Exam 1 New Flashcards
eukaryotes
protists, algae, fungi
structural characteristics of bacteria
- no organelles
- no nucleus, instead have nucleoid (no membrane)
- peptidogylcan in cell wall
- 70s ribosome (50s + 30s)
- much smaller than eukaryotes (~1 micro meter long)
prokaryotes
bacteria, archea
what do bacteria do?
-They consume and grow
Organism of the week: Vibrio fischeri
Marine bacterium, squid symbiont, luciferase (enzyme), produces light only at high cell density, “quorum sensing”
4 requirements for microscopy
- magnification: relative increase in size
- resolution: the ability to distinguish two points that are close together
- light quality: sets limit of resolution
- contrast: the ability to detect objects against a background
phase contrast microscopy
uses a trick of light (interference) to generate contrast
-no staining necessary
fluorescence microscopy
- light does not pass through specimen
- light excites fluorescent molecules which then emit light themselves
Green fluorescent protein (GFP)
-Aqueoria victoria (jellyfish)
Fluorescence vs. luminescence
Fluorescence: emits light after photoexcitation (ex. GFP)
Luminescence: produces light by chemical reaction–no light source (ex. luciferase)
Resolution is ultimately limited by light quality
the lower the wavelength, the higher the resolution
two types of electron microscope
- transmission
- scanning
scanning electron microscopy
high resolution but cells are stained and dead
Transmission electron microscopy
- cells stained and dead
- takes long time to prepare sample
- looks like a sliced cell
Electron cryotomography
- freeze sample in ice
- take pictures as you tilt the stage
- recombine images in 3-dimensions
- cells are still alive!
Atomic force microscopy
- does not use light!
- and ultra fine probe taps over objects
- cells can be alive
Species
-an interbreeding population that is reproductively isolated
Classification strategies for bacteria
- numerical taxonomy (traits)
- DNA-DNA hybridization (genome comparison)
- phylogenetics (molecular chonometer)
- polyphasic approaches (combinations)
- naming by disease
Numerical taxonomy
Naming bacteria based on traits. Order is based on a similarity coefficient. The disadvantages are that trait choice is arbitrary, all traits are weighted equally, traits vary in complexity, traits aren’t necessarily related to each other.
DNA-DNA hybridization
Entire genomes are compared, >70% is the same species. It’s the only formal definition of bacterial species, however, it’s technically cumbersome and only very close comparisons work.
phylogenetics
`Sequences of a molecular chronometer is compared. Organisms with fewer changes in sequences are more closely related. Molecular chronometers must be found in all representatives, have the same function, have sufficient similarities, and also sufficient differences.
molecular chronometers
must be the following criteria:
- found in all representatives of the group studied
- function must be same in all representatives
- sufficient similarity between molecules so that the sequences can be alligned
- sufficient differences such that each sequence has its own signature
-ex. 16S rRNA (nucleic acid) all molecules have this
polyphasic approach
A combination in which 16s rRNA is sequenced and compared to a database to find the closest relative. Physiological traits are then compared to the closest “type strain.” The genome is then compared to the type strain to determine if it’s the same or different species.
wild type vs. type strain
Wild type: we think of them having no mutations
type strain: lab mutated species we look at in lab, and compare it back to the wild type
Organism of the week: Epulopiscium fishelsoni
Found in the gut of the surgeon fish. 1 million times larger than E coli. Very unusual cell division, cannot be cultured in the lab. The new daughter cells grow inside the mother cell and the mother cell is killed when daughters are released.
naming by disease:
Naming by disease - pathogen species named by the disease they cause
Organism of the week: Escherichia coli
E. coli K12: used in lab, doesn’t make you sick
-harmless symbiont
E. Coli O157:H7: makes you sick
- dangerous pathogen
- due to phage infection
identical 16s rRNA but chromosomes are only 75% similar
(human and chimps are 98% similar)
phages
Phages are composed of proteins and nucleic acids. They are viruses that infect bacteria. They insert into the bacterial genome which then replicates and expresses the phage genes like its own.
