Microbiology Rutgers NB (390) Dr.R Midterm Study Guide

structure and function

microorganism features

A DNA genome, ribosomes and a cell membrane.

All cells have which 4 cellular structures in common?

1) a plasma membrane, an outer covering that separates the cell's interior from its surrounding environment;

2) cytoplasm, consisting of a jelly-like region within the cell in which other cellular components are found;

3) DNA, the genetic material of the cell; and

4) ribosomes, particles that synthesize proteins.

microbe

Microbes is an overarching term. Microbes include microorganisms and viruses.

A microbe is an agent that interacts with the environment.

Virus

- obligate parasites that only replicate within host cell

- not cells- do not carry out metabolism; take over infected cells to replicate

- have small genomes of double-stranded or single-stranded D N A or R N A

- classified based on structure, genome composition, and host specificity (e.g., bacteriophages)

Cell membrane

allows for transport- is a selective fluid layer, also plays a role in cell metabolism. ALL CELLS HAVE A MEMBRANE BUT NOT ALL CELLS HAVE A CELL WALL.

3 domains of the tree of life

bacteria, archaea, eukaryotes

Characteristics of Prokaryotes

-cells are smaller and simpler

-cells have DNA, but not inside a nucleus

-no organelles

-single-celled

- most are 0.5 to 10 micrometers long

Eg: Bacteria and Archaea

Characteristics of Eukaryotes

-cells are larger and more complex

-DNA enclosed in a membrane-bound nucleus

-mitochondria and chloroplasts

-can be unicellular or multicellular

-typically 5 to 100 micrometers long

examples: plants, animals, algae, protozoa, fungi

LUCA

Last Universal Common Ancestor. The shared ancestor that multiple organisms diverged from.

- Had RNA and a membrane, might've had some DNA.

Bacteriophage

- Why are they beneficial

viruses that affect bacteria

They kill bacterial cells. Eg: Everyone has Propionibacterium (acne) but in addition we also have the phages that inject them and keep the bacteria from overgrowing (regulate bacterial growth)

How will climate change affect microbial populations?

Different microbes have diff optimal temperatures so their functioning gets affected. This could affect oxygen levels

Why do we need viruses and where they come from?

Life started from a single RNA molecule according to the RNA world hypothesis. We believe viruses could be leftovers.

We need them because they keep populations in check and in order to drive genetic change. Some viruses mutate. So they're still around because we need them to drive evolution.

What is bacterial growth

Refers to the increase in the number of cells in the population.

Properties of ALL cells

1. Structure: all cells have a cytoplasmic membrane, cytoplasm, a genome made of DNA and ribosomes.

2. Metabolism: all cells use the info encoded in DNA to make RNA and protein. All cells take up nutrients, transform them, conserve energy and expel wastes. (catabolism and anabolism)

2. Growth: Info from DNA --> protein. proteins convert nutrients from the environment to new cells.

3. Evolution: chance mutations in dna causes new cells to have new properties

Properties of some cells

1. Differentiation: some cells can form new cell structures like spores.

2. Communication: cells interact with eachother through chemical messages (biofilms). Tooth plaque, slippery rocks, sometimes forms on medical equipment

3. Horizontal gene transfer: mechanism to exchange genes. Conjugation- moving dna between cells.

4. Motility: come cells are capable of self-propulsion.

Differentiation

process in which cells become specialized in structure and function

SOME bacteria and archea do this. Bacterial endospores are a survival strategy for bacteria. Spores survive high heat, disinfectants, environments with very low water content etc.

Communication

Biofilms have these small layers where microbes grow and work together. Tooth plaque, slippery rocks, sometimes forms on medical equipment.

Size

Bacteria and archea are measured in micrometers. Viruses in nanometers. Average size of a bacterial cell is between 0.5-2 micrometers.

Biggest bacterial cell: Thiomargarita magnifica (800 nm +)

Smallest becaterial cell: Mycoplasma genitalium 200-300nm

Biggest virus: Megavirus chilensis (400 nm)

Smallest virus: Phi-X174 (20nm)

The cytoplasmic membrane

A phospholipid bilayer embedded with proteins that surrounds the cytoplasm and defines the boundary of the cell.

Surrounds cytoplasm (mixture of macromolecules and small molecules)

• Separates it from environment

• Main function: selective permeability (nutrients transported in and waste products out)

- Membrane proteins facilitate these reactions and function in energy metabolism

- Membranes are fluid and membrane structure match the environment they're in.

