Eukaryotic Microbiology (Lectures 2-11)

Antoni van Leeuwenhoek

• Made his own microscope and lens at around 30-100X

• Observed what he called “animalcules” from pond water and stool

August Johann Rosel von Rosenhof

• Described amoebae in 1755

Nicolas Theodore de Saussure

• Noted division of cultured ciliates in 1769

Carl Theodor von Siebold

• Redefined the term protozoa as not animalcules but “early animals” in 1845

John Hogg

• Coined the term protoctista in 1861 (would include multicellular algae given the exceptions)

• All eukaryotes except…

• Animals from blastula

• Plants from embryonic stages

• fungi without flagellate stage

Ernst Haeckel

• Defined the term Protist in 1866

• Now all single celled eukaryotes

Lynn Margulis

• Coined the theory of Endosymbiosis in 1967

• Overpowering evidence, but extreme resistance from the field

Protozoa

• Animal like eukaryotic microbes

• locomotion

• ingest other organisms

Algae

• Plant like eukaryotic microbes

• Photosynthetic - light energy to organic compounds (CO2 → Glucose/Cellulose

• Sedentary

Yeast/Mould

• Fungi like eukaryotic microbes

• Absorb small organic molecules from environment directly over cell wall and membrane

Euglena

• Flagellated (like protozoa)

• Photosynthetic (like algae)

• Moves to find light not food… so protozoa or algae?

• EM revealed closer relation to non photosynthetic flagellates like Trypanosomes

• Developed the ability to photosynthesis how, through secondary endosymbiosis?

Protoctist

• Would include single celled eukaryotes plus multicellular algae given the exceptions

• All eukaryotes except…

• Animals from blastula

• Plants from embryonic stages

• fungi without flagellate stage

Eukaryote vs Prokaryote nucleus

Eukaryote: nuclear material surrounded by nuclear membrane with pores for RNA transfer, where material can leave to be transcribed by ribosomes in cytoplasm

Prokaryote: fibrous masses of nuclear material not surrounded by membrane (Bacteria and Archaea)

Eukaryote vs Prokaryote genetic material

Eukaryote:

• Nuclear material across multiple linear chromosomes

• DNA in nucleus wrapped around histones (protein spools)

• DNA with many introns which must be removed from mRNA but important for transcription

• Cellular and nuclear division by Mitosis

Prokaryote:

• Nuclear material is circular

• DNA association with non- histone proteins

• no introns mRNA ready

• single chromosome is replicated followed by cellular division

Introns

• Non coding regions of pre-mRNA removed before translation

Eukaryote vs Prokaryote organelles

Eukaryotes:

• Many membrane bound organelles that partition metabolic and physiological processes

• partitioning increases efficiency of cellular processes

Prokaryotes:

• No membrane bound organelles

• processes occur in cytoplasm, inclusions (similar to organelles but covered by protein or lipid laters and process specific) , PM or internal extensions of PM

Eukaryote vs Prokaryote cytoskeleton

Eukaryotes:

• composed of microtubules and microfilaments that support and maintain the form of a cell which increases its complexity and dynamic capacity

Prokaryotes:

• Elements that stabilize nucleoide but much less structured

Eukaryote vs Prokaryote locomotion

Eukaryotes:

• flagella (propeller like movement) and cilia (undulating hairs)

• movement and feeding

Prokaryotes:

• flagella that consists of monomers of flagellin

Eukaryote vs Prokaryote reproduction

Eukaryotes:

• A3exual: Mitosis for replication diploid

• S3xual: Meiosis that results in gametes from reduction in chromosome #s by separating twice to haploid instead of diploid

Prokaryotes:

• Binary fission: As3xual enlargement of the cell, replication of the DNA,and division of the cell to form two identical cells (very quick)

• Conjugation: transfer of small sections of DNA from one bacterium(donor) to another recipient) via a s3x pilus (a tube of pieces of DNA)

Both Eukaryotes and Prokaryotes

• DNA transcription

• ATP usage

• Genetic code

• Same metabolic pathways (glycolysis)

Cellular organisation

• Number of specialized organelles to compensate for unicellularity

• Secondary loss of organelles (mitochondria since no oxygen)