macromolecules
- polysaccharides
- nucleic acids
- proteins
- lipids
polysaccharides
-polymers of carbohydrates
-sugar names are determined by: number of carbons & orientation of hydroxyl groups
-polysaccharides are joined together by alpha and beta linkages
beta linkages are harder to digest (lactose, cellulose)
-beta linkages are linear
alpha linkages have kinks in structure
functions of carbohydrates
- structural component (cell wall material, capsule)
- energy source (found in environment, intracellular storage molecules)
- information (sugar pattern on cell surface can identify bacteria, intracellular trafficking and recognition in eukayotes)
important 5 carbon sugars:
ribose & deoxyribose (precursors to DNA and RNA)
Where are polysaccharides found
cell wall
capsule
Nucleic acids
- carry genetic information (required for replication and transcription)
- polymers of nucleotides
DNA & RNA
- polymers of nucleotides
- RNA short term information storage (A, D, G, U)
- DNA long term information storage (A, D, G, T)
- antiparallel strands
- forms double helix
- major groove (more info)
- minor groove (less info)
- certain proteins only bind to major/minor groove
Polymerization
- nucleotides are activated with tri-phosphate
- added to 3’ end of a growing chain
- polymerized 5’ to 3’
Base pairing
Purines: A, G
Pyrimidines: T, C
A pairs with T: two hydrogen bonds
G pairs with C: three hydrogen bonds
DNA/RNA differences
DNA is doublestranded, has long term information storage, has deoxyribose sugars. RNA is single stranded, has short term information storage and has ribose sugar (extra OH group attached to the 2’ carbon).
Functions of nucleic acids
- information storage (genetic material, information conversion, signaling)
- structure (ribosomal rRNA)
- energy intermediate (energy currency ATP/NADH)
Where are nucleic acids found?
cytoplasm
chromosome
ribosome
Protein structure and function
- very complex structure
- shape of protein determines what function it has
- proteins are made of amino acids
- important for protein folding (4 levels)
Amino acids
-proteins are polymers of amino acids
4 parts to amino acid:
- carboxyl group
- variable group “R” (carries information)
- amino group
- proton
Bond between amino acids:
- peptide bond
- very strong
- have an orientation “N terminus to C terminus”
primary structure
- linear series of amino acids
- contains all information for protein folding and function
secondary structure
- folding in two basic motifs
- alpha helix
- beta sheets
tertiary structure
- three dimensional interaction of alpha helices and beta sheets
- tertiary structure folding is driven and stabilized by hydrophobic interactions
Quaternary structure
-multiple proteins assemble to form a super-structure
protein naming
- three letter prefix, one letter suffix
- usually gives hints about protein function
functions of proteins
- enzymes
2. structure
where are proteins found?
cytoplasm membrane nucleoid ribosomes secreted
cytoplasm membrane nucleoid ribosomes secrete
one glycerol
-up to three fatty acids
composition of a phospholipid
multiple fatty acid chains, glycerol backbone, phosphate and head group
where are lipids found:
-membrane
macromolecules in a typical bacterium
- proteins contribute the most dry weight and are the most diverse
- DNA is the largest macromolecule in the cell
- lipids are the more numerous macromolecule
peptidogylcan
- an exoskeleton that protects cell from bursting
- hybrid of protein and polysaccharide
sugar component: NAG & NAM (two sugars joined together by B linkages)
protein component: chains crosslinked by AA
strength contributors:
B linkages connect sugars
D-amino acids
crosslinking–> DAP (permits crosslinking, can form 2 peptide bonds)
antibiotics that target peptidogylcan structure
penicillin: antibiotic that prevents crosslinking
lysozyme: cleaves B linkages between sugars of the chain
gram positive cells;
- thick external layer of peptidogylcan
- plasma membrane (under it)
- rigid external wall protects membrane from osmotic pressure
-surface negatively charged by techoic acids
techoic acids: negatively charged gylcerol phosphate polymers
-makes surface very hydrophillic
-repels hydrophobic detergents from inserting into membrane
gram negative cells
- outer membrane
- thin layer of peptidogylcan
- periplasm
- inner membrane/plasma membrane
special features of the gram-negative envelope
- outermembrane
- lipopolysaccharide
- braun’s lipoprotein
- porins
- periplasm
outer membrane of gram (-) cells
-two leaflets of OM are different
lipopoylsaccharide sits ontop of membrane
brauns sits below OM
lipopolysaccharide (LPS)
Composed of an O-antigen, core, and Lipid A. It’s negatively charged to repel hydrophobic molecules. Each gram neg species has a unique O-antigen. The core is the charge barrier and the lipid A anchors the LPS to the outer membrane. The lipid A is very hydrophobic.
how is lipid A different from phospholipid?
- phospho-sugar backbone instead of glycerol
- 6 fatty acids instead of two
Very hydrophobic!
Brauns lipoprotein
- attaches to OM to peptidogylcan
- structural anchor for outer membrane
porins
- outer membrane is a diffusion barrier
- porins allow small molecules to diffuse into cell
- passive transport
periplasm
- not a structure but a space
- contains thick gel spanning plasma and outer membranes
- chemical defense containing detox enzymes and chemical binding proteins
organism of the week: mycoplasma pneumoniae
Causes walking pneumonia. One of the smallest bacteria. Has the smallest genome of about 500 genes. No peptidoglycan
extracellular polysaccharides (EPS)
extra, non-essential layers beyond the peptidogylcan
-not all bacteria have an EPS
ex. capsule (anchored), slime layer (loose)
EPS advantages
- adhesive (cells stick to surfaces)
- cohesive (cells stick to cells)
- desiccation resistance
- protection from immune system
- clog circulatory system of hosts (in plants)
organism of the week: myobacterium tuberculosis
1 infectious bacterial pathogen, infects the lungs. 3 million deaths/year worldwide. Unusual architecture with a thick waxy cuticle of mycolic acid.
