BI276 midterm 2

C, N, P, S, K, Mg, Ca, Na, Fe

Macronutrients required for growth (x8) + ___ for siderophore sequestration

Growth factors

Ex: vitamins (organic composition)

Chemically defined, undefined

The 2 broad classes of culture media

Chemically defined

A broad class of culture media
Exact chemical composition is known
- Add precise amounts of growth requirements

Undefined

A broad class of culture media
Uses digests of animal or plant products, exact composition not entirely known
Ex: yeasts extract, tryptone

Selective medium

Contains compounds that selectively inhibit the growth of some microorganisms and not others
ex: Add antibiotics = only antibiotic resistant bacteria will survive (trait selective)

Differential medium

Contains an indicator which distinguishes b/t chemical rxns generated by different species of bacteria
Ex: reactive dyes

Pure culture

Contains one single type of microorganism

Semisolid, morphological

Use of ______ culture allows for formation of colonies.
Colonies can have specific ______ characteristics,

Streak plates

1. Start with confluent growth in first streak

2. Work towards getting isolated colonies Ex: Pour plate, spread plate

Amphibolic pathways

Reaction pathways what utilize roles of both catabolism and anabolism

Energy

Ability to do work

Free energy

Energy release that is available to do USEFUL work

Activation energy

Amount of energy needed to disrupt the stable arrangement of a given molecule; minimum energy required for chemical reaction to begin

Catalysts

Facilitates reaction without being consumed as a reactant
Usually required to overcome the activation energy barrier by lowering it
Speeds up the reaction rate
Act on a specific substrate
Equilibrium is unaffected

Reaction rate

Frequency of collisions containing sufficient energy to create a reaction

Enzyme substrate complex

Created by the temporary binding of enzyme and reactants
Lowers the activation energy of a particular reaction

Enzymes

BIOLOGICAL CATALYSTS

• typically proteins

• highly specific

• Larger than substrate

• Rely on weak bonds (HB, Van Der Waals, hydrophobic interactions)

• Increase rate of reaction by 10^8 to 10^20 times the spontaneous rate

• Many contains small non protein, non substrate molecules that participate in catalysis Ex: prosthetic groups, co-enzymes

Prothetic groups

Tightly bound usually covalently and permanently to enzymes

Coenzymes

Loosely, transiently bound to enzymes
Most are derivatives of vitamins, can be metals

Enzyme-substrate, bonds , coupling, reversible, reverse

_______-_______ complex aligns reactive groups and strains specific ______, reducing the Ea.
To catalyze endergonic rxns, _______ is used between endergonic and exergonic rxns.
______ rxns have ____ or _____ free energy change
All enzymes theoretically are ______ but highly exergonic or endergonic rxns catalysis usually goes in one direction.
Another enzyme is needed for the ____ rxn.

Active site

Region of enzyme that binds substrate

substrate binding, position of substrate, coenzyme and prosthetic groups

Catalysis is dependent on 3 factors:

Temperature, pH, substrate concentration, inhibitors

Enzyme activity can be affected by 4 things:

Competitive inhibitors

Binds to the active site of an enzyme which means the substrate no longer can

Non-competitive inhibitors

Binds to the allosteric site of an enzyme which changes its conformation, meaning the substrate can no longer bind to the enzyme

Feedback inhibition

Prevents wasteful use of resources/energy
End product inhibits an earlier process

Ex: Last enzyme of a pathway creates an end product which can act as a non-competitive inhibitor to a previous enzyme in the pathway which shuts it down

Nutrients

Supply of elements required for cell growth

Micronutrients

Nutrients requires in small amounts
Ex: Trace metals (inorganic) and growth factors (vitamins: organic)

Heterotrophs

Obtain carbons from breakdown of organic polymers or uptake of monomers (amino acids, fatty acids, organic acids, sugars, nitrogen bases....)

Autotroph

Synthesizes organics from Carbon dioxide (CO2 = carbon source)
AKA primary producers: synthesize organic matter from inorganic carbon

nitrogenous bases (DNA), amino acids

Nitrogen is needed by the cell for: (x2)

Usually used in the form of ammonia (NH3) or nitrate (NO3 -)
**Nitrogen fixers can convert N2 to usable form (NO3 -) by other organisms

Nucleic acids, phospholipids

Phosphorus is required by the cell for: (x2)
Usually in the form of inorganic phosphate (PO4 3-)

Amino acids (cysteine, methionine), vitamins (thiamine, biotin, lipoic acid)

Sulfur is required by the cell for: (x2)
Usually in the form of sulfate(SO4 2-), sulphide (H2S) or organics

enzymes

Potassium is required by the cell for: (x1)

Co-factor, stabilize ribosomes, membranes, nucleic acids

Magnesium is required by the cell for: (x4)

Co-factors

Elements that enhance enzymatic activity and increase enzymatic efficiency
Inorganic usually metals

marine

Calcium and sodium is usually required by _____ microbes

Cellular respiration, oxidation reduction rxns

Iron is required by the cell for: (x2)

