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How can we determine evolutionary relationships among microbes?
Difficulties of elucidating the evolutionary relationships of microbes
• Few distinguishing morphological features in microbes as compared to larger organisms (plants, animals, etc).
• Microbes have been traditionally classified based on physiology and phenotype
• Bergey’s Manual largely based on phenotype
• HOWEVER, physiology (e.g., respiration) rarely tracks with phylogeny (i.e., genotype) in microbes.
Which ones are more closely related?
Escherishia coli Nitrosopumilus maritimus
Homo sapiens subsp. Gaga
Ernst Haeckel (1866)
- unicellular - “prokaryotic”
- unicellular - eukaryotic
PLANTAE FUNGI ANIMALIA
• Classification scheme based on morphological similarities and energy sources
• Highest taxonomic group = KINGDOM
• Identified 5 kingdoms - Monera - Protista - Animalia - Plantae - Fungi
Robert Whittaker: The 5 Kingdoms
1969
“Molecules as documents of evolutionary history” Zuckerkandl and Pauling (1965)
J. Theoret. Biol., 8:357-366
• developmental homologies
• molecular homologies
• universal genetic code
• eukaryote chromosomes same structure
• chlorophyll a
• cytochrome C
• nucleic acid and amino acid comparisons
• much more about this in a few weeks
gill bars
post-anal
tail
Hemoglobin
• Present in all organisms
• Homologous (common evolutionary origin)
• Not transferred horizontally (vertically inherited)
• Readily obtained & sequenced
Requirements of a gene “proxy” for an organismal phylogeny
Carl Woese (U-Illinois)
Used ribosomal RNA (rRNA) genes to determine “natural” or evolutionary relationships among organisms.
Small subunit ribosomal RNA • 16S rRNA in
archaea & bacteria
• 18S rRNA in eucaryotes
• Contains regions with 100% conservation in all organisms
Natural taxonomy: based on evolutionary relatedness i.e, allows phylogeny to be predictive
--> Related organisms should have similar properties (not that we always know “which” properties those are)
Woese, 1977
Amann (1995) Microbial Reviews 59: 143-169
Sediments 0.25% Soil 0.3% Seawater 0.001-0.1% Freshwater 0.25% Activated sludge 1-15%
Most microorganisms cannot be grown in the lab!
Norman Pace: Cultivation-independent identification of microorganisms
• Use molecular biology (PCR) to obtain and sequence rRNA genes directly from environment
• Enables cataloging of microbial species in any environment
• Revolutionized our view of microbiology
BACTERIA
ARCHAEA
Kirk Harris
Bacterial Diversity: 20 years later
You are here
THE BIG
TREE
Lessons from the “Big Tree” • All cellular life is related (one origin of Life!)
• Three domains of life, not two: Bacteria, Archaea, Eukarya
• Most life is microbial (still)
• Root is on bacterial line
• Eukaryotic nuclear line of descent as old as Archaea
• Bacterial origins of both mitochondrion and chloroplast
mitochondria
chloroplasts
You are here
Thermophilic Origin of Life?
• The organisms closest to the “Root” of the Big Tree are all thermophiles
• This suggests that Life may have originated in a high temperature environment
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THE ARCHAEA
• Archaea are “old” or “primitive”
• Archaea are obligate “extremophiles”
• Archaea are basically the “same” as bacteria (morphology, size)
• Archaea “turned into” eucarya?
Preconceptions about archaea
• Metabolism is like bacteria (e.g., energy metabolism, central metabolism).
• Cellular machinery (replication, cell division, transcription, translation, etc.) is much more closely related to machinery in eucaryotes.
• Lipids are unlike those in either bacteria or eucaryotes.