Membrane in bacteria and archaea is used to generate energy- electron transport chain- because they don't have mitochondria.

- What would a thermophile do in a very hot temperature?

-What would happen to a thermophile at room temperature/human environment?

- They are adapted to survive this so their membranes would change their composition to withstand these temperatures (change the amount of phospholipids and proteins)

- They would not function.

Bacteria/Eukaryotic membrane

- Hydrophobic and phallic bi layers

- Fatty acid tail

- Ester linkages

Archeal membrane

- Bi layer AND mono layer (Monolayer: provides more stability- may find in thermophiles)

- Phytanyl and Isoprene subunits

- Ether linkages

The cell wall

- Outside of membrane. Not all bacteria have it. If they don't have a cell wall they'll have some modifications to the membrane

- Environmental changes are more rapid for single celled. Cell wall provides structure and rigidity.

- Bacteria have peptidoglycan (AKA murein) in their cell wall. Protein sugar.

- Peptidoglycan are alternating for NAM-NAG. Made inside the cell and transported out. Super rigid.

- Archea: has a pseudomurein cell wall.

Gram positive cell wall

Thick peptidoglycan- 90%

Teichoic acids and Lipoteichoic

Gram negative cell wall

Thin peptidoglycan

Outer membrane

- Cell wall = outer membrane and 10% peptidoglycan

Porin

Periplasmic space more significant

* More resistant to antibiotics. Eg: E.coli, Salmonella

Lipopolysaccharide (LPS)

Molecule that makes up the outer layer of the outer membrane of Gram-negative bacteria.

- Mostly made up of lipid A (endotoxins). If the cell dies lipid A may be released.

- ONLY gram negative have endotoxins

Exotoxins also exist- secreted into the environment.

Gram stain

A staining method that distinguishes between two different kinds of bacterial cell walls.

- Dehydrates the PG layer trapping the stain in the positive and in the negative it washes the outer membrane away.

gram positive stain

retain crystal violet and stain purple

Eg: streptococcus, Staph aureus.

gram negative stain

red/pink

acid fast stain

a differential stain used to identify bacteria that are not decolorized by acid-alcohol

1. Mycobacteria (TB) Mycolic acid

Mycobacteria are acid fast because you need acid alcohol to stain it because of the extra mycolic acid layer. NOT stained with gram stain.

Positive: red and Negative: Blue

2. Mycoplasma --> ONLY plasma membrane, no cell wall

If you stain it'll show as gram negative. Causes atypical pneumonia and you can't treat penicillin because pen attacks Peptidoglycan and mycoplasma has none.

Archaeal Cell Walls

contain polysaccharides and proteins but lack peptidoglycan.

Not considered positive or negative. Cell was has psuedomuerin. Some also have S-layer.

S-layer: alternate cell wall made of knobby protein balls. Either have S-layer OR psuedomuerin.

Eukaryotic cell wall

The ones that have cell wall have one have it made of cellulose or chitin (peptidoglycan equivalent)

Cell Surface Structures: Capsules and Slime Layers

capsule: if tightly attached, tight matrix; visible if treated with India ink.

Are a virulence factor and keeps cell from drying out. - If an organism has a capsule itll be less likely to be seen by the immune system. Allows it to multiply.

Slime layer: -sticky polysaccharide coat outside cell envelope. Provides additional protection. Environmental isolates usually have slime layers.

Pili

thin filamentous protein structures

▪ Enable organisms to stick to surfaces or form pellicles (thin sheets of cells on a liquid surface) or biofilms

Fimbriae

short pili mediating attachment

▪ Produced by all gram-negatives and many gram-positives

▪ Conjugative/sex pili facilitate genetic exchange between cells (conjugation)

▪ Electrically conductive pili conduct electrons

Flagella/archaella:

structure that assists in swimming in Bacteria and Archaea, respectively (tiny rotating machines)

Chemotaxis

Cell movement that occurs in response to chemical stimulus

VIRULENCE

severity of infection

Virulence factors

traits of a microbe that promote pathogenicity. Eg: Capsules, Fimbriae, Pili, and Hami (hami are in archaea)

Endospores

A resistant, dormant structure formed inside of some bacteria that can withstand adverse conditions

- Virulence factor

Fungal Spores vs. Bacterial Endospores

Fungal spores - are for reproduction. Endospores are for survival

- only found in gram positive because the peptidoglycan helps them make their dormant feature. Clostridium and Bacillus form endospores.

