of chloroplasts
      • endosymbiotic events leading to generation of chloroplasts appear to have occurred several times (at least 6) and were more recent than those leading to generation of mitochondria
    • proposed, but not well-supported, hypotheses include the ideas that:
      • spirochetes which attached themselves to the exterior of primitive eukaryotes and established an ectosymbiosis with those eukaryotes eventually evolved into undilipodia (flagella and cilia); examples of modern-day eukaryotes with ectosymbiotic spirochetes that provide motility to the consortium include Trychonympha
      • the origin of the nucleus itself may have involved a series of similar events that originated from refinement of associations of consortia of mutualistic prokaryotes into the combined reproductive/metabolic units that we now think of as nucleated (eukaryotic) cells

  Did ya know ...?

  The National Medal of Science, established by Congress in 1959, was awarded to these scientists for the year 2000:

  Jared Diamond, physiology professor at the UCLA School of Medicine, for research that applied Darwinian evolutionary approaches to physiology, ecology, conservation biology and human history.

  Lynn Margulis, geosciences professor at the University of Massachusetts, Amherst, for contributions to the understanding of the structure and evolution of living cells. Dr. Margulis first postulated the endosymbiotic theory of organelle formation.