• Highly variable size and shapes

Protozoa Cell Membrane

• Many have only a plasma membrane (outer covering sans other supports)

• Glycocalyx exterior to PM contains receptor proteins to selectively absorb solutes from medium

• Pellicle (extra stiffness through more lipid membranes) below plasma membrane reinforced by microtubules

• Trypanosomes: can switch glycoprotein markers to hide from immune system

• 
  

Pellicle of PM

• Extra stiffness through more lipid membranes

• Below plasma membrane reinforced by microtubules

Test outer covering

• Dome shaped from organic material with an opening for pseudopodia for feeding and movement

• Composed of silicon dioxide (silica) or chitin (polysaccharide carbohydrate)

• Ex. Arcella ameobae

Shell outer covering

• shells made of calcium carbonate (foraminiferans)

• silicon dioxide shells (two of diatoms)

Cell Wall of algae/ fungi

• Composed of cellulose, pectin other compounds like plants

• Ex. Red algae is harvested for carrageenan (carbohydrates added to thicken soups) and agar

• fungi cell walls are composed of chitin

Ciliates - Nucleus

• Two nuclei

1. Macronucleus: polyploid, undergoes mitotic division to make many copies of the same gene

2. Micronucleus: diploid, does not partake in transcription but “sexual reproduction” with other ciliates

Endoplasmic reticulum (ER)

• Synthesis and transportation of proteins

Golgi apparatus

• Transport of proteins from the ER to outside of the cell PM

Lysosome

• Contain digestive enzymes, fuse with phagosomes to digest material from exterior of cell etc.

Vacuoles

• Water-filled

• Empty waste outside of cell

• Ex. if living in freshwater constant influx of H2O since a salty organism that must osmoregulate

Peroxisomes

• Contain peroxidase which breaks down oxygen byproducts

• Ex. Peroxides can cause break down of cellular membranes if not removed

Trypanosome - Glycosomes

• Large vesicles that contain enzymes to metabolize glucose to pyruvate

• Form when anaerobic inside of insect gut

Hydrogenosomes

• Ferment pyruvate through oxidization (glycolysis) into acetate, CO2 and H2

• makes 4>2 ATP

Apicomplexan - apicoplast

• Essential for metabolism

• Developing drugs to target and disable the organism

Extrusomes

• Like a harpoon that lies beneath the PM

• Trichocyst: Used for defence

• Toxicyst: Used to subdue prey

Mitochondria

• Remnant of symbiotic bacterium ~ 32 ATP

• Outer and inner membrane

• Site of oxidative cellular respiration

• ETC arranged on cristae (increase SA to increase space for e-)

Mitochondria - 3 types cristae

• Shape and arrangement differs based on groups and is therefor useful fro taxonomic characterization

Lamellar cristae: most eukaryotes (curtain formations)

Tubular cristae: many protozoa ciliates, amoebae (finger like projections)

Discoid cristae: some protozoa, euglenozoans (pea shaped projections)

Chloroplasts

• Remnant of Cyanobacteria symbiont which provided the ability to photosynthesize

• Possesses an outer and inner membrane

• Site of photosynthesis in photosynthetic protists Ex. chromists, dinoflagellates, euglenozoans, green and red algae

• light reactions occur on membrane bound thylakoids (membrane extensions through chloroplasts

Types of Chloroplasts

Rhodophyta:

• Phycobilisomes covering the thylakoids which assist in light harvesting at different wavelengths to make energy

• Thylakoids in rope like strings

Chloroplastida:

• Surrounding thylakoid stacks

• Starch is synthesized at the pyrenoid which is the site of CO2 fixation

Stramenopiles/Chromists:

• Thylakoids in groups of threes always

• Chlorphyll pigment is variable

• The outer membrane is continuous with the ER abdominal nuclear membrane

Why was complex cytoskeleton development an important advancement in eukaryotic evolution?

