exam1 – Microbiology – Flashcards

Bounded by a plasma membrane

Contain cytoplasm

Utilize energy and raw materials through metabolism

Have both DNA and RNA

Reproduce by cell division processes

Have: no (or few) internal membranes

 Many processes that are associated with organelles in eukaryotes (e.g. Respiration, photosynthesis) are mediated by specialized regions (folds, etc.) of the plasma membrane in prokaryotes

There is no membrane-bound nucleus in prokaryotes. Instead the DNA is located within a specialized region of the cytoplasm of the cell called the nucleoid region.

There is no nuclear membrane surrounding the nucleoid.

Includes: the bacteria & archaea

the terms “prokaryotic cell” and “bacterial cell” often are used interchangeably

Shapes & Arrangements: See shapes handout Sizes Typically ~ 0.1 - 20 m (with some exceptions)

Typical coccus: ~ 1 m (e.g. Staphylococcus) Typical short rod: ~ 1 x 5 m (e.g. E. coli)

Barely within the best resolution of a good compound light microscope

Plasma membrane

The cytoplasmic matrix

Ribosomes

Cytoplasmic inclusions

Nucleoid Prokaryotic cell walls

Capsules, slime layers, and S-layers

Bacterial flagella and motility

Bacterial spores

Maintain Cell Integrity

Regulate Transport

Specialized Functions in Bacteria

Phospholipid (know the structure of a phospholipid) Bilayer with Associated Proteins Cholesterol is absent (except in the mycoplasma group)

Hopanoids are often present in place of cholesterol. Similar in molecular structure to cholesterol.

Some archaea have plasma membranes with unusual lipids and monolayer structures

folds of the plasma membrane

Respiratory and Photosynthetic folds

Composition: Viscous aqueous suspension of proteins, nucleic acid, dissolved organic compounds, mineral salts

Network of protein fibers similar to the eukaryotic cytoskeleton

Sites of protein synthesis

Typically several thousand ribosomes per bacterial cell, depending on the state of its metabolic activity

Smaller than eukaryotic ribosomes

Glycogen Granules similar in structure to starch Poly--hydroxybutyrate granules

Lipid droplets – mostly di and triglycerides

Gas vacuoles -

Metachromatic granules – common in chorynebacterium (Phosphate crystals or volutin granules)

Sulfur Granules

Chromosomal DNA

Typically, one chromosome per bacterial cell Consists of double-stranded, circular DNA

A few recently discovered groups have >1 chromosome per cell and linear chromosomes Plasmid DNA – smaller dbl stranded molecule that encode extra functions

R-Plasmids – encodes antibiotic resistance in a cell.

Antibiotic = substance secreted by one type of microbe to inhibit or kill another type of microbe Ex. PCN inhibits cell wall production

F-Plasmids – F for fertility. Carry the genes for the process of conjugation (transfer of DNA from one cell to another)

Composition

A Polysaccharide Composed of alternating units of N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM) both are 6C sugars

Peptide crosslinking between NAM units: Tetrapeptide or pentapeptide chains attached to NAM may “crosslink” adjacent PG strands This gives PG (pentaglycine) a net-like or mesh-like structure that contains the cell wall.

Indirect cross linking: Found in Gram-positive bacteria TP chains of adjacent PG strands are linked by pentapeptide chains

Direct crosslinking: Found in both Gm + and Gm - bacteria TP chains are directly attached to each other Proteins always contain the L-alanine amino acids

Thick layer of Peptidoglycan 20-80 nm in thickness

Extensively crosslinked, both with indirect & direct links

Teichoic Acids

• Polymers of glycerol or ribitol Inserted into the PG layer
• Sometimes attached to plasma membrane lipids
• May help stabilize the cell wall
• May play a roll in bacterial adhesion, has been associated with dental plaque buildup.