Metabolism

the chemical processes that occur within a living organism in order to maintain life.

Includes catabolism and anabolism- Relies on electron donors directing electrons to electron acceptors

reducing power

ability to donate electrons during electron transfer reactions

Redox reactions

include two half reactions.

▪ Electron donor: transfers electrons (oxidized)

▪ Electron acceptor: adds electrons (reduced)

▪ e.g., Aerobic respiration of glucose

Classification based on metabolism

Reduction potential

affinity of substance for electrons

NAD-/NADH

The coenzyme is, therefore, found in two forms in cells: NAD+ is an oxidizing agent - it accepts electrons from other molecules and becomes reduced. This reaction forms NADH, which can then be used as a reducing agent to donate electrons. These electron transfer reactions are the main function of NAD.

Mechanisms of Energy Conservation

A T P generated through 1 of 3 mechanisms

1. Substrate-level phosphorylation:

2. Oxidative phosphorylation

3. Photophosphorylation

Substrate-level phosphorylation

energy-rich substrate bond hydrolyzed directly to drive ATP formation (e.g., hydrolysis of phosphoenolpyruvate)

Oxidative phosphorylation

Movement of electrons generates proton motive force (electrochemical gradient) used to synthesize ATP

Photophosphorylation

light used to form proton motive force

Principles of Fermentation

Fermentation of glucose involves substrate-level phosphorylation and redox balance via pyruvate reduction + excretion as waste

All fermentations must do two things:

▪ Conserve energy

▪ Redox balance

Need to produce compounds containing high-energy bonds for A T P synthesis

fermentation (2)

• uses substrate level ONLY which is why cells don't grow a lot.

- purpose is to recycle NADH back to NAD+. For some cells it's a survival mechanism (they switch once they run out of oxygen)

Side products: alcohol, carbon dioxide, Acid

Respiration

electrons transferred from reduced electron donors to external electron acceptors (eg: O2). Respiration is oxidative phosphorylation.

NADH and FADH2 produced in glycolysis and citric acidcycle must be reoxidized for redox balance

- In respiration, reoxidation occurs during electron transport

▪ Occurs in cytoplasmic membrane

▪ Forms electrochemical gradient (usually protons) that conserves energy through A T P synthesis

- Aerobic respiration- will always make more energy. Eukaryotes don't do ANEROBIC RESP.

Glycolysis

Need glucose --> forms pyruvate. Needs 2 ATP. Also produces 4 ATP (Net gain of 2 ATP). Occurs in the cytoplasm. (THIS IS SUBSTRATE LEVEL). Also generates NADH.

What usually comes after glycolysis is the Krebs (TCA, citric acid) cycle (an intermediate step to keep carrying on the oxidation of the molecule- making more NADH). Not all prokaryotes have it. They pass right to the electron transport chain instead.

Why does metabolism continue? Why aren't there cells that solely require glycolysis?

Because they need to recycle the NAD+ (electron carrier). The NAD+ is taken in by the cell (from food) and also made (but not very energy efficient to make it which is why its recycled). NAD+ is the limiting factor and if a cell was doing glycolysis only it would run out.

What is the point of the electron transport?

To make ATP, and to recycle NADH into NAD+. More energetically favorable.

Phototrophy

- Uses light to generate proton motive force

- A T P synthase makes A T P by photophosphorylation

▪ oxygenic (e.g., cyanobacteria, algae, plants),forming as waste product, or anoxygenic(many Bacteria)

▪ anoxygenic phototrophs evolved first, more metabolic diversity

Anoxygenic phototrophs

• Energy from light but reducing power from organic or inorganic sources

• The first phototrophs to live on Earth

• Habitat - anoxic environments that are exposed to light

• Highly diverse: Phylogeny, pigments, electron donors, photosystems, mechanism of CO2 fixation

Anabolism

Biosynthesis of cellular macromolecules

- • Cells require carbon and nitrogen to perform biosynthesis

• Atmospheric sources (CO2 and N2) must be chemically reduced for assimilation (CO2 fixation and N2 fixation)

• Requires A T P and reducing power

Nitrogen Fixation

- Nitrogen needed for proteins, nucleic acids, other organics

- Most microbes obtain this nitrogen from "fixed"nitrogen (ammonia NH3, or nitrate NO3)

- Many prokaryotes can conduct nitrogen fixation:form ammonia (NH3) from gaseous dinitrogen (N2)

Nitrogen-fixing

Diazotrophs: reduce nitrogen to ammonia for cellular assimilation

▪ Form symbioses with higher organisms

Enzymes

proteins that act as biological catalysts.