Culture media

Nutrient solutions used to grow microbes in the lab
Typically sterilized in an AUTOCLAVE

Solid media

Prepared by addition of the gelling/solidifying agent: agar to liquid media
When grown on this, cells form isolated colonies

Colony morphology

Physical appearance of colony
Can help determine if its contaminated, mixed or pure
Sometimes used to identify microorganisms

Microscopic cell count

Observing and enumerating ALL cells (total cell count) present on slide once dried or on liquid sample
Counting chambers (grid) etched on a slide is used for liquid samples
COUNTS ALL CELLS DEAD AND ALIVE
COUNTING ERROS ARE COMMON
Often used on natural samples
Often used to visualize and provide phylogenetic or metabolic properties
Stains can help differentiate live and dead cells

Phylogenetic stain

Can determine proportions of bacterial and archaeal cells in a sample

Viable (alive) count

Measurement of ONLY living, reproducing cells in a population 2 methods:

• Spread plate

• Pour plate ** Can only count on plates with 30-300 colonies Reported in CFU/mL

Colony forming units

CFU stands for:

not reliable, agar, increase, separate

If there are too few colonies on a spread plate: _______
If there are too many colonies on a spread plate: ______ can't support all colonies, ________ in counting error, not all colonies are _______.

Food, dairy, medical, aquatic biology, waste water analysis

Examples of application of the plate count
+ Can also target particular species in mixed samples (e.g staphylococcus)

Plate bias (different organisms have different requirements for growth), underestimate dead to live cell ration (viable count vs. total count)

The great plate count anomaly: Direct microscopic counts of natural samples reveal far more organisms than those recoverable on plates. WHY?

turbid, more, more, more turbid

Cells in suspensions are ______ (cloudy) because cells scatter light.
The ____ cells are in suspension = the _____ light is scattered = the ____ _____ the suspension

Turbidity measurements, optical density (OD), turbidity, optical density (OD)

Rapid and widely used for estimation of the number of cells Measured by spectrophotometer in units of ________ at specified wavelengths ** Not a viable count ** Standard curve must be established to relate direct cell count to a _____ value ***For unicellular organisms ________ is proportional to the number of cells present

clumps, biofilms, debris

Common issues with turbidimetric measures: microbes that form _____ (UNDERESTIMATES in measure) and/or _____ in liquid medium and/or ________ (NON ACCURATE MEASURES: INCREASES OPACITY BUT IT ISNT CELLS)

Negative

An exergonic rxn has a ____ Delta G

Positive

An endergonic rxn has a _____ Delta G

Septum

Partition (structure) b/t dividing cells, pinching off b/t two daughter cells

Generation time

Time required for microbial cells to double in number
AKA DOUBLING TIME
Differs for microbes and varies depending on conditions

Batch culture

A closed system microbial culture of fixed volume

Lag, exponential, stationary, death

Typical growth curve for population of cells grown in a closed system has 4 phases:

Lag phase

Period of little to no cell division
Typically when cells are freshly inoculated into new media
New conditions = altering metabolic state
Intense metabolic activity including synthesis of enzymes + metabolites required for growth

Exponential phase

Once cell divisions begins, a period of ____ growth follows during which generation time is constant (constant doubling of cells)
Logarithmic plot results in a straight line
Cells are more sensitive to adverse conditions during this period

Stationary phase

Growth rate slows during this period (in response to physical or chemical limitation: limited nutrients, increase in toxic byproduct waste)
Number of cell deaths balance number of new cells produced

Death phase

When the number of deaths in the population exceeds the number of new cells formed
AKA logarithmic decline phase
Continues until population dies out or greatly diminishes

media, conditions, organisms (itself)

Exponential phase vary greatly due to: (x3)

Cryptic growth

subpopulations adapt

Continuous culture

An open system for microbial culture

Chemostat

Most common type of continuous culture device; known volume added while spent medium is removed at the same rate which results in a STEADY STATE (cell density and substrate concentration DO NOT change overtime)
** Can maintain exponential phase of growth for weeks/months
*** Study physiology, microbial ecology, evolution , enrichment, isolation of bacteria from nature

dilution rate, growth yield

Growth rate and population density of culture can be controlled independently and simultaneously depending on: _____ (determined by flow rate and volume of culture) and _____ (controlled by concentration of a limiting nutrient)

growth rate, growth yield

ONLY AT LOW NUTRIENT CONCENTRATIONS ARE BOTH _____ ____ AND _____ ____

Temperature, pH, water availability, oxygen exposure

Factors that affect microbial growth: (x4)

Cardinal temperatures

Minimum, optimum and maximum temperatures at which an organism grows
Differ dramatically b/t species
Range typically

Psychrophile

low, found in cold environments
max:

