04 - cells

what makes a good antimicrobial drug?

selective toxicity, specificity, low risk of resistance, few side effects, good pharmacological properties

why do certain antibiotics only work agains some species of bacteria? (ex: penicillin is not as effective against salmonella but works against streptococcus pyogenes)

differences in cell walls, outer membranes, or resistance enzymes affect whether the drug can reach and bind its target

what are the main parts of the eukaryotic cell?

nucleus, nucleolus, mitochondria, chloroplasts

what does the nucleus contain?

cell’s genetic material

what happens at the nucleolus?

where ribosomes are synthesized

what does the mitochondria do?

carry out “cellular respiration” to generate ATP

what does the mitochondria contain?

their own dna and 70S ribosomes

what happens at the chloroplasts?

photosynthesis takes place in plant cells

what is the role of the endomembrane system (secretory pathway)?

trafficking (moving) of proteins and membranes between organelles using vesicles

what are the two endoplasmic reticulums (ER)? what happens there?

smooth and rough; its where membrane proteins or secreted proteins are synthesized

what happens at the golgi apparatus?

where the membrane or secreted proteins are chemically modified and sorted either to the lysosome or the cell surface

what does the lysosome contain?

contains low pH and degradative enzymes to destroy proteins and lipids

that is the role of the cytoskeleton?

important for the structure of the cell, for movement, for moving vesicles in the endomembrane system and moving chromosomes during cell division; microtubules and microfilaments

what is the cell wall?

rigis polysaccharide surrounding the plasma membrane of plants (contains cellulose) or fungi cells (contains chitin)

what is the flagella?

whip like structures made of tubulin that allows cells to swim; much larger than the flagella of prokaryotic cells

is the flagella of prokaryotes or eukaryotes larger?

eukaryotes

what is the cilia?

numerous, short hair like structures on the outside of cells that occur in groups; coordinated beating motion to help cells move to or move particles along the surface of the cell

what is the role of centrioles?

aid in chromosome movement during mitosis

what is the parts of a prokaryotic cell?

cell wall, nuceloid, cell membrane, polyribosomes, capsule, flagella, and pilli

what is the bacterial cell (cytoplasmic) membrane?

the boundary between the inside and the outside of the cell

what is the bacterial cell membrane made of?

a phospholipid bilayer that has lots of proteins (70% of its mass)

how is the bacterial cell membrane similar to eukaryotes?

performs many functions that, in eukaryotes, are carried out by organelles and the plasma membrane

what do polymyxins do?

disrupt the bacterial cell membrane

what are the two main types of solute transport across the cell membrane?

passive transport (no energy) and active transport (requires energy)

what is passive transport?

movement by diffusion without energy

what is active transport? what is its role?

transport that requires energy; mediates energy of almost all nutrients

where is the electron transport chain located in prokaryotes?

in the cell membrane

what type of lipids are found in bacterial cell membranes?

phospholipids with glycerol esters

what type of lipids are found in archaeal cell membranes?

glycerol ethers (isoprenoids attached via ether bonds)

do archaeal membranes always contain phosphate?

no; they may or may not be phospholipids

what unique structure can archaeal membranes form?

lipid monolayers with diglycerol tetraethers

do most bacteria have cell walls?

yes to protect their membranes

what are the two main functions of the bacterial cell wall?

determines bacterial shape and protects from osmotic stress

what is osmotic stress?

stress on a cell caused by water moving in or out due to differences in solute concentration

JUMP TO SLIDE 31

Q: What is a capsule or slime layer usually made of?

A: A layer of slime usually made of polysaccharide.

Q: Give an example of a capsule/slime layer used as a food additive and thickening agent.

A: Xanthan gum.

Q: What is one function of the capsule/slime layer related to water?

A: It retains water and protects the cell from drying.

Q: How does the capsule/slime layer help bacteria attach?

A: It helps the cell adhere to surfaces.

Q: What does an “antiphagocytic capsule” do?

A: It protects bacteria from phagocytosis.

Q: How does the capsule of anthrax bacteria help them survive in the body?

A: The capsule prevents the bacteria from being phagocytosed efficiently.

what are the three types of flagella?

single polar (monotrichous), multiple polar (lophotrichous), and peritrichous

Q: What is the filament of a bacterial flagellum made of, and how does it assemble?

A: It is a helical, hollow structure of flagellin proteins that assemble spontaneously.

Q: How are archaeal flagella different from bacterial flagella in structure?

A: Archaeal flagella are structurally similar to bacterial pili.

Q: Despite structural differences, how do archaeal flagella function like bacterial flagella?

A: Archaeal flagella rotate like bacterial flagella.

Q: What is convergent evolution

A: an evolutionary concept explains the similarity in function between archaeal and bacterial flagella

Q: What are pili made of?

A: Tiny, hollow projections made of pilin proteins.

Q: What does conjugation pili do?

type of pili are used for DNA transfer between cells

Q: What are fimbriae?

A: A type of pili involved in attachment.

Q: How do attachment pili work?

A: They use adhesins at the tips or sides to attach to surfaces.

Q: What type of motility involves polymerization and depolymerization at the pilus base?

A: Twitching motility.

Q: In which group of bacteria are pili common?

gram negative bacteria

Q: Are pili common in Gram-positive bacteria?

A: No, they are uncommon in Gram positives.

in class question: given that it takes 40 minutes for E. Coli to replicate its genome, is it possible for E. coli ells to divide every 30 minutes? why or why not?