S-layer (surface layer)
A crystalline shell made of protein beyond the peptidoglycan. Their function is unknown.
shape of a rod
- MreB: protein that forms spiral cables
- found only in rod shaped bacteria
- coupled to wall biosynthesis: helps cell find its middle and helps peptidoglycan synthesize
shape of a vibrio
- CreS: crescetin
- found only in vibrio
- may bend MreB cables
shape of a coccus
- lacks MreB
- lacks CreS
- it is unknown how a coccus cell finds its middle
nucleoid structure
- well defined structure but not membrane bound
- structure seems to be result of DNA binding proteins
- SMC protein “structural maintenence of chromosomes”
- bends DNA into loops
- ->analogous to histones in Euk, they compact DNA
advantages of a nucleoid:
Rapid signaling, permits coupled transcription and translation, allows bacteria to respond faster.
advantages of a nucleus:
- less room for error–transcription is checked for errors before leaving the nucleus
- protects RNA from degradation
bacterial membrane invaginations
- extensions of the plasma membrane
- increase SA and decrease volume
- provides protection
- increases probability of signaling & increases exchange of nutrients
- more surface to mount transporters or light harvesting centers
- more interaction with envrionment
They increase surface area and decrease volume. They create more surface to mount transporters and light harvesting centers. They allow more interaction with the environment
storage granules
Energy storage, element storage and damaged proteins. They have reserve materials.
organism of the week: magnetospirillum magnetotacticum
Magnetosomes are storage granules composed of magnetite. Used to orient motility in a magnetic field. They’re membrane invaginations.
magnetosomes:
- magnetosomes are actually membrane invaginations
- MamK organizes magnetosomes inside of cell
endospores:
Only some bacteria make spores. They contain a copy of the chromosome. They have extremely low water content, they’re metabolically dormant, DNA is tightly compacted, and they’re highly resistant to dessication, mechanical stress, heat and radiation.
Microbiology
The study of small (microscopic) organisms
- bacteria
- archea
- protists
- algea
- fungi
- viruses
What are the differences between bacteria and eukaryotes?
Bacteria have no organelles, they have nucleoids (no membrane), they’re 100x smaller than eukaryoktes, they have a single circular chromosome
Different types of symbiosis
Bacteria+Bacteria, Bacteria+Animal, Bacteria+Plant, Bacteria+Fungi
Understand the Bright Field Microscope; does it use light or electrons? -They use light
They use light
What is refraction? How do lenses use this property?
Refraction is when light bends when passing from one medium to another, because light travels more slowly in a denser medium. Lenses use this property because they refract visible light to focus on a single point.
What is magnification? What is the relationship with focal length?
Magnification is a relative increase in image size. As focal length decreases magnification increases.
What is resolution? How does the numerical aperture change with resolution?
Resolution is the ability to distinguish between two points close together. Numerical aperture is the widest angle of light captured. As it increases, resolution increases.
Why would a scientist stain cells? What does staining enhance? Name at least one type of staining technique. Are cells alive or dead when stained?
Staining cells enhances contrast. Cells are dead when stained. Types of staining techniques include gram stain and flagellar stain.
Know phase contrast microscopy, fluorescence microscopy, electron microscopy (transmission and scanning), atomic force microscopy -
Phase contrast microscopy: uses interference to generate contrast, no staining necessary
Fluorescence microscopy: Light excites fluorescent molecules which then emit light themselves. Light doesn’t pass through the specimen
Electron microscopy: Uses a beam of electrons with a very short wavelength, allowing high resolution.
Atomic force microscopy: An ultra fine probe taps over objects, doesn’t use light or require staining
What is GFP? How do scientists use it?
Green Fluorescent Protein. Fuse 2 gene sequences to make a hybrid protein, which can then be detected with fluorescence microscopy. Protein location informs protein function.
What is 16sRNA comprised of? Why is RNA important? Where can it be found?
It can be found in the 30S component of the 70S ribosome. It is important because it is in all cellular life. It is composed of nucleic acids.
What are the types of linkages that can occur between sugars?
They can be linked by alpha or beta linkages
Where does polymerization of nucleotides occur?
Nucleoid
What sugars are found in DNA and RNA?
