MCB 2610 Exam 2

Why might people be interested in cultivating bacteria?

- model systems (stress response)

- people for health benefits

What do microorganisms generally need to grow?

- macronutrients

- micronutrients (trace elements)

Macronutrients Needed by Microorganisms

carbon, nitrogen, phosphorus, sulfur, oxygen, and hydrogen

Macronutrients needed by enzymes as cofactors

potassium, sodium, magnesium, calcium, and iron

Micronutrients needed by microorganisms

Mn, Zn, Co, Mo, Ni, and Cu

are trace elements often added to media?

typically don't have to; sometimes they contaminate other parts of your media, they can also leach out of glassware

What are micronutrients often used for?

enzyme cofactors

Elements that make up proteins

C, H, O, N, S

elements that make up lipids

CHOP

elements that make up carbohydrates

C, H, O

elements that make up nucleic acids

C, H, O, N, P

only macromolecules that contain phosphorus

lipids and nucleic acids

all organisms require:

carbon, hydrogen, oxygen, and electrons

what is the backbone of all organic components present in cell?

carbon

electron role in macromolecules

energy production

ATP is

the biological currency of energy

redox reactions

chemical reactions that transfer electrons between reactants

autotrophs

assimilate carbon from inorganic sources

heterotrophs

assimilate carbon in preexisting organic form

phototroph

an organisms that captures light energy to produce ATP

chemotrophs

organisms that capture energy from oxidation of reduced organic or inorganic compounds

organotroph

microorganism that acquires electrons from organic molecules (glucose, sodium)

lithotroph

an organism that uses reduced inorganic compounds as its electron source (elemental sulfur, hydrogen gas, ferric iron) -- "rock eaters"

what does it mean to be organic?

CHO; CO2 is not organic

Why is carbon needed?

for nutrients

Acquisition of Energy Subtypes:

Phototrophs or Chemotrophs

Electron Acquisition Subtypes

organotrophs or lithotrophs

mixotrophs

some organisms have great metabolic flexibility and alter their metabolism in response to environmental changes

- can be advantageous

Why isn't everyone a mixotroph?

genome has to encode additional pathways so more genomic space taken up, more resources are needed and it may slow the growth

Where are mixotrophs typically found?

- unstable, dynamic environments

Where do we find nitrogen?

amino acids, amine groups, purines, pyrimidines, some carbohydrates, lipids, enzyme cofactors, etc

How is nitrogen supplied?

- Metabolism of amino acids, nitrates, ammonia from the atmosphere

- Nitrogen fixation

nitrogen fixation

Process of converting nitrogen gas into ammonia

nitrogenase

an enzyme of nitrogen-fixing microorganisms that catalyzes the conversion of nitrogen to ammonia to allow the ability to rip apart ammonia and turn into any macromolecule you need

enzyme needed to undergo nitrogen fixation

nitrogenase

how do the majority of microorganisms obtain nitrogen?

from the environment

where do we find phosphorus?

nucleic acids, phospholipids (membranes), coenzymes, some proteins (not a lot)

Common Phosphorus Sources

- inorganic phosphate

- organic molecules containing a phosphoryl group (pull apart and release phosphate)

sulfur sources

amino acids, coenzymes

where is sulfur obtained from?

1. amino acids cysteine and methionine

2. Sulfate

assimilatory sulfate reduction

sulfate to sulfite to hydrogen sulfide

growth factors

organic compounds that cannot be synthesized by an organism but are essential for growth

what are sterols important for?

membrane formation

microplasma

lack a cell wall

what is added to media for those with microplasmas

sterol like molecules to help growth

3 classes of growth factors

amino acids, purines and pyrimidines, vitamins

amino acid growth factors

protein synthesis

purines/pyrimidine growth factors

nucleic acid synthesis

vitamin growth factors

enzyme cofactors, needed in small amounts

hemen

important for cytochrome function -- needed for electron transport chains and generating ATP

hemofluous influenza

unable to make hemen (can't make ATP) so won't grow; needed hemen to be added to media

environmental factors that play a role on growth of microorganisms

nutrients, oxygen, reactive oxygen species, enzymes that protect against O2 toxicity, pH, temperature, solutes and water

nutrient concentration

-Growth rate will depend on the amounts of nutrients in the environment.