LOOK AT IMAGE 1: even though a single replication cycle takes 40 minutes, the coordination of overlapping replication cycles allows E. coli to maintain a division time of 20–30 minutes under optimal conditions (you have a round of dna replication starting before the last one ended with more replication forks)

Q: Where do prokaryotes keep their DNA?

in the nucleoid (not nucleus)

Q: Do prokaryotes undergo mitosis? Why or why not?

no because they don’t have a nucleus

Q: What is the typical structure of a prokaryotic genome?

a singular circular chromosome (not the same thing as a plasmid)

what are plasmids?

A: Small DNA circles, separate from the chromosome, that carry non-essential genes.

Q: How long is the DNA in the E. coli genome compared to the cell length?

A: ~1000× the length of the cell.

Q: If E. coli DNA were the thickness of spaghetti, what would it equal?

A: About 200 platefuls of spaghetti.

Q: What is the state of the nucleoid in prokaryotes?

A: Highly supercoiled, condensed, and compacted.

Q: What enzyme adds negative supercoils to DNA in prokaryotes?

A: DNA gyrase (topoisomerase II).

Q: Which antibiotics inhibit DNA gyrase?

A: Quinolones such as ciprofloxacin and norfloxacin.

Q: Where do transcription and translation occur in prokaryotes?

A: At the nucleoid–cytoplasm interface.

what is rifampin?

antibiotic that inhibits RNA polymerase in prokaryotes

Q: which type of bacteria have more nucleoids per cell?

fast growing bacteria

Q: Why do fast-growing bacteria have more nucleoids?

A: Because more than one round of replication can occur at the same time.

Q: What can happen to a newborn E. coli cell during rapid growth?

A: It may receive a chromosome that is still undergoing replication.

Q: How does gene dosage vary in rapidly growing bacteria?

A: Genes near the origin of replication have higher dosage.

which of the following could directly determine a serotype (can be seen by the immune system)?: ribosomes, capsule. pili, dna polymerase, flagellin, lipopolysaccharide (LPS)

capsule, pili, flagellin, lipopolysaccharide (LPS)

where is lipopolysaccharide?

outer leaf lip in outer membrane in gram negative bacteria

what is a multifork replication?

overlapping rounds of replication in prokaryotes

what is the appearance of bacterial cytoplasm? what is the reason for its appearance?

granular because its packed with ribosomes, proteins, and rna

Q: What is the texture/consistency of the cytoplasm?

viscous

what is a membrane protein?

a protein that spans or sticks to cell membrane

what is a soluble protein?

protein that is not stuck to the cell membrane

Q: What is the size of the prokaryotic ribosome, and what are its subunits?

A: 70S, composed of 30S and 50S subunits.

Q: How does the size of prokaryotic ribosomes compare to eukaryotic ribosomes?

A: Prokaryotic = 70S, Eukaryotic = 80S.

Q: What is the 16S rRNA gene used for in microbiology?

A: Taxonomic studies (comparing sequences).

Q: What special feature links transcription and translation in prokaryotes?

they are occupied and occur simultaneously

Q: Why are bacterial ribosomes important medical targets?

A: They are a major target for antibiotics.

Q: Name some antibiotics that target bacterial ribosomes.

A: Aminoglycosides (streptomycin, kanamycin, neomycin, gentamicin, amikacin), tetracyclines, chloramphenicol, erythromycin.

Q: Why is determining the subcellular localization of a protein important?

A: It helps in understanding the protein’s function.

Q: What might a protein localized to the periplasm or cell membrane be involved in?

A: Transporting nutrients into the cell.

Q: What might a protein localized to the cell membrane be involved in?

A: The electron transport chain.

Q: What might a protein localized to the cytoplasm be involved in?

A: Transcription or replication.

Q: What are the four main possible locations for proteins in E. coli?

A: Cytoplasm, inner membrane (cell/cytoplasmic membrane), periplasm, outer membrane.

Q: Which three locations are usually the focus when distinguishing protein localization experimentally?

A: Cytoplasm, inner membrane, and periplasm.

what two parts come from centrifugation?

pellet and supernatant

what technique can you use to distinguish between soluble vs membrane protein?

cell lysis and high speed centrifugation

what is the process of cell lysis and high speed centrifugation?

prepare cell lysate (breaks open cell in buffer); centrifuge (pellet will be membrane fraction and supernatant will be soluble fraction); then test the soluble fraction and membrane fraction for your protein of interest (ie: use immunoblot)

what technique can you use to distinguish protein in the periplasm?

spheroplasting and centrifugation

what is the process for spheroplasting and centrifugation?

treat cells with EDTA and lysozyme to make spheroplasts (permeabilizes he outer membrane and digest the cell wall); centrifuge (periplasm fraction will be supernatant that contains periplasmic proteins and pellet will be spheroplast fraction that contains membranes and cytoplasm); then test the periplasm fraction and the spheroplast fraction for your protein of interest (ie: immunoblot)

Q: What is the first step in immunoblotting?

A: Load a gel with a sample of each fraction.

Q: How are proteins separated in immunoblotting? what is it separated by?

A: By electrophoresis through a gel and they are separated by size

Q: After electrophoresis, where are proteins transferred?

A: To a nitrocellulose blot.

Q: How are specific proteins visualized in immunoblotting?

A: Using labeled antibodies that bind to the proteins of interest.

Q: In an immunoblot after spheroplasting and centrifugation, what do T, S, and P stand for?

• T = Total (whole cell extract)

• S = Supernatant

• P = Pellet