Periplasmic Space

• Space between the PG layer and the plasma membrane
• Parplasmic space in Gm+ bactieria is much smaller than in Gm- bacteria -- significance questioned

Outer Membrane 7 - 8 nm in thickness

Bilayer of lipopolysaccharide and phospholipid, with outer membrane proteins Lipopolysaccharide contains: *

•  Lipid A: A dimer of glucosamine with 6 fatty chains *
• Core Polysaccharide: About 10 monosaccharide units *
• O-side chain (O antigen) different antigens have different O-side chains. It is the antigen in Gm- bacteria.
• H antigen is the antigen in flagellates.

Lipid A is the bacterial endotoxin: triggers inflammatory effects and hemorrhaging Outer Membrane

Proteins:

• Porin Protein: 3 porin molecules form a channel for transport/diffusion
• Peptidoglycan Layer Thinner than gm positive 1 - 3 nm thick Less extensively crosslinked
• Anchored to outer membrane via Braun's lipoprotein

Periplasmic Space

• Fluid or gel-filled space
• Much larger in Gm negative cells: possibly 20 - 40% of cell volume
• Periplasmic proteins:
  • Hydrolytic enzymes ; Transport proteins (proteases, lipases, bacterial amylases)
  • Bind to target molecules and carry them to transport proteins in the plasma membrane

Acid-fast Cell Walls

Many genera in the ;High GC gram-positive; bacterial group contain mycolic acids (very hydrophobic molecule, long aliphatic chain), embedded in the peptidoglycan

Mycolic acids are a class of waxy, extremely hydrophobic lipids due to long hydrocarbon chains. Certain genera contain very large amounts of this lipid, and are difficult to gram stain These genera may be identified by the ;acid-fast; staining technique

• Includes Mycobacterium (one type causes leprocy) and Nocardia Mycoplasmas

Bacteria that naturally have no cell walls,

• phylogenetically similar to Gm+ bacteria, with no PG.
• Includes Mycoplasma and Ureaplasma

Archaea Have archaea cell walls with no peptidoglycan, often found in extreme environments. Many have cell walls containing pseudomurein, a polysaccharide similar to peptidoglycan but containing N-acetylglucosamine and N-acetyltalosaminuronic acid

Species and strain specific

Structure of capsules ; slime layers

• Species specific
• Polysaccharide or polypeptide layer outside cell wall May be tightly or loosely bound tightly bound is a capsule, loosely called slime layer.
• Detected by negative staining techniques.
• Prepare heat fixed smear, one stain for the bacteria followed by another that stains the glass.
• Halo around the cell represents the capsule.

Structure of S-layers

• Found on surfaces of some bacteria and archaea
• Protein layer on exterior of cell
• Regular ;floor tile; pattern
• Function not clear -- Stability?

Functions of capsules & slime layers

• Attachment
• Resistance to desiccation
• Nutrient Storage
• Evasion of phagocytosis e.g. in Streptococcus pneumoniae
• S strain (wild type) is encapsulated & virulent
• R strain (R for rough) is non-encapsulated & non-virulent experiments on R-type helped to identify DNA as the genetic material.

Short, hair-like filaments of protein on certain bacterial cells

Believed to function in attachment In a few species, specialized pili (sex pili, encoded by genes on the F plasmid. F pili are responsible for conjugation, F+ E. coli have F pili) enable the transfer of DNA from one cell to another (conjugation)

Motility

Almost all motile bacteria are motile by means of flagella

Motile vs. nonmotile bacteria

• Detected by flagella staining or by motility agar using an inoculating needle.
• If non-motile, growth will only occur on the surface of the inoculation orifice.
• Different species have different flagella arrangements (Monotrichous, lophotrichous, amphitrichous, peritrichous)

Bacterial flagella are completely different in structure and mechanism than eukaryotic flagella. Filament Composed of the protein flagellin.

Hook & Rotor Assembly Permits rotational "spinning" movement.

Protein shaft mounted in a protein bushing.

True wheel and axle movement, possibly the only example in the natural world.

Energy comes from a H ion gradient across the plasma membrane via cellular respiration down the e- transport chain. See fig on slide 7. Know fig. on slide 6.

“Run and Tumble” Movement controlled by the direction of the flagellar spin

• Counterclockwise spin = Straight Run
• Clockwise spin = Random
• Tumble Direction of spin seems to be determined by a molecular timer.