Mesophile

Midrange, most commonly studied

Thermophile

high, found in hot environments
optima b/t 45oC and 80oC

Hyperthermophile

very high, found in extremely hot habitats such as hot springs and deep-sea hydrothermal vents
Optima >80oC
Very low generation time (1 hour compared to 20mins for e-coli)
High prokaryote diversity (bacteria, archaea)

psychrophile, mesolphile, thermophile, hyperthermophile

The 4 broad classes related to growth temperature optima are:

Psychrotolerant

Can grow at 0oC but have an optima lower than usual phsycrophiles at 20oC not 40oC
More widely distributed in nature than psychrophiles
Isolated from soils and water in temperate climates and food at 4oC

enzymes, alpha, beta, polar, hydrophobic, weak, unsaturated, cold shock, cryoprotectants, exopolysaccharide

Molecular adaptations to life in the cold:

• production of ______ that function optimally in the cold

• More ____ helices than ____ sheets which confers greater flexibility for catalysis in the cold

• More _____ and fewer ____ amino acids

• Fewer _____ bonds

• Cytoplasmic membrane function at low temps

• Higher ______ and shorter-chain fatty acid content

• Some polyunsaturated fatty acids which remain flexible at low temps

• _____ ____ proteins

• ________ (ex: antifreeze proteins, certain solutes) to prevent formation of ice crystals

• _______ cell surface slime

Methanopyrus

Most thermophilic bacteria

amino acid, ionic, hydrophobic, lipid, saturated, unsaturated

Protein and membrane stability at high temps

• Enzymes and proteins more heat-stable and function optimally at high temps

• heat stability from subtle ____ ____ substitutions resist _______

• Increased _____ bonding and highly ______ interior

• Production of solutes (di-inositol phosphate, diglycerol phosphate, mannosylglycerate) help stabilize proteins.

• Cytoplasmic membranes must be heat stable

• Bacteria have ____ rich in long-chain and ______ aftty acids, fewer _____ fatty acids

• Most hyperthermophiles (archaea) have C40 hydrocarbons which form lipid monolayer not bilayer

Neutrophiles

grow optimally at pH 5.5-7.9 (neutral pH)

Acidophiles

grow best at moderately acidic or very low pH
Governed by stability of cytoplasmic membrane: at neutral pH, membranes of strong _____ lyse; protons required for stability (high H+ environment = more acidic)

Alkaliphiles

High pH optima for growth pH>8
Found in highly alkaline habitats: soda lakes, high carbonate soils
Secretes proteases and lipases that are added to laundry detergents to remove proteins and fats from cloting
Some have an Na+ motive force rather than an H+ motive force bc H+ environment confer acidity

higher, inside

Typically the cytoplasm has a ___ solute concentration that the surrounding environment, thus, the tendency for water to move _____ the cell (+ water balance)

Halophiles

Grow best at aw=0.98 (water activity)
Have a specific requirement for NaCl (salt)
Ex: Sea water
Range from _____ tolerant (can live but rather not) to extreme _____ which require very high levels (15-30%) of NaCl often unable to grow at lower concentrations.

Osmophiles

Live in environment high in sugar

Xerophiles

Able to grow in very dry environments
Lowest aw=0.61 for life; physiochemical constraints on obtaining water at lower aw.

Aerobes

Grow at full O2 tension (21%) and respire O2
Cellular respiration for ATP

Microaerophiles

Can use O2 only at levels reduced (5%) from that in the air (microbic) due to limited respiration or oxygen sensitivities

Anaerobes

Cannot respire oxygen
Ex: anoxic habitats: mud, bogs, marshes, animal intestines...

Aerotolerant anaerobes

Tolerate oxygen and grow in its presence even tho it cannot do cellular respiration

Superoxide anion (O2-), hydrogen peroxide (H2O2), hydroxyl radical (OH.)

Why is Oxygen toxic?

• Metabolic rxn intermediates can be toxic

• Exposure to oxygen yields toxic byproducts Ex: (x3)

• Enzymes neutralize these intermediates

*** Molecular O2 is not toxic

catalase, peroxidase, superoxide dismutase

______: H2O2 +H2O2 = 2H2O + O2
______: H2O2 + NADH +H+ = 2H2O + NAD+
______: O2- + O2- + 2H+ = H2O2 + O2 (requires pairing w/ other enzyme)

Catabolism

Breakdown molecules and generate ATP
Spontaneous

Anabolism

Build up starting material and use the ATP generated by catabolic pathways
Not spontaneous

Reducing power

Stems from NADH or NADPH
In the form of a reduced electron: source of electron to carry out anabolic rxns

Oxidation

Removal of electrons from an atom or molecule

Reduction

Addition of one or more electron to a molecule

Electron donor

Substance that is being oxidized is the:
AKA THE REDUCTANT (what reduces a molecule)

Electron acceptor

Substance that is being reduced is the:

Metabolism

All biochemical rxns needed for life
Includes: catabolism to obtain energy, anabolism to make cellular material
Relies on electron donors and acceptors