Micrbio Midterm 6-10
The primary function of a bacteria cell wall is to protect the cell from harsh external conditions.
Made up of:
Peptidoglycan (or murein) - alternating (NAG) N-acetyl glucosamine and (NAM) N-acetylmuramic acid connected two dimensionally by peptide bridges.
Gram-Positive cross linked directly by tetra peptide chains
Gram-Negative those tetrapeptide chains are additionally linked by pentaglycine cross-bridges.
*** Peptidoglycan is unique to bacteria - antibiotics can target and weaken cell walls exposing them to osmotic pressure. Additionally, the human immune system recognizes this peptidoglycan, engulfs and then destroys the cells.
Staining techniques -
Gram Positive are more simple and contain TA (teichoic acid) carbohydrate chains extending through the peptidoglycan layers increasing stability.
Acid Fast contains an external layer made of waxy mycolic acid.
Gram Negative are more complex, made of 3 layers: see figure below
Inner membrane
Thin peptidoglycan layer
Outer membrane containing lipopolysaccharide
**Notice the size difference in the peptidoglycan layers - gram negative = >4nm thick
From the Image above we can see that the inner membranes are pretty similar with typical phospholipids and membrane proteins, but we observe many additional characteristics in gram negative such as:
Lipopolysaccharide (LPS) - endotoxin responsible for fever, hemorrhaging and septic shock. Part of the router membrane and composed of Lipid A, A core polysaccharide and the O antigen.
Lipoprotein
Lipid A
O antigen
Porins
Periplasmic space - gel like matrix between plasma membrane and peptidoglycan layer responsible for sensing environmental signals important for the protection and function of the cell, protein folding as well as transport of some molecules.
Some prokaryotic cells have an additional cell envelopes which help them adhere to each other and surfaces and afford a small level of protection:
S-Layer - structural and glycoproteins - serves as the cell wall - regular highly ordered paracrystalline array.
Glycocalyx - a sugar coat which can be capsules (organized layer of polysaccharides or protein or a slime layer (less organized and can be washed off)
Fimbriae - short bristle like proteins important for adhering to surfaces and mobility
Pili - longer less numerous protrusions important in transfer of genetic material between members of the same generation.
Flagella - propeller(s) help cell move in aqueous environments composed of the basal body embedded in the plasma membrane by a hook portion.
The flagella propel the cell in two ways:
Bacterial Cell Structures Module 7
See the above modules for most of this information- I've added new concepts from this module where I believe they fit best with previous content
***Common misconceptions:
Eukaryotes have organelles because they are multicellular, nope, many eukaryotes are unicellular and still have organelles and some bacteria are multicellular and lack organelles.
Eukaryotes have organelles because they are more complex - nope, metabolic complexity of bacteria is much greater than eukaryotes. Eukaryotes might be more complex in structure, but more simple in metabolism.
The hypothesis for why eukaryotes are so large is because they needed to be large to eat common C and N sources on earth at the time.
Bacterial Cell Division Module 8
Binary Fission - common bacteria cell replication - once the cell reaches biomass critical level t= (Cell grows in size) → increases number of cell components → single origin of replication and continues in opposite direction until terminus is reached → center of cell constricts until 2 daughter cells form with complete copy of parental genome + a portion of cytoplasm (cytokinesis)
Cytokinesis = FtsZ (similar to eukaryotic tubulin) defines the cell division plane via polymerization and is anchored by FtsZ-binding proteins assembling into a Z-ring on cytoplasm membrane → Additional proteins are added to form the divisome which is activated to produce the peptidoglycan septum → cells outer layers are remodeled to complete division
FtsZ - uses autolysis to create holes in the peptidoglycan layer by targeting glycosidic bonds.
Bactoprenol - carries NAG and NAM to these sites to form new peptidoglycan.
FtsI - drives transpeptidation creating new cell wall to form septum
Penicillin inhibits cell division by inhibiting FtsI
The Min System - How does FtsZ know where the middle of the cell is?
MinD is a membrane bound anchor protein that activates MinC → MinC inhibits Ftsz →
MinD and MinC are localized to the poles, so the inhibition of FtsZ by this complex forces the FtsZ to be localized where Minc&MinD are absent (the center of the cell).
MinC&MinD are localized to the poles by MinE - MinE is localized to the center of the cell and blocks them from the center.
MinC&D- form a spiral structure on cytoplasmic membrane and oscillate from pole to pole.
MinE allows DNA to pass to the center of the cell.
Nucleoid Occlusion System (NOC)- this protein coats the chromosome, inhibiting FtsZ from polymerizing on chromosomes. As the new chromosomes migrate to the poles of the cell, that NOC protein is attached and moves with it, thus leaving the center of the cell open for FtsZ to for the Z-ring.
NOC and Min systems work together a follows in the diagram.
Generation time - Time between the same points in the life cycle between two successive generations.
-Humans = about 25 yrs – E.coli = 20 mins in lab conditions
-Also called doubling time for Prokaryotes - time it takes for 1 round of binary fission.
Calculating number of cells over time during a period at constant rate.