Archaea have mixed characteristics
Archaeal Lipids • Different from bacterial, eukaryotic lipids
– L-glycerol, not D-glycerol – Ether links, not ester – Branched chains of lipids
• Made from isoprene units • No unsaturations in lipid
isoprene unit
OO
Major Lipids of Archaea and Membrane Structure
PHYTANYL: 20 Carbons = C20 (½ membrane width)
BIPHYTANYL: 40 Carbons = C40 (full membrane width)
Archaeal Lipids: Glycerol Dialkyl Glycerol Tetraethers (GDGTs)
Archaeal groups defined phylogenetically
• Euryarchaeota
• Crenarchaeota
• Nanoarchaeota
• Korarchaeota
• Thaumarchaeota
• Many uncultivated lineages
CULTIVATED ARCHAEA
The Euryarchaeota
• Large physiological diversity
• Thermophiles, hyperthermophiles
• Mesophiles & psychrophiles
• Methanogens
• Extreme halophiles
• Many uncultivated lineages
Euryarchaeota: Methanogens • Phylogenetically diverse group with a common metabolism
• Produce methane (CH4) from a variety of compounds (CO2, acetate)
• All methanogens are strict anaerobes
• Group includes psychrophiles, mesophiles and thermophiles
• Commonly found in swamps & sediments
Methanobrevibacter arboriphus Methanospirillum
hungatei
Methanocaldococcus jannaschii
Methanopyrus kandleri
Euryarchaeota: Extreme halophiles
• Require > 1.5M NaCl (up to 5M) • Some live in soda lakes (pH > 9) • Many unusual morphologies
Haloquadratum
Seawater Evaporating Ponds Near San Francisco Bay, California
Euryarchaeota: Thermoplasma/Ferroplasma
• Acidophiles, capable of growing below pH=0
• Lack cell walls - similar to mycoplasmas
• Oven obtained from coal refuse piles (contain pyrite FeS2) that heat up spontaneously
• Ferroplasma (35°C), also in mine tailings, coal refuse
Korarchaeota
• Originally defined phylogenetically based on environmental sequences
• One representative cultivated in consortium (mixture of 3 organisms)
• Hyperthermophile (>85˚C)
• Ferments peptides
Cell Preparation and Genome Sequencing. It was observed thatfilamentous cells hybridizing to probes KR515R/KR565R remainedintact in the presence of high concentrations of SDS (up to 1%) inthe hybridization buffer. This feature allowed highly enriched cellpreparations to be made by exposing the Obsidian Pool enrichmentculture to 0.2% (wt/vol) SDS (without cell fixation) followed byseveral washing steps and filtration through 0.45-!m syringe filters.PCR-amplified SSU rDNA sequences from SDS-treated filteredcell preparations showed that !99% of the clones sequenced (n "180) were identical to the SSU rDNA sequence of pOPF!08 (seeFig. S3). Phase-contrast (Fig. 1B) and EM (Fig. 1C) showed thesamples to be highly enriched for ultrathin filamentous cells with adiameter of 0.16–0.18 !m. DNA clone libraries were constructedfrom both SDS- and nonSDS- (libraries BHXI and BFPP, respec-tively) treated enrichment culture filtrates. A total of 23,000 and11,520 quality sequencing reads from libraries BHXI and BFPP,respectively, were binned based on %GC content and read depth.Overlapping fosmid sequences containing the pOPF!08 SSU rRNAgene (Fig. S4) were used to guide the WGS assembly. Five largescaffolds with a read depth of #8.4–9.9 were closed by PCR(further details are provided in SI Text). Single-nucleotide poly-morphisms occur at a rate of $0.2% across the genome.
General Features. The complete genome consists of a circularchromosome 1,590,757 bp in length with an average G%C contentof 49% (Table 1). A single operon was identified that contains genesfor the SSU and LSU rRNAs. Forty-five tRNAs were identified byusing tRNAscan-SE (18). A total of 1,617 protein-coding geneswere predicted with an average size of 870 bp. Of the predictedprotein-coding genes, 72.4% included AUG; 17.6%, UUG; and10% had GUG for start codons. The archaeal Clusters of Ortholo-gous Groups (arCOGs) analysis (see below), combined with addi-tional database searches, allowed the assignment of a specific
biological function to 998 (62%) predicted proteins; for another 246proteins (15%), biochemical activity but not biological functioncould be predicted, and for the remaining 373 (23%) proteins, nofunctional prediction was possible, although many of these areconserved in some other archaea and/or bacteria.