Cytoskeleton:

• Supports cells and organelles in functions

• Supports cellular movements

Importance:

• Phagocytosis resulted from these advancements which unlike bacteria, allowed them to exploit other food sources

• Fine movement which maintaining encourage of organelles

• increased size by up to 1mm

Composition of cytoskeleton

• microtubules, microfilaments, intermediate filaments, spasms filaments

Microtubules

• ling hollow fibrils (24nm diameter)

• helical polymers of tubulin a primary protein inside living cells that join together to build support structures

Microfilaments

• Thin filaments (7nm diameter)

• two strings of actin polymers wound around each other

• actin and myosin complexes are involved in cellular locomotion

Intermediate filaments

• intermediate diameter (10nm)

• composed of 70 closely related proteins, help shape cells

Spasmin filaments

• Present in some ciliates with quick contractile movements

• enables quick contraction of stalks

Pre Eukaryotic Life

4 billion years ago…

• no oxygen therefore life was anaerobic bacteria, some evolved to synthesize porphyrin pigments which absorb light energy resulting in photosynthetic cells which used water as e- donor and released O2

• Some bacteria remained anaerobic living in muds

• Some bacteria became O2 utilizers of e- transfer and oxidative phosphorylation

• Bacteria and archaea fused to form a pre eukaryote

Direct Filiation Theory

• Nuclear envelope surrounding genetic material was formed through an invagination of the PM

• These invaginations surrounded plasmids and chromosomal DNA acquired from different bacteria

Endosymbiotic Theory

• An early eukaryote likely through fusion of 2 anaerobic bacteria/archaea developed phagocytic abilities (complex cytoskeleton)

• That early eukaryote ingested aerobic bacterium that can make upwards of 32 ATP from one glucose such as mitochondria

• Members of some photosynthetic lineages ingested photosynthetic bacteria and kept them as chloroplasts

Endosymbiosis - Cellular Fusion Hypothesis

• Fusion of bacterium (maintained metabolic and lipid biosynthetic genes) and archaean (maintained genes involved with transcription/translation) = pre eukaryote

• Formation of anaerobic amoeba-like-pre-eukaryote that developed a nucleus (somehow)

• likely phagocytksed prokaryotes as food…

• Mitochondria: from endosymbiosis of aerobic bacterium where the end-membrane system resulted in partitioning

• Chloroplasts: engulfed symbiotic cyanobacteria

Evidence of Endosymbiosis - Mitochondria and Chloroplast

• size = similar to bacteria

• DNA = single loop

• Similarity of DNA genome… mitochondria = proteobacteria lineage, chloroplasts = cyanobacteria lineage

• 70S ribosomes > 50S ribosomes in cytoplasm

• binary fission and division is independent of cells

• double membrane structure indicative of G- bacteria (inner wall=PM, outer wall= ?, no peptidoglycan)

Criticism of Endosymbiosis Theory

How did nucleus arise?

• development of internal membrane similar to direct filiation ?

• maybe internal membrane wrapped around genetic material

Assume first pre-eukaryote was anaerobic?

• this would be more advantageous otherwise what reason to take up bacteria?

ATP translocate from bacterial symbiont into eukaryote?

• Mitochondria to cell… how did it originally occur

Secondary and Tertiary Endosymbiosis

1. fusion event Bacteria and Archean both anaerobic = anaerobic pre- eukaryote

2. engulfs aerobic protobacteria = aerobic eukaryote

3. Ciliates: forms micronucleus 3. Specialization of flagella 3. engulf cyanobacteria = algae cell

4. algae cell engulfed by flagellate (secondary endosymbiosis)

5. ciliate engulfs secondary endosymbiont (tertiary endosymbiosis)

Biogeography

• Not well studied, the niches of protists

• More than 1/3 of all described spp. of soil protists reside in Scotland, therefore… not that many and widespread?

