extreme biology lecture 1- bacteria

Question 1

what are extremophiles?

Answer 1

-Latin extremus (extreme) & Greek philiā (love)

-Organisms that grow optimally in environments considered extreme

-Dominant in earth’s evolutionary history
-an extreme environment 40 M years ago!
-life may even have begun in deep sea hydrothermal vents

-Have continued to thrive in extreme conditions
-One of most abundant life forms
-Biological “dark matter” - many yet to be discovered

Question 2

are there extreme bacteria that are not extremophiles?

Answer 2

yes..
Very slow/fast growing
Very large/small
Very large genome
Extreme cellular complexity
Some are not really bacteria - Archaea

Question 3

what are e.coli stats?

Answer 3

-2 µm long
-0.7 µm3 volume
-1 chromosome
-4.6 Mbp genome
-4,300 protein coding sequences
-20 minute doubling time

Question 4

what are Small: Candidatus Pelagibacter ubique

Answer 4

-In salt- & fresh-water globally
Discovered in Sargasso Sea - v. low in nutrients - relatively devoid of life

-Possibly most numerous bacterium globally
~ 2 x 1028 Candidatus P. communis & relatives
Total weight > all fish in sea

-One of smallest self-replicating cells known
An ultramicrobacterium (V < 0.1 µm3), 0.4-0.9 µm x 0.1-0.2 µm
Genome 1.3 Mbp, 1,354 protein genes, 35 RNA genes

-Understanding ultramicrobacteria important for synthetic biology
e.g. creation of self-replicating synthetic organisms based on minimal genomes

Question 5

what are Large & extreme polyploidy: Epulopiscium spp

Answer 5

Most bacteria & archaea rely on diffusion to move metabolites & biomolecules short distances

-Exception: bacterial symbiont of surgeonfish - Epulopiscium spp.
Latin: “a guest at a banquet of fish”!
L > 600 µm, V ~ 3 x 106 µm3
Homozygous: 10s of 1000s genome copies

-How does it maintain large size?
Highly folded cell membrane increases effective surface area
Arrangement of genomes around cell periphery may permit regional responses to local stimuli

-Abundance of genome copies may permit unstable genetic feature common in eukaryotes, rare in bacteria
long (10 bp) poly(A) repeat in dnaA
essential gene encoding DnaA which activates DNA replication initiation
functional significance of mononucleotide repeat unknown

-Epulopiscium has acquired some characteristics & advantages of eukaryotic cells!

Question 6

what are Bacterial heterozygosity: Achromatium

Answer 6

Large (125 µm), hyperpolyploid & only known heterozygous bacterium
~300 different chromosomes
Allelic diversity far exceeds that of typical bacterium
Common worldwide, spanning T, salinity, pH & depth ranges normally resulting in bacterial speciation
Achromatium from differing ecosystems (freshwater, saline) equally functionally equipped but differ in gene expression patterns by transcribing only relevant genes
Hypothesis: environmental adaptation occurs by increasing copy number of relevant genes across chromosomes, without losing irrelevant ones, maintaining ability to survive any ecosystem

Question 7

what are fast and slow bacterial growth rates?

Answer 7

fast= several times/ hour in nutrient rich lab conditions
slow = once/year in nutrient poor subglacial lakes

Question 8

what limits growth rates?

Answer 8

-Many organisms vary ribosomal abundance as function of growth conditions

-Translation time / ribosome (~7 min) places inherent limit on bacterial growth rate

-Could be surpassed only if cell could
increase polypeptide elongation rate
reduce total mass of ribosomal protein & rRNA

Question 9

what are normal environment stats?

Answer 9

Temperature ~ 10-37°C
Relative humidity ~ 30-50%
Water activity ~ 0.75
[salt] ~ 0.15-0.5 M
pH ~ 7
Pressure ~ 0.1 MPa
Ionising radiation ~ 0.6 mSv/year

Question 10

how do we study bacteria?

Answer 10

Collect them!
e.g. from “Door to Hell” crater in Tukmenistan
Culture them!
Examine them!
e.g. custom-built chamber to warm thermophile & acidophile Sulfolobus acidocaldarius
Extract & sequence genomic material
Knock out key genes & look at effect on organism
Express key genes in workhorse bacteria
Solve structures of key proteins

Question 11

what is the bacterium?

Answer 11

Deinococcus radiodurans
Polyextremophile
Survives cold, dehydration, vacuum, acid, ionising radiation, UV
General UV resistance strategy
Mn accumulation, reduced Fe levels
Antioxidants (glutathione)
Chaperones
DNA-repair proteins
Discovered in experiments in 1950s to determine if food could be sterilised with g radiation - it survived!
“Unique” ability to repair ss- and ds-DNA
One of most radiation-resistant organisms known
Survived 3 years in outer space on ISS!!
Deinococcus radiodurans
Polyextremophile
Survives cold, dehydration, vacuum, acid, ionising radiation, UV
General UV resistance strategy
Mn accumulation, reduced Fe levels
Antioxidants (glutathione)
Chaperones
DNA-repair proteins
Discovered in experiments in 1950s to determine if food could be sterilised with g adiation - it survived!
“Unique” ability to repair ss- and ds-DNA
One of most radiation-resistant organisms known
Survived 3 years in outer space on ISS!!

Question 12

why do we study extreme bacteria?

Answer 12

-PCR
-Biosensing e.g. of As in water
-Synthetic biology
-Bioprocessing enzymes e.g. lignocellulose processing
-Astrobiology

Question 13

what are acidophiles?