arCOGs. The predicted proteins were assigned to arCOGs (19) (seeSI Text, Dataset S1]. Of the 1,617 annotated proteins, 1,382 (85%)were found to belong to the arCOGs, a coverage that is slightlylower than the mean coverage of 88% for other archaea and muchgreater than the lowest coverage obtained for Nanoarchaeumequitans (72%) and Cenarchaeum symbiosum (58%). When thegene complement was compared with the strictly defined core genesets for the Euryarchaeota and Crenarchaeota (i.e., genes that arerepresented in all sequenced genomes from the respective division,with the possible exception for C. symbiosum in the case of theCrenarchaeota, but that are missing in at least some organisms of theother division), a strong affinity with the Crenarchaeota was readilyapparent. Specifically, Ca. K. cryptofilum possesses 169 of the 201genes from the crenarchaeal core (84%) but only 33 of the 52 genesfrom the euryarchaeal core (63%). When the core gene sets weredefined more liberally, i.e., as genes present in more than two-thirdsof the genomes from one division and absent in the other division,the korarchaeote actually shared more genes with the Euryarcha-eota than with Crenarchaeota (Table 2, Table S1). Seven proteinshad readily identifiable bacterial but not archaeal orthologs, asdetermined by assigning proteins to bacterial COGs (20) (TableS2). Conceivably, the respective genes were captured via indepen-dent horizontal gene transfer (HGT) events from various bacteria.By contrast, no proteins were specifically shared with eukaryotes,to the exclusion of other archaea. The organism lacks only five genesthat are represented in all sequenced archaeal genomes, namely,diphthamide synthase subunit DPH2, diphthamide biosynthesismethyltransferase, predicted ATPase of PP-loop superfamily; pre-dicted Zn-ribbon RNA-binding protein, and small-conductancemechanosensitive channel.
Energy Metabolism. The predicted gene set suggests that Ca. K.cryptofilum grows heterotrophically, using a variety of peptideand amino acid degradation pathways. At least four ABC-typeoligopeptide transporters and an OPT-type symporter couldimport short peptides, which more than a dozen peptidasescould hydrolyze into amino acids. As in Pyrococcus spp.,pyridoxal 5&-phosphate-dependent aminotransferases can con-vert amino acids to 2-oxoacids, while scavenging amines with"-keto-glutarate to form glutamate. Four ferredoxin-dependent oxidoreductases (specific for indolepyruvate, pyru-vate, 2-oxoglutarate, or 2-oxoisovalerate) could oxidize anddecarboxylate the 2-oxoacids, producing acyl-CoA molecules.
Fig. 1. Microscopy of Ca. K. cryptofilum. (A) FISH analysis with Korarchaeota-specific Cy3-labeled oligonucleotide probes KR515R/KR565R. The undulatedcell shape results from drying of the specimen on gelatin coated slides beforehybridization. (Scale bar, 5 !m.) (B) Phase-contrast image of korarchaealfilaments after physical enrichment. (Scale bar, 5 !m.) (C) Scanning electronmicrograph of purified cells. (D) Transmission electron micrograph after neg-ative staining with uranyl acetate displaying the paracrystalline S layer. Cellsare flattened, which increases their apparent thickness.
Table 1. General features of the Ca. K. cryptofilum genome
Genome feature Value
Total number of bases 1,590,757Coding density, % 90.7G % C content, % 49.0Total number of predicted genes 1,665Protein coding genes 1,617Average ORF length, bp 870rRNA genes* 3tRNA genes 45Genes assigned to COGs 1,401Genes assigned to arCOGs 1,382Genes with function prediction 998Genes with biochemical prediction only 246Genes with unknown function or activity 373
*16S, 23S, and 5S rRNA.