• There are also many unique spp. found in each area of the world

• Parasitic protozoa are distributed with their hosts (unless able to switch hosts)

Future Biodiversity Protozoa

• Define ranges of species

• Descriptions and naming more regulated

• Survey new habitats and hosts

• globalization of diversity studied as spreading incidence of disease-causing agents and accidental transportations

Ciliate - cortex

• cytoskeletal elements will stain black which form a fine scaffolding called a cortex not found in any other group

Axopodia

• Radiolarians - skeletal elements

• Stiff fingerlike spikes that give structure

• small organisms stick to them and get sucked down into the cell for ingestion

Cysts

• Obligatory life stage in gut/soil parasites

• Consequence of sexual activity

• Resting stage for protection against drought (unfavourable conditions)

• Provides ability to disperse by sticking onto organism or being carried away

• Protists in aquatic environments, walls will be thinner as consistently wet environment

• Composed of chitin (nitrogen) cellulose (fibres)

• viability from short time - 15 years dormancy

Encystment

• Entering into dormant stage, proteins send message to nucleus to express genes for making a cyst from signals in the environment

Stimuli for encystment

• Changes in PH (acidic/basic environment)

• Changes in temperature (winter/summer moving away from thermal optima)

• Drought conditions

• Low/high O2 levels (aerobic/anaerobic protist)

• Accumulation of end products from high population density (waste from other organisms from excess biological activity)

• Depletion/excess of food

Excystment

• Requires stimulation from the environment (chemical, electric, mechanical etc.) much is unknown about the relationship

• Cyst wall disintegrates due to enzymatic activity

• Operculum a hole in the cyst, is used as an “escape hatch” for the protist

Holdfast

• Adhere permanently or temporarily to substrate (rock, intestinal cell etc.)

• Many different structures but same function

• Ex. Stentor = ciliate attaches to substrate by secreting mucus, when cilia beats drags food in

• Ex. Vorticella = ciliate many cells share a stock that is fastened to substrate/organism in a pond, contractile stock filled with spasmin that contracts quickly so if disturbed they contract into the stock

• Ex. Gregarina = apicomplexan that uses epimerite that sticks in and attaches to intestinal epithelial cell, damaging the cell and holding onto the wall of the insect gut

Stentor

• Ciliate attaches to substrate by secreting mucus, when cilia beats drags food in

Vorticella

• Ciliate many cells share a stock that is fastened to substrate/organism in a pond, contractile stock filled with spasmin that contracts quickly so if disturbed they contract into the stock

Gregarina

• Apicomplexan that uses epimerite that sticks in and attaches to intestinal epithelial cell, damaging the cell and holding onto the wall of the insect gut

Extrusome - Trichocyst

• Shoot out of the cell, tip is located in pellicle just below PM

• Spindle shaped like a stinger, in some cases as long as cell

• When organism is disturbed (chemical, mechanical, electric stimuli)

• Made of collagen like proteins that are quite stiff

• Used to repel predators, and is capable of regenerating them

Extrusome - Mucocysts

• Coats cells with mucus to…

• Enter first stage of encystment

• Makes the cell sticky to capture prey

• Used for locomotion on a slime trail

Extrusome - Rhoptries

• Apicomplexan parasites must invade cells in their life stages

• When they detect cells as part of the apical complex will discharge and insert proteins into the cellular membrane of the host cell (docking proteins) and enter into cell

Extrusome - Pigmentocysts

• Extrude pigment granuals onto surface of cells

• makes the cell look green, purple, blue, brown etc.

• filled with a type of toxin that when predators eat them, will not want to eat others again due to foul taste or induce illness

• Absorb suns dangerous energy like a sunscreen and protects from ultraviolet rays

Extrusome - Toxicysts

• thick long tubes that are poisonous/ venomous and disable prey

• they can protect against predators but mainly used to kill prey

Haptoglosssa - penetration apparatuses/ gun cell

• Specialized organelles that contain cytoplasm with massive vacuole

• Contain a vacuole that can rapidly fill with water and can push this force/propulsion of contents into another organism

• after rotifer/nematode makes contact, cellular osmotic pressure causes sudden eversion of projectile into animal, organism now inside other animal, tends not to cause long lasting harm, becomes multicellular inside the animal

Excretion

• nitrogenous wastes diffuse out of cells

Osmoregulation - amoebae

• Ions actively transported by contractile vacuoles (use of ATP)

• Excess water follows the osmotic gradient

• Vacuoles fuse with the cell membrane and release waste outside of cell

Osmoregualtion - ciliates

• Water is collected by the ER and emptied into feeder canals that leas to vacuoles

• The vacuoles contract and expel the excess water from the protist