- drive life. Determines an organisms ability to function.

Why are we limited as humans?

- We can only use glucose and oxygen to make energy (ATP)

Electron donor

the substance oxidized in a redox reaction. - ie. it loses an electron. (electrons come from the bonds).

- Need electron carriers like NAD+ (NADH).

Electron acceptor

- reduced.

- Microbes don't need electron acceptors

Energy source (naming)

- chemotroph: Uses chemical energy. Eg: humans

- Phototrophs: Use sunlight.

Electron donor (naming)

- Organic comp: organotroph

- Inorganic compound: lithotroph

carbon source (naming)

- Organic compound: heterotroph

- Carbondioxide: autotroph

Aerobic

- oxygen is electron acceptor

binary fission

A form of asexual reproduction in single-celled organisms by which one cell divides into two cells of the same size. Microbes grow in the number of cells. - (not mitosis- that is eukaryotic)

incubation temperature

35C and 25C are the temps we usually use for microbes at lab

Batch culture

- a closed system microbial culture- nothing we add to it

Growth curve

(only in closed system) has starting and end point. # of cells (or growth) on x axis and time on y axis. Time depends on organism. Eg: ecoli is 20 mins

lag phase

intense activity preparing for population growth, but no increase in population

Log phase

The period of exponential growth of bacterial population. - this is where you do all your experiments. Why? Because you have sufficient sample and cells are healthy and dividing.

Stationary phase

- They stop dividing. Cells dividing= cells dying. Because you reach carrying capacity, run out of nutrients and oxygen.

Do you think there's always a lag phase?

- No. When you transfer log to log or from log grown culture into same medium or into even richer nutrients- lag phase is very unlikely.

Continuous culture

- we take microbes out and add stuff. Growth is continuous at a steady rate.

Defined media

- exact chemical comp. known.

Complex media

Composed of digest of microbial, animal or plant products. You don't know exact composition. Tryptic soy agar, tryptic soy broth etc

Selective medium

a culture medium with an ingredient that inhibits the growth of microbes other than the one being sought. Antibiotics, change in pH, salt conc.

Differential medium

- contains an indicator, usually a dye that detects metabolic reactions during growth. You can tell apart. ALWAYS select first then differentiate.

Mannitol salt agar

- You can tell its selective because of salt (high percent). We inoculate with Staph aureus, E.coli, and staph epi. Which will not grow? E.coli because it isn't halotolerant.

Sessile growth

attached to surface

can develop into biofilms

attached polysaccharide matrix containing embedded bacteria

advantage: anchors them down, microbes can communicate with eachother (virulence factors) through quorum sensing (when microbes get together and secrete molecules and trigger a collective genetic response).

Natural biofilms (also virulence factor)

- dense organized structures with many kinds of microbes. Advantages: stability, have access to nutrient source, safety in numbers, extracellular matrix provides protection.

Psychrophile

- love low temp. Never pathogen.

Mesophiles

moderate temperature loving microbes

Psychotrophs

human pathogens- Eg: listeria. Organisms that grow between 0-30 degrees celsius and are responsible for most food spoilage while in the refrigerator

Acidophile

an organism that grows best at low pH; typically below pH 5

Nuetrophile

Grows pH 5-9.0

(Nuetral Loving)

alkalophile

grows optimally at a pH above 9

Halotolerant

can survive at higher salt concentrations but grow best at low or zero concentations. - Staph aureus. Grows on ur skin is tolerant to salt in sweat.

Halophile

"salt-loving" archaea that live in environments that have very high salt concentrations

extreme halophiles

An organism that lives in a highly saline environment, such as the Great Salt Lake or the Dead Sea.

Sterile

no living microbes

Aseptic

Prevents cross contamination.

Pasteurization

the process of treating a substance with heat to destroy or slow the growth of pathogens. doesn't chemically change stuff but makes it safe to consume.

Autoclaving

High pressure, high heat. At 115 degrees Celsius. For 15 mins. To check if it worked, you'd incubate it. They use endospores because they're highly resistant.