Elkins et al. PNAS ! June 10, 2008 ! vol. 105 ! no. 23 ! 8103
MIC
ROBI
OLO
GY
Nanoarcheaota • Nanonarcheum equitans
• Obligate symbiont (parasite?) of Ignicoccus
• 0.4µm diameter
• Smallest genome (0.5Mb) – contains only core genes for
molecular processes
Nanoarchaeota
• Phylogenetic placement is currently hotly debated
• May be highly derived members of the Crenarchaeota
Crenarchaeota
• Cultivated Crenarchaeota are primarily hyperthermophiles & anaerobes
• Many are also acidophiles (pH 3-4)
Sulfolobus solfataricus
Solfatara in Naples
• Hot sulfur-rich enviroments • Tom Brock • Lobed coccus • Chemolithotroph • Chemoorganotroph • Growth 75-87°C • pH 2-3 • Good genetics!
Pyrodictium strain 121 • Current record for life’s temp limit • Grows at 121°C (in an autoclave!) • Withstands temperatures up to 130°C • Lives in walls of black smoker • Chemolithotroph
Crenarchaeota
• The vast majority of Crenarchaeota are from uncultivated lineages.
WHAT DO THEY DO?
Marine archaea: Discovery of the Thaumarchaeota
Karner, DeLong & Karl (2001), Nature 409:507-10
Marine archaea at the Hawaii Ocean Time Series (HOTS)
Temp.: 24˚C [NH4
+]: ~0.5 µM
gravel substratum
Marine archaea “eat” ammonia and convert CO2 into biomass
NH3 + 1½ O2 NO2- + H+ + H2O
humans…
C6H12O6 +
6 O2
6 CO2 +
6 H2O
The Nitrogen Cycle
0.1
Cenarchaeum symbiosumCandidatus Nitrosopumilus maritimus
Candidatus Korarchaeum cryptofilumThermofilum pendens
Caldivirga maquilingensisPyrobaculum calidifontis
Pyrobaculum islandicumPyrobaculum aerophilum
Pyrobaculum arsenaticumIgnicoccus hospitalisStaphylothermus marinus
Aeropyrum pernixHyperthermus butylicus
Metallosphaera sedulaSulfolobus solfataricus
Sulfolobus acidocaldariusSulfolobus tokodaii
Nanoarchaeum equitansThermococcus gammatoleransThermococcus kodakarensis
Pyrococcus furiosusPyrococcus abyssiPyrococcus horikoshii
Methanopyrus kandleriMethanosphaera stadtmanae
Methanothermobacter thermautotrophicusMethanocaldococcus jannaschii
Methanococcus aeolicusMethanococcus maripaludisMethanococcus vannielii
Picrophilus torridusFerroplasma acidarmanus
Thermoplasma acidophilumThermoplasma volcanium
Archaeoglobus fulgidusHalobacterium sp
Natronomonas pharaonisHaloarcula marismortui
Halorubrum lacusprofundiHaloquadratum walsbyi
Haloferax volcaniiMethanocorpusculum labreanum
Methanoculleus marisnigriCandidatus Methanoregula
Methanospirillum hungateiMethanosaeta thermophila
Methanococcoides burtoniiMethanosarcina barkeriMethanosarcina acetivoransMethanosarcina mazei
Giardia lambliaEntamoeba histolytica
Leishmania majorTrypanosoma cruziTrypanosoma brucei
Cryptosporidium parvumTheileria parvaPlasmodium yoelii
Plasmodium falciparumArabidopsis thaliana
Oryza sativaDictyostelium discoideum
Homo sapiensAnopheles gambiae
Saccharomyces cerevisiaeSchizosaccharomyces pombe
94
7061
90
52
55
71
69
8275
61
88
95
38
78
97
96
90
100
100100
100100
100
100
100
100
100
100
100100
100
100
100100
100
100
100100
100100
100
100
100100
100100
100
100
8651
97
64
100100
100100
100
100
100
100
100
