Microbiology, Lec 8.

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I put some questions from an old exam on the web.

MCDB A112

nrpace@colorado.edu

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A. In terms of their fundamental properties, Archaea are as different from Bacteria as they are from Eukarya; indeed Archaea in many regards are more similar to euks than to Bacteria.

2. The relationships of Archaea to other "kinds" of organisms only became evident in the late 1970s, with the revelation of the Big Tree (handout) through studies of ribosomal RNA sequences by Carl Woese.

A. In essence, the tree is a quantitative map of evolutionary relatedness, a comprehensive map (albeit rough) of biological diversity.

B. Note that the text deals with this tree as a model for "classification" a la "5-kingdoms" or "prokaryote/eukaryote" "taxonomic" models. The Big Tree is more than a model/idea/hypothesis: it is a map.

C. Indeed, the tree is a quantitative estimate of that slippery concept of "biological diversity" - you can put a number on how "alike" organisms are, and how "different".

3. A few large-scale lessons from the Big Tree:

A. There was a single origin for the terrestrial type of life -- all lifeforms are related

B. Three "primary lines of evolutionary descent" -- "Domains" -- "ur-kingdoms" -- "Empires"

1) Sometimes see referred to as "kingdoms," but usage in this context is probably not a good idea -- too historically loaded.

C. The eucaryote nuclear line of descent is as old as the ÒprocaryoteÓ lines.

D. "Prokaryotes" is not a "kind" of organism -- There are two primary lineages of "procaryotes," Bacteria (formerly eubacteria) and Archaea (formerly archaebacteria -- try to avoid using this term; they arenÕt bacteria. No one in the field who I know-of uses the term "archaeobacteria," the text/BergeyÕs usage.)

1) Are there still more domain-level divergences to be discovered??

E. Note that lines connecting organisms to nodes are not all the same length -- the evolutionary clock is not constant between different lineages (e.g. Haloferax vs. Methanopyrus, Aquifex vs. Bacillus, Eucarya in general vs. any representative of Archaea or Bacteria)

1) Rate of evolution not necessarily the same for a particular lineage at all stages in the evolution of the line, e.g. Agrobacterium vs. mitochondrion

2) Note domain-level tendencies:

Eucarya -- fast clocks

Archaea -- slow clocks

Bacteria -- intermediate rates of evolution

3) Because of variable rates, estimating time from sequence change is chancy--even fatuous--without some sort of calibration.

F. Note that the phylogenetic space occupied by multicellular eucarya is shallow and limited, but enormously diverse in morphological (less biochemical) phenotype. A consequence of large, highly plastic genomes?

1) Note that the "typical" eucaryote is microbial and has a small genome, e.g. Saccharomyces cerevisiae at 13.5 x 10^6 bps.
E. Coli at ~4.2 x 10^6 bps
Calothix [a cyanobact.] at ~12.5 x 10^6 bps
human at ~3.2 x 10^9 bps
Methanococcus jannaschii at ~1.7 x 10^6 bps.

G. The rRNA (and other molecular) data prove that mitochondria and chloroplasts were of bacterial origin (bacterial "divisions" Proteobacteria and "cyanobacteria," respectively)

H. Note how deeply divergent are Giardia, Trichomonas and Vairimorpha in the eucaryotic line. These organisms lack mitochondria, so may have diverged from the main eucaryal line of descent before the mitos came in.

1) It turns out they have a few bacterial genes, but it is not clear where they got them.

A. Phylogenetic analyses using molecules other than rRNA show that the "root" (origin) of the Big Tree is (presumably deep) on the bacterial line.

B. This means also that Eucarya and Archaea shared common history after divergence from Bacteria

1) This explains many similarities between archaeal and eucaryal machineries, to the exclusion of reps of Bacteria.

e.g. similar transcription machineries; Archaea and Eucarya use TATA-binding proteins whereas Bacteria use s factors for specification of transcription initiation.

e.g. Archaeal and eucaryal DNA-synthetic machineries far more like one another than either is to bacteria (for good overview, Bernander, "Archaea and the cell cycle," Molec. Microbiol. 29:955-961[1998])

2) But there are many systematic differences between Archaea and euks; the nature of the cellular machinery was not yet fully developed at the stage of the Archaea/Eucarya split. E.g., the two kinds of organisms have different membrane chemistries, ester-linked (us) vs. ether-linked (Archaea) lipids!! (The chemistry of the membrane must not have yet been chosen at the time of that split.)

C. The topology of the BT means that the traditional classification system prokaryote/eukaryote is meaningless: there are two different kinds of proks, as different from one another as either is from euks.

(Norm Pace has stopped using the word prokaryote. It carries incorrect conceptual implication)

5. Note that the Big Tree shown is a limited set of specific organisms: ca. 15,000 16S sequences are now available.

A. One database of rRNA sequences -- Ribosomal Database Project:
You can download trees, carry out functions, get programs, etc.