Deoxyribose and Ribose
Know the subunit of each macromolecule: Polysaccharides, nucleic acids, proteins, and lipids.
Polysaccharides are composed of carbohydrates, nucleic acids are composed of nucleotides, proteins are composed of amino acids, lipids are composed of fatty acids
What is the major/minor groove?
There is more info in the major groove than the minor groove. Both are on the “sides” of the bases and carry information
What are teichoic acids?
They are in gram positive bacteria. They’re negatively charged glycerol phosphate polymers and are attached to the peptidoglycan. They protect the membrane because they are highly hydrophilic and repel hydrophobic detergents.
Where are proteins found in the cell?
They can be secreted, cytoplasm, membrane, nucleoid, and ribosomes
How many levels of protein folding are there? What are they? What is the purpose of each level?
There are 4 levels, primary, secondary, tertiary and quaternary. Primary structure is the linear sequence of amino acids. Secondary structure is the folding of the two basic motifs, alpha helix or beta sheet. Tertiary structure is the 3 dimensional interactions between helices and sheets. Quaternary structure is when multiple proteins assemble.
What is the “R” group?
The variable group (different for each protein)
What is the primary component of cell membranes?
Lipids
How do you name fatty acids?
CX:Y, X= number of carbons, Y= number of double bonds
What’s the difference between saturated and unsaturated?
Unsaturated fatty acids have double bonds and saturated ones do not
What is a micelle?
A circle of phospholipids with the polar head groups on the outside and the hydrophobic tails on the inside
Are fatty acids amphiphatic? If they are, then why?
Yes because they have a polar head and a hydrophobic tail
What is a ribosome? What purpose does it serve?
It’s a massive RNA/protein complex. They translate mRNA into new proteins.
What is the difference between a capsule and a slime layer?
Capsules have anchored extracellular polysaccharides (EPS) and slime layers have loose EPS
Can cells protect themselves from detergents?
The two architectures that protect from detergents are gram positive and gram negative
What is the difference between gram positive and gram negative? Draw the differences.
Gram positive has peptidoglycan on the outside as a cell wall and then a plasma membrane on the inside. Gram negative has an outer membrane, then a periplasmic space, then the peptidoglycan, and finally a plasma membrane on the inside.
How do detergents work in killing the cell?
They insert into membranes and destabilize it.
What is the most abundant macromolecule in an E. coli cell?
Lipids
How do cells adjust the membrane for changes in temperature?
They make it more fluid when it is cold and less fluid when it is warm. Fluidity can be increased by unsaturation/branching/shorter chains.
How do cells adjust for osmotic damage?
The peptidoglycan creates an exoskeleton that protects the cell from bursting
What is NAG? NAM?
Polysaccharide polymers
What type of linkage is present between NAG and NAM?
Beta linkage
What is a D-amino acid? Where is it used in the cell?
It is used in peptidoglycan and increases resistance to proteases
What are Pili usually involved in?
They’re rigid fibers made of protein that are typically involved in adhesion.
What are the different cell shapes?
Coccus, spirillum, rod, spirochete, vibrio, prosthecate.
What is the protein needed for the rod shape in bacteria?
MreB
What is the protein needed for the vibrio shape in bacteria?
CreS
Why must eukaryotes compartmentalize?
They must reduce volume in order to restrict diffusion, concentrate reactants, and isolate incompatible reactions
What is DAP?
Diaminopimelic acid. It’s an unusual amino acid not found in proteins, it permits crosslinking (in peptidoglycans) because it can form 2 peptide bonds.
What are the 5 special features of the gram negative envelope? Explain what they do.
1) Outer membrane
2) LPS (lipopolysaccharide) - Composed of an O-antigen, core, and Lipid A. It’s negatively charged to repel hydrophobic molecules. Each gram neg species has a unique O-antigen. The core is the charge barrier and the lipid A anchors the LPS to the outer membrane. The lipid A is very hydrophobic.
3) Braun’s lipoprotein - Attaches the outer membrane to the peptidoglycan, it’s a structural anchor.
4) Porins - Permeability gates in the outer membrane that allow small molecules to diffuse into the cell.
5) Periplasm - Thick gel that spans the outer and plasma membrane. It contains detox enzymes and chemical binding proteins
What does Penicillin and lysozyme target? How do they work?
They both target peptidoglycan. Penicillin prevents chain crosslinking. Lysozyme is an enzyme that cleaves the beta linkage between sugars.
What are advantages of having an EPS?
Adhesive (cells stick to surfaces), cohesive (cells stick together), desiccation resistant, and protection from the immune system (prevents phagocytosis and shields antigens from antibodies).
What is a peptide bond?
Linkage caused by the dehydration of a carboxyl group and an amino group