100
Eucarya
ThaumarchaeotaKorarchaeota
Sulfolobales
Desulfurococcales
Thermoproteales
Nanoarchaeota
Thermococcales
MethanopyralesMethanobacteriales
Methanococcales
ThermoplasmatalesArchaeoglobales
Halobacteriales
Methanomicrobiales
Methanosarcinales
Crenarchaeota
Euryarchaeota
0.1
Crenarchaeota
Euryarchaeota
Saccharomyces cerevisiaeDictyostelium discoideum
Oryza sativa1.00Candidatus Korarchaeum cryptofilum
Candidatus Nitrosopumilus maritimusCenarchaeum symbiosum1.00
Pyrobaculum aerophilumThermofilum pendens1.00
Aeropyrum pernixHyperthermus butylicus
1.00
Sulfolobus acidocaldariusMetallosphaera sedula1.00
1.00
1.00
0.88
Nanoarchaeum equitansThermococcus kodakarensis
Pyrococcus abyssi1.00Methanopyrus kandleri
Methanothermobacter thermautotrophicusMethanosphaera stadtmanae1.00
1.00
Methanocaldococcus jannaschiiMethanococcus maripaludis1.00
Thermoplasma volcaniumFerroplasma acidarmanus1.00
Archaeoglobus fulgidusHaloarcula marismortuiNatronomonas pharaonis1.00
Methanosarcina mazeiMethanosaeta thermophila
1.00
Methanospirillum hungateiMethanocorpusculum labreanum1.00
0.70
1.00
1.00
1.00
1.00
1.00
1.00
1.00
0.88
1.00
Eucarya
ThaumarchaeotaKorarchaeota
Sulfolobales
Desulfurococcales
Thermoproteales
NanoarchaeotaThermococcalesMethanopyralesMethanobacteriales
Methanococcales
ThermoplasmatalesArchaeoglobalesHalobacteriales
Methanomicrobiales
Methanosarcinales
Figure S4. (A) Maximum-likelihood phylogeny of Group 1 Archaea. The phylogeny was inferred using an alignment of concatenated R-proteins (66 taxa, 6,142 positions). WAG+Inv+Gamma (4 classes). 100 replicates. (B) Bayesian tree of mesophilic Group 1 Archaea inferred using an alignment of concatenated R-proteins (29 taxa, 6,142 positions). Mixed model + Gamma (4 classes). 100 replicates.
Supplemental Figure S4
Thermophilic Origin of Life?
Obsidian Pool, Yellowstone National Park
75 – 80 ˚C pH 4.5 – 6.0
Heart Lake 1 70 - 80°C pH 8.3 NH4+ 95 µM NO2- 3 µM NO3- 174 µM
Nitrosocaldus yellowstonii
200 nm
2 µm
Emily Tung
Nitrosocaldus yellowstonii
Growth of Nitrosocaldus yellowstonii HL72
N. yellowstonii has no intracellular compartments
Photo by Yuichi Suwa
Nitrosomonas europaea Nitrosocaldus yellowstonii
Watson et al. (1989)
Nitrosococcus oceanus
Photo by Emily Tung
100 nm
Nitrosocaldus yellowstonii Genome Size 1.43 Mbp
G+C Content 37%
Number of Genes 1605 Protein coding 1550 16S rRNA 1 23S rRNA 1 tRNAs 45
Percentage coding 91%
Many interesting features: – Fla, Che, Ure, Topo, Cdv, FtsZ
Eucarya Korarchaeota Crenarchaeota Thaumarchaeota Euryarchaeota Bacteria
N. yellowstonii BLASTP
Hits
N. yellowstonii
N. maritimus
C. symbiosum
188 679 genes
302 genes
364 genes
22
10 149
Nitrosocaldus yellowstonii
1.4 Mb 1600 genes
Nitrosocaldus yellowstonii
Nitrosopumilus maritimus
Cenarchaeum symbiosum
188
679
302 364
22
10 149
Function unknown/ Poorly characterized: 120 (80%)
Metabolism
Cellular Processes & Signaling
Information Storage & Processing
Hope Gray
Tengchong, China
Lassen NP, CA
Great Basin, NV
Yellowstone National Park, WY