Response to the concentration of chemical attractants (nutrient molecules) and repellants

As a bacterium approaches an attractant: the lengths of the straight runs increase

As a bacterium approaches a repellant (nitrogenous waste products): the lengths of the straight runs decrease

Mechanism of chemotaxis: Stimulation of chemotactic receptors in the PM: this triggers a “cascade” of enzymatic activity that alters the timer setting of the flagella rotors

To permit the organism to survive during conditions of desiccation, nutrient depletion, and waste buildup

Bacterial spores are NOT a reproductive structure, like plant or fungal spores.

Some of the most resistant and durable structures in biology.

Produced by very few genera of bacteria

Major examples

• Bacillus (can represent a descriptive term of rod shaped bacteria, or a genus name, genus name is italicized and capitalized)
• Clostridium (botulism and blah)
• Both are very common spoilage bacteria.

Spores are resistant to killing

Cannot be killed by moist heat at 100°C (boiling) Killing spores by moist heat requires heating to 120°C for 15-20 min (autoclaving or pressure cooking)

see fig on slide 43. The process of spore formation

• Governed by genetic mechanism
• A copy of the bacterial chromosome is surrounded by a thick, durable spore coat
• This forms an endospore within a vegetative cell
• When the vegetative cell dies and ruptures, the free spore is released

When a spore encounters favorable growth conditions The spore coat ruptures and a new vegetative cell is formed

Have: complex internal  membrane system compartmentalization 

membrane-enclosed organelles

DNA is enclosed in a membrane-bound nucleus Includes: animal & plant cells, fungi, & protists (protozoa & algae)

Thousands are located suspended in the cytoplasm and attached to the rough endoplasmic reticulum

Major process: Protein synthesis (translation) Ribosomes in the eukaryotic cytoplasm are larger than prokaryotic ribosomes

Ribosomes are also found within mitochondria and chloroplasts; the ribosomes of these organelles are very similar in structure & size to prokaryotic ribosomes

Folded sacks of membranes within the cytoplasm Carry out processing and export of the cell’s proteins

Major components:

• Endoplasmic reticulum (rough and smooth)
• Golgi apparatus
• Transport vesicles
• Lysosomes

Located in the cell’s cytoplasm

Major process: cellular respiration

The mitochondria oxidize nutrient molecules with the help of oxygen

Some of the energy is conserved in the form of chemical energy (energy-containing chemical compounds) that can be used for biological processes

Evolved from bacteria by a process called endosymbiosis

Located in the cytoplasm of plant cells, algae cells, and certain protozoan cells

Major process: photosynthesis

Using the energy from light, CO2 is converted into carbohydrates such as glucose

Evolved from bacteria by endosymbiosis

maintain structure and shape, and allow for cytoplasmic streaming.

Microfilaments – actin monomers, involved in muscle contractions.

Microtubules – tubulin monomers, involved in intracellular transport.

Intermediate filaments – Keratin for ex.

Noncellular Biological Entity Contains either DNA or RNA (not both)

Nucleic Acid is surrounded or coated by a protein shell (capsid)

Some viruses possess a membrane-like envelope surrounding the particle

No independent metabolism or replication Replicate only inside an infected host cell

Do not replicate via a process of cell division Replicate via a process of:

Attachment and Penetration

Disassembly (uncoating)

Synthesis of Viral Protein and Nucleic Acid Reassembly of new viral particles

Release of new viral particles

Phototroph Uses light as an energy source Chemotroph Uses energy from the oxidation of reduced chemical compounds

Organotroph Uses reduced organic compounds as a source for reduction potential

Lithotroph – ex. Thiobacillis – reduces sulfur to sulfuric acid. Uses reduced inorganic compounds as a source for reduction potential.

Amino acids

Nucleotide bases

Enzymatic cofactors or “vitamins”

Some bacteria are able to manufacture all of their own essential biomolecules with only simple carbohydrates as a nutrient source. Thiobacillis is able to do this with only CO2.