-One key nutrient, available in the lowest amount, will dictate how much growth can occur over time (i.e., it will be a limiting factor).

nutrients added in excess except for one

the one that is limited will limit the overall growth potential of the culture

High Nutrient Content results in:

a faster growth rate until it plateaus

type of oxygen that refers to what a macromolecule needs

atmospheric

aerobes

grow in presence of oxygen

obligate aerobes

require oxygen; will not grow if no oxygen

microaerophiles

grow best when there is less oxygen than normal

Where are microaerophiles typically found?

high altitudes, underground (not too low), in water

Anaerobic growth

occurs without oxygen

aerotolerant anaerobes

do not utilize oxygen but can survive and grow in its presence

obligate anaerobes

organisms that cannot live where molecular oxygen is present; oxygen will kill

facultative anaerobes

CAN use oxygen but can also grow in the absence of oxygen (prefers oxygen but don't have to have it)

obligate aerobe

facultative anaerobe

aerotolerant anaerobe

strict anaerobe

Microaerophiles

enzyme content of obligate aerobes

+SOD

+Catalase

enzyme content of facultative anaerobe

+SOD

+Catalase

aerotolerant enzyme content

+SOD

- Catalase

enzyme content of strict anaerobe

-SOD

-Catalase

does NOT have protective enzymes

Enzyme content of a microaerophile

+SOD

+/- Catalase (low levels)

effects of oxygen on microbial growth are often determined by:

what defenses are available against oxygen's negative effects in the cell

toxic oxygen species

- singlet oxygen

- superoxide anion (O2-)

- Hydroxyl radical (OH)

- Hydrogen peroxide (H2O2)

what does H2O2 tend to react with?

iron -- will produce hydroxyl ions

what do toxic oxygen species do to cells?

damage DNA (or any of the 4 macromolecules; amino acids)

ROS

reactive oxygen species

enzymes that help to neutralize H2O2

catalase and peroxidase, superoxide dismutase

________________ produce protective enzymes

aerobes; encode neutralizing enzymes in genome

superoxide dismutase

neutralizes superoxide

catalase

an enzyme that catalyzes the reduction of hydrogen peroxide.

peroxidase

an enzyme that destroys hydrogen peroxide

things that help to neutralize singlet oxygen species

antioxidants, pigments, enzymes

strict anaerobic microorganisms lack

superoxide dismutase and catalase

how are anaerobes grown in a lab?

- work station with an incubator

- gaspak anaerobic system

should we shake a test tube when growing anaerobes?

NO

Labs that Contain a work station with an incubator

labs that work with anaerobic organisms all time time (permanent device)

gaspak anaerobic system labs

- labs that don't work with anaerobes a lot

- much less expensive

anaerobic workstation

Contains incubator, work glove hole, and airlock

Seals area and pumps in N and H to purge oxygen from system

Need palladium crystal for reducing agent

Catalize left over oxygen

what does the vacuum pump do in an anaerobic workstation?

pumps nitrogen and hydrogen in

GasPak Anaerobic System

Lid, lockscrew, clamp, catalyst chamber, gas generator envelope, rubber gasket seal, anaerobic indicator strip

oxygen removed from chamber by combining with hydrogen to form water -- catalyzed by the palladium pellets

catalyst chamber of the GasPak anaerobic system

contains palladium pellets -- reducing agents that bind to any oxygen in the system

how to determine the GasPak is cleared of oxygen?

anaerobic indicator strip

anaerobic indicator strip of The GasPak

methylene blue becomes colorless in absence of O2

gas generator envelope

Water is added to chemicals in envelope to generate H2 and CO2. Carbon dioxide promotes more rapid growth of microorganisms

why does carbon dioxide promote more growth in the GasPak?

a lot of anaerobic systems use inorganic sources of carbon

dessicant of GasPak

soaks up the H2O so ensure oxygen is removed

effects of pH on microbial growth

pH affects macromolecule structures and transmembrane electrochemical gradients

- microbes have an optimal pH range for growth