Answer 13

Acetobacter acetiproduces acetic acid from oxidation of ethanol
Acidithiobacillus thiooxidans main component of snotitles - extremophile matshang in caves - nasal mucus consistency

-General acidophile (pH < 2) survival strategy
Membrane highly impermeable to H+
K+ antiporter releases H+ to extracellular medium for pH homeostasis
Chaperones
DNA-repair proteins

Question 14

where are high pressure bacteria? High Pressure!! Deepest ocean bottomMariana Trench, Challenger Deep, 11 km

Answer 14

-High hydrostatic pressure (HHP)
Obligate barophilic/piezophilic bacteria isolated from sediment
Samples kept at 100 MPa, optimal growth at 70-80 MPa

General HHP survival strategy
Shift between different respiratory components with similar functions according to pressure conditions
a.a. composition, motility & metabolic pathways differ from shallow water strains

D-alanine:D-alanine ligase involved in peptidoglycan biosynthesis - may form extra linkage between flagellar components & peptidoglycan to stabilise structure & reinforce motility
Shewanella benthica & Moritella yayanosii have D-alanine:D-alanine ligase gene

Toxin-antitoxin (TA) system protects bacterial population from predating bacteriophage (also populous at depth!)
-Shewanella benthica has 4 x TA systems
-Moritella yayanosii has 2

Question 15

what are alkaliphiles?

Answer 15

Alkalihalobacillus halodurans produces bacteriocin (haloduracin) - may be useful as a lantibiotic
Bacillus KSM-19, -64 & -520 produce alkaline cellulases - used in laundry detergents

General alkaliphile (pH > 8.5-11) survival strategy
Electrogenic antiporters for H+ accumulation
Na+ uptake system
Cytochrome c-552 e- & H+ accumulation

Question 16

what are psychrophiles? 2 km deep in (Ant)arctic ice

Answer 16

Cell fluctuations reveal prokaryotic response to long-term climatic & environmental processes

North Greenland Eemian Ice Drilling core
2 km deep = 80,000 years ago, 3.6 x 105 cells/ml
85% viable!!, But hard to culture, 0.00028%
success rate

4 isolates cultured- Acinetobacter, Firmicutes x 2
Gammaproteobacteria

General psychrophile cold tolerance strategies
High level of unsaturated & short chain fatty acids (SCFA)
Cold shock proteins (CSP)
Nucleic acid chaperones - prevent formation of mRNA 2° structures at low Tthus, facilitate translation initiation
Protein chaperones (assist folding/unfolding)
Anti-freeze proteins (AFP) restrict ice growth on protein surfaces
Mannitol & other solutes accumulate in cytoplasm as cryo-protectants - prevents protein aggregation
Carotenoids (★) support membrane fluidity & prevent UV damage

Question 17

what are thermophiles?

Answer 17

Thrive at 41°C (moderate) - 122°C (hyperthermophiles)
Hyperthermophilic & mesophilic enzymes highly similar
-a.a. sequences typically 40-85% similar
-3D structures superimposable
-same catalytic mechanisms

Main source of industrially important thermostable enzymes
Thermus aquaticus Taq DNA polymerase I
Used in PCR to amplify short segments of DNA
Thermophilic Clostridium spp
Used to produce biobutanol from corn

Hypothesis: hyperthermophilic enzymes more rigid than mesophilic homologues at mesophilic T, rigidity prerequisite for high protein thermostability
Rigidity arises from more ion-pairs
e.g. in glutamate dehydrogenase (GDH)
45 / subunit in Pyrococcus furiosus
26 / subunit in Clostridium symbiosum
High melting temperature (Tm), long half-life at high T

Question 18

what are xerotolerance?

Answer 18

Atacama Desert, Chile
Oldest desert ~ 90 M y.o.
Ave. rainfall < 5 mm/year - hyper-arid
pH extremes
Highest UV levels on earth
Mars-like soil

Importance of extremophile research
Medicine: S. atacamensis synthesises ansamycin & 22-membered macrolactones- antibacterial & antitumor activities
Climate change: response to drought by bacteria important to crops
Bioremediation: of soils in extreme dry conditions
Space science: survival on Mars!

General xerotolerant survival strategies
evasion of environmental stress - spore formation
adaptative mechanisms
preventing H2O loss & increasing H2O retention through osmoprotectant accumulation
trehalose, L-glutamate, glycine betaine
increasing H2O retention through cell membrane modifications
production of extracellular polymeric substances (EPS)
synthesis of DNA-repair proteins

Question 18

what are archaea (prokaryotes but not bacteria)

Answer 18

Like bacteria- size & shape, reproduce asexually by binary fission, fragmentation or budding

Unlike bacteria- genes & metabolic pathways, more closely related to eukaryotes, membrane lipid e.g. archaeol, (not e.g.phosphatidylcholine)
do not form endospores

Major part of life on Earth – key to animal microbiota
Not pathogens/parasites – mutualists & commensals
Hard to grow in laboratory

First observed archaea were extremophiles
E.g. Haloquadratum walsbyi
discovered in brine pool Sinai, Egypt
phototrophic halophile
survives [NaCl] ~ 3 M, [MgCl2] ~ 2 M
among most haloresistant organisms known
General halophile high-salt strategy
increase internal osmolarity by accumulating K+ ions in cytoplasm, requires 2 x ATP / K+
General halophile low-salt strategy
de novo synthesis / uptake of osmoprotectants maintain osmotic balance & establish proper turgor pressure