Nn=N0 2n
Nn= number of cells at any generation
N0 = initial number of cells
n = number of generations
Example : A cell divides every 30 mins for 24 hours. Start by finding the number of generations in that time frame → 24x2=48 generations. If we start with 1 cell then N0 = 1
Nn=N0 2n → Nn= 1 x 248 = 2.8 x 1014
Bacterial Growth: Module 9
Terms:
Generation/doubling time: the time it takes for the population to double through one round of binary fission (book defintion)
-time required to form 2 separate cells (slide definition)
Culture density: number of cells per unit volume
Stringent response: response during starvation when amino acids run low
Exponential growth: cells doubling at a constant rate with each generation
Batch culture: Microorganisms grown in closed culture
Growth divisome: protein complex in bacteria that is responsible for cell division
Objectives:
Differentiate growth rate and generation time
Growth rate is the increase in # of cells while generation time is the time required to form 2 separate cells
Growth rate is the inverse of generation time: k= 1/g and k= n/∆t
Growth rate (k)= # of generation/hr
Growth rate (k)= 1/generation time
Generation time (g)= hrs/ # of generations
Explain several lab methods used to determine viable cell counts & total cell counts in population undergoing exponential growth
Direct counting: cells must be spread evenly & works for any sample
Ex: counting chamber
Viable counting: measuring the # of viable cells/ spreading cultures on petri plates
Counting colony forming units (CFU)
Counting rule: <30 = insignificant >300+ = TOO CROWDED
Ex: serial dilution
Spectrophotometry: measuring turbidity of culture (by using light)
More dense= more light absorbed
0.1= NOT DENSE 1.0= QUITE DENSE
OD
To indirectly count: OD600 = gives relative (qualitative growth)
INDIRECTLY COUNTING =OD600 + viable + direct counting
Flow cytometry: counting individual cells w/ a laser
Bacteria flows through tube and are individually counted
Like spectrophotometer→ counts 1 cell at a time
Explain what limits bacterial growth:Growth can be limited by:
type of species or strain
type of nutrients
unfit temperature
Identify and describe the activities of microorganisms undergoing exponential growth:
Lag Phase: cells are gearing up for the next phase of growth
Log Phase: the cells are actively dividing by binary fission and their number increases exponentially
Stationary Phase: The total number of live cells reaches a plateau
Death Phase: the number of dying cells exceeds the number of dividing cells, leading to an exponential decrease in the number of cells
Explain how stringent response effects cell physiology:
ppGpp triggers changes in cell physiology such as:
Decreased cell size
Increased cell wall crosslinking
Decreased membrane fluidity
Compacted nucleoid
Increase detox enzymes in periplasm
Proteins recycled
Increased motility
Sporulation (in SOME bacteria)
Cells get smaller & more resistant
Metabolism: Module 10
Terms:
Metabolism: all of the chemical reactions inside a cell (anabolism + catabolism)
Catabolism: exergonic pathways that break down complex molecules into simpler ones (book)
- rxn that break down larger molecules into smaller ones + energy is released (slides)
Anabolism: those endergonic metabolic pathways involved in biosynthesis, converting simple molecular building blocks into more complex molecules, and fueled by the use of cellular energy (book)
-rxn that assembles small molecules into macromolecules and biomass→ requires energy input (slides)
Endergonic: require energy to proceed
Exergonic: Reactions that are spontaneous and release energy
Objectives:
Compare and contrast autotrophs and heterotrophs
Autotrophs: Organisms that convert inorganic carbon dioxide (CO2) into organic carbon compounds
Heterotrophs: rely on more complex organic carbon compounds as nutrients; these are provided to them initially by autotrophs
Explain how enzymes make metabolism possible
-its a biological catalyst that lowers activation energy of a chemical rxn inside the cell
Describe the importance of oxidation-reduction reactions in metabolism
- most of the energy stored in atoms and used to fuel cell functions is in the form of high-energy electrons → The transfer of energy in the form of electrons allows the cell to transfer and use energy incrementally avoiding the risk of destructive bursting
List 3 ways cells store energy:
Proton Motive Force (PMF): energy as artificial gradient
made stronger by electrical attraction of positive and negative charges (△Ψ)
-PMF= ∆p + ∆Ψ
Gradient coupled to other rxns
Charges line up at HIGH conc. but VERY localized
Protein machines can be plugged in & powered by PMF
NADH ( “reducing equivalents”) : Energy as excited electrons
Stores HIGH-energy electrons
FAVORABLE electron DONOR
Essential for catabolism + anabolism
ATP: energy as chemical disequilibrium
ATP= HIGH conc. In cell→ VERY FAR from equilibrium
ATP⇨ ADP + Pi , cell spends energy so that: ATP⇨ ADP + Pi
High Energy Bonds:
Free energy of P~O > O-H bond that forms AFTER hydrolysis
Phosphate Groups: negative charge + repel each other
⋆Energy needed to get them close enough to bon dis stored in P~O bond
Describe why ATP, FAD, NAD+, and NADP+ are important in a cell
-Both NAD+/NADH and FAD/FADH2 are extensively used in energy extraction from sugars during catabolism in chemoheterotrophs, whereas NADP+/NADPH plays an important role in anabolic reactions and photosynthesis.
-ATP can be used to fill any energy need of the cell
Identify the structure and structural components of an enzyme
-serve as catalysts for biochemical reactions inside cells; lowers activation energy
-an enzyme binds to its substrate(s), the enzyme structure changes slightly to find the best fit between the transition state (a structural intermediate between the substrate and product) and the active site & molds to a hand inserted into it.
- high temps cause enzymes to denature
Describe the differences between competitive and noncompetitive enzyme inhibitors
Competitive: A molecule similar enough to a substrate that it can compete with the substrate for binding to the active site by simply blocking the substrate from binding
Non-competitive: binds to the enzyme at an allosteric site (a location other than the active site), and still manages to block substrate binding to the active site by inducing a conformational change that reduces the affinity of the enzyme for its substrate
Describe why Proton Motive force, NADH, and ATP are important in a cell
They are all 1 of 3 ways cells store energy
Given an energy source and a carbon source, determine the metabolic lifestyle of an organ