Movement of substances directly across a phospholipid bilayer, with no need for a transport protein

Movement from high  low concentration

No energy expenditure (e.g. ATP) from cell

Small uncharged molecules may be transported via this process, e.g. H2O, O2, CO2

Movement of substances across a membrane with the assistance of a transport protein Movement from high  low concentration

No energy expenditure (e.g. ATP) from cell

Two mechanisms: Channel & Carrier Proteins

Movement of substances across a membrane with the assistance of a transport protein Movement from low  high concentration

Energy expenditure (e.g. ATP or ion gradients) from cell

Active transport pumps are usually carrier proteins

The target binds to a soluble cassette protein (in periplasm of gram-negative bacterium, or located bound to outer leaflet of plasma membrane in gram-positive bacterium).

The target-cassette complex then binds to an integral membrane ATPase pump that transports the target across the plasma membrane.

Transport of one substance from a low  high concentration as another substance is simultaneously transported from high  low. For example: lactose permease in E. coli: As hydrogen ions are moved from a high concentration outside  low concentration inside, lactose is moved from a low concentration outside high concentration inside

Low molecular weight organic molecules that are secreted by bacteria to bind to ferric iron (Fe3+); necessary due to low solubility of iron;

Fe3+- siderophore complex is then transported via ABC transporter

A medium to which has been added a gelling agent

Agar (most commonly used) doesn’t melt until it reaches 100o C

Gelatin – is easily broken down by bacteria.

Silica gel (used when a non-organic gelling agent is required)

Lag stage

Logarithmic (exponential) growth

Stationary stage

Death stage

Calibrated “Petroff-Hausser counting chamber,” similar to hemacytometer, can be used

Generally very difficult for bacteria since cells tend to move in and out of counting field

Can be useful for organisms that can’t be cultured

Special stains (e.g. serological stains or stains for viable cells) can be used for specific purposes

Also know as “viable cell counts”

Concentrated samples are diluted by serial dilution

The diluted samples can be either plated by spread plating or by pour plating

Diluted samples are spread onto media in petri dishes and incubated

Colonies are counted.

The concentration of bacteria in the original sample is calculated (from plates with 25 – 250 colonies, from the FDA Bacteriological Analytical Manual).

A simple calculation, with a single plate falling into the statistically valid range, is given below:

Serial dilution (cont.) If there is more than one plate in the statistically valid range of 25 – 250 colonies, the viable cell count is determined by the following formula:

Where: C = Sum of all colonies on all plates between 25 - 250 n1= number of plates counted at dilution 1 (least diluted plate counted)

n2= number of plates counted at dilution 2 (dilution 2 = 0.1 of dilution 1)

d1= dilution factor of dilution 1

V= Volume plated per plate

Used for samples with low microbial concentration

A measured volume (usually 1 to 100 ml) of sample is filtered through a membrane filter (typically with a 0.45 μm pore size)

The filter is placed on a nutrient agar medium and incubated

Colonies grow on the filter and can be counted

Cells are removed from a broth culture by centrifugation and weighed to determine the “wet mass.”

The cells can be dried out and weighed to determine the “dry mass.”

is an open system in which fresh media is continuously added to the culture at a constant rate, and old broth is removed at the same rate. This method is accomplished in a device called a chemostat.

Typically, the concentration of cells will reach an equilibrium level that remains constant as long as the nutrient feed is maintained.

means that the organism can grow under the condition, but doesn’t require it

The term “facultative” is often applied to sub-optimal conditions

A facultative thermophile may grow in either elevated temperatures or lower temperatures

As the bacteria in the population grow, they secrete a quorum signaling molecule into the environment (for example, in many gram-negative bacteria the signal is an acyl homoserine lactone, HSL)

When the quorum signal reaches a high enough concentration, it triggers specific receptor proteins that usually act as transcriptional inducers, turning on quorum-sensitive genes

Tetrapeptide or pentapeptide chains attached to NAM may “crosslink” adjacent PG strands This gives PG (pentaglycine) a net-like or mesh-like structure that contains the cell wall.

Found in Gram-positive bacteria

TP chains of adjacent PG strands are linked by pentapeptide chains

Found in both Gm + and Gm - bacteria TP chains are directly attached to each other

Proteins always contain the L-alanine amino acids