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7. Overview of the archaeal tree (handout):

A. Two main lineages represented by cultivars, Crenarchaeota and Euryarchaeota.

1) Crenarchaeota: All cultivated types are high-temperature, tend to be "chemoautotrophs." BUT, UNcultivated low-temp. crens are abundant in the environment (detected by cloning rRNA genes)

a) Name cren- from the Greek for spring or fount, referring to the ostensible similarity of such organisms to the earliest life (high temperature, using geothermal compounds for energy, e.g. H2 / S more later)

2) Euryarchaeota: methanogens, extreme halophiles, many heterotrophs (more later)

a) Name from Greek eury meaning varied, referring to variable phenotypes, compared to cultivated crenarchaeota.

3) Representatives of potentially a third kingdom of Archaea, Korarchaeota (kor is greek for youth) have only been detected in high-temperature environments by rRNA gene-cloning. No rep has been pure-cultured.

4. Studies of rRNA genes isolated directly from the environment now show that cultured reps of Archaea substantially under-represent the kinds of organisms that occur in the real world.

A. Ether-linked membrane lipids (Whoa! Real different!)

B. No archaeon makes the common bacterial envelope-component peptidoglycan. However some (notably methanogens) make a similar cell-wall material, pseudopeptidoglycan with some different linkages. The enzymologies for those compounds are pretty different, i.e. evolved independently.

C. Walls of Archaea generally are composed of chemically crosslinked glycoproteins, often as an S-layer, a crosslinked crystalline array of proteins.

D. Archaeal mechanisms, like eucaryal are resistant to the usual bacterial antibiotics (penicillin, chloramphenicol, tetracycline, rifampin); they are hit by the usual eucaryal inhibitors (aphidicolin).

1. It may be a good thing that there are no known archaeal pathogens!

E. All primary productivity in Archaea is through chemoautotrophy, generally H2-oxidation, transferring electrons to CO2 or some oxidized S- compound. There is no real photosynthesis; the archaeal bacteriorhodopsin wonÕt support growth on light alone.

F. Have many cellular properties with homologs in Eucarya; e.g. histones and chromatin, many nuclear-specific genes.

A. Methanogens:

1. Hard-core anaerobes, so not discovered until fairly late (1950s). In a review article in the mid-70s only 13 species (cultures) were known!

2. Metabolically uniform: chemolithotrophic (H2/CO2 or H2/acetate, making methane = natural gas).

3.. Simple genomes: 1500-2000 genes in Methanococcus jannaschii)

4. Commonly occur as symbionts:

with bacteria (e.g. Syntrophus)

with anaerobic protists (e.g. Pelomyxa)

with animals (e.g. cows. We have but probably don't need)

B. Halophilic Archaea:

1. Usually thought-of in the context of brines: Dead Sea (high mono and divalent cations); Salted fish - was an early source for Halobacterium spp.; floor of Red Sea??

2. But note - many microenvironments may also be high salt - e.g. drying soil surfaces; e.g. a drying armpit or a used sneaker.

3. Note common occurrence in environment; e.g. evaporation ponds in south S.F. Bay, flying over Great Salt Lake.

4. Invented (bacterio)rhodopsin; serves to generate membrane potential using light, but supposedly can't make enough ATP to grow by photosynthesis. Most cultivated archaeal extreme halophiles are considered chemoheterotrophs. Some can do methanogenesis.

5. Note that halophiles are derived from a particular lineage of methanogens (the Methanosarcina, Methanospirillum group).

10. Some factoids on Crenarchaeota

A. All cultivated instances are sulfur-dependent and thermophilic. However, molecular analyses show that uncultivated low-temp. Crenarchaeota are abundant in all/most[?] environments.

B. Based on cultivars (only a few dozen, all high-temp.), metabolic diversity in the Crenarchaeota is pretty limited: H2/S° is common (e.g. Pyrodictium, Pyrobaculum, probably Sulfolobus). Some will also oxidize organics, using about anything as electron acceptor. Sulfolobus will even breath molybdate!

C. Pyrodictium, Pyrolobus - growth at the highest temps in culture, 110-113°C. Metabolism by H2/S°, grow on surface of sulfur globs in a mineral salts mixture under a few At. of H2/CO2. Hard country to live-in!

D. A pretty inconspicuously boring bunch based on cultivars -- but in fact they are common all around us: rRNA clone analysis has detected crenarchaeotes in soils, marine and freshwaters, etc.

1. Typically, rRNA gene abundance indicates that they are a few percent of most environments, in some places much higher.

2. Mesophilic Crens are ca. 50% of marine microbes below ca. 100 m. (Globally thatÕs a LOT of biosphere! What are all those Crenarchaeota out in the soils and sediments, and in the open ocean-column doing? Not known.

3. One crenarchaeote has been detected as a common symbiont in sponges. What is it doing there?

4. The deeply branching, slowly evolving Archaea are the modern organisms that are most closely related to the last common ancestor of all life, in terms of rRNA sequence-divergence.

a. Note that in tree calculations low-temp lines spin-off shorter, high-temp lines. It is argued that the short lines (high-temp organisms) are the ancestral , that the earliest life was thermophilic.

Some names:

Methanococcus, Methanopyrus - methanogens (eury)

Thermococcus, Pyrococcus - non-methanogenic Euryarchaeota, anaerobic heterotrophs with sulfur, most commonly cultivated organisms at 90 - 100°C

Archaeoglobis - Euryarchaeota, H2-driven sulfate-reducers

Pyrodictium, Thermophilum, Pyrobaculum, Sulfolobus -- Crenarchaeota