-Alexander Fleming discovered Penicillin in 1928.

Antibiotics

-can be either bactericidal or bacteriostatic.

-Must target the cellular machinery of the pathogen but not the host.

-exploit differences between the structures of bacteria and eukaryotes.

-cell walls

-cell membranes

-DNA replication

-Transcription

-Translation

-inhibit the formation of peptidoglycan

-irreversibly bind to enzymes that form cross links

-have a beta lac-tam ring

-includes penicillin's and cephalosporins

-prevents peptidoglycan subunts from being transported across the inner membrane

-only used topically

-enters the membrane and forms a cation channel

-cannot be ingested, it also works on eukaryotic membranes

What is Rifamycin B?

-Blocks the initiation of transcription

-Binds to RNA polymerase

-Binds to subunits of the 30s ribosome.

-can prevent the 50s subunit from binding

-or it can cause mis translation

-Binds to the 30S ribosome

-Prevents binding tRNA  Asite of the ribosome.

-Binds to the 50s subunit of the ribosome.

Pulls tRNA out of the Psite while stopping peptidlytransferase from connecting the amino acid.

-binds to the peptidyltransferase of the 50s ribosome

-inhibits the formation of peptide bonds.

-an antibiotics effectiviness is organism dependent.

Steps:

1.Minimarl Inhabitory Concentration= lowest concentration of the drug that will prevent the growth of an organism.

2.Kriby Bauer Testing= filter paper with antibiotics are dispensed onto an agar plate.

1)mutate the target

2)Pump the antibiotic out

3)Degrade the antibiotic

4)Inactivate the antibiotic

What is the Antibiotic Eflux pump?

What is Beta lactamase?

-removes the drug from the cell as it diffuse in, usually with a proton gradient

-some pumps can remove mutiple drugs.

-Beta Lactamase= cleaves the Beta lac-tam ring of antibiotics

1)Evolution

2)Gene transfer

-Transformation(plasmids)

-Transduction(viruses)

-Conjugation(direct contact)

Prokaryotic

1)circular chromosome

2)580-9400kpb

3)little noncoding DNA

4)operons

Eukaryote

1)Linear Chromosome 2)2900-4,000,000

3)more noncoding DNA 4)exons and introns

-A cluster of genes cotranscribed from a comon promter

-related genes will tend to be on the same operon

lacz:B-Galactose -cleaves lactose into glucose and galactose.

Lacy:-Lactose Permease-transports lactose through the membrane

lacA:Thiolgalactoside-Transaceylase.

1)Radiation(gamma rays, Xrays, UV)

2)Chemicals(benzene, tobacco smoke)

3(Reactive O₂ species

4)DNA replication erros

*overall rate: about 1 in 10¹¹

Silent mutation causes no change in phenotype.

1)can result in the same amino acid

2)can occur in noncoding regions

3)can mutate in the promoter region with altering its strength.

What is a misense mutation?

What is a nonsense mutation?

What is inversion?

Misense mutations- change in an amino acid

Nonsense Mutation-change to a stop codon

Inversion=rotatation of amino sequences(literally sections of DNA flip sides)

What is insertion?

What is deletion?

What is a mutagen?

What are carcinogens?

-Insertion is addition of new bases 

Deletion= removal of bases

Mutagen=a chemical that causes an increase in mutation rates.

Carcinogen= a chemical that can cause cancer.

*not all mutagens are carcinogens, not all carcinogens are mutagens.

-uses bacteria to determine if a chemical compound is a mutagen

AmesTest:a strain of salmonella with a mutant histidine synthesis gene.

-this strain requres histidine to grow

-Salmonella are plated on a minimal media

-Some salmonella will revert to wild type

-adding a mutagen will increase the rate of reversion

-chemicals that cuase reversions in an ames test tend to cause cancer in rodents

*bacteria have a sinlge chromosome

Plasmids: small peices of circular DNA

-replicated independently

-range from 1 to 50 copies

-size of 2kpb to 50 kpb

-can be transfered between cells

1)Antibiotic resistance

2)Metabolic enzymes

3)Virulence

4)Toxins

- Small piece of DNA that can insert them selves into a chromosome.

-First discovered in corn in the 1930's by Barbara McClintok.

-Can lead inside of genes and incativate them

-can increase the frequency of mutations

-size range from .1kpb to 17 kpb

-has short inverted repeates at each end

-includes transposase

5-'AATCAT.......ATCGATT-2'(inverted repeats)

3'-TTAGCTA......TAGCTAA-5'

1)Transposase binds to the inverted repeat and target sequence.

2)The transposon attaches to the target

3)DNA pol1 and ligase fill the gap

4)the Transposon can jump out later

- when the transposon jumps out of a gene, the duplicated DNA will stil be there

-The duplication can either be three, five or nine base pairs.

-occasionally the transposon will remove the duplicated DNA as it jumps out

or

-it can perform aberrant excision, where it removes additional bases.(this can disrupt multiple genes)

Nonreplicative Transposition= The transposable element is moved

Replicative Transpostion- The transposable element is duplicated.

- Genes that confer antibiotic resistance can be found in transposons.

-Transposons can jump on and off plasmids

-this aids in the transfer of anitbiotics.

-some bacteria speices can import DNA ad plasmids from other dead bacteria

-Bacteria that cannot take up DNA can be artificially transformed in a laboratory enviorment.

What does calcium chloride do as far as artificial transformation?

1)Calcium cholride- cells are chilled in CaCl₂ , making the membrane more permeable. THe bacterai then heat schoked at 42 C.

2)Electroporation- cells are placed in an electric field which creates holes in the cell membrane and allows DNA to enter.

Fimbriea=-thin solid protein tubes

-Allow bacteria to adhere to surfaces

-grwos from the base of the cell

Pili(pilus)

-hollow tubes

-grow from the tip

-plasmid that is 95 kpb

- has genes for special type of pilus

-Sex pilus= transfers DNA between bacteria by forming a

conjugal bridge between bacteria.

-This usually occurs in groups of 50 or 100 cells, and each F+ has three to four sex pilli.

1) the sex pilus attaches the two cells.

2)It then contracts to draw them together

3)the plasmid is nicked at the origin

4)the 5' end enters and replication begins

5)both ends replicate and circularize

The F factor replicates slowly and can be lost during cell division.

-Viruses can infect bacteria when they interact through the sex pilus.

-An F factor plasmid can integrate into the bacterial chromosome by recombination

-Bacteria that have integrated an F factor are called HFR(high frequency recombination)

-HFR strains ca transfer part of their chromosomes into other cells

-Transferring the entire chromosome can take 100 min.

- The F factor in an HFR cell can be lost through recombination.

-Sometimes the F factor will take DNA from bacterial chromosome with it

-THe new F factor with DNA from both plasmid and bacterial chromosome is refered to as an F' factor

-Genes for antibiotic resistance can be transferred by F factor plasmids

-They can also be transfered by other plasmids during conjugation.

-and transposons wth antibiotic resistance can be on plasmids.

-Bacterial conjugation can occur between different species and genera.

-Microbes must monitor the status of both the cytoplasm and the environment in order to properly regulate expression.

-Regulation of gene expression primarly occurs at the level of transcription

1)copy number(plasmids)

2)Promoter sequence Aternation

3) Regulatory Proteins

4) Operons

5)Gene Inversion

6)Rnases

7) Translational Control 

8) Post translational control

-Specific -35 and -10 sequences can vary

-Mutation can make it easier or harder for RNA polymerase to bind to the promoter.

-Essential proteins will tend to ahve -35 and -10 sequences that alow better binding

-Some proteins can assist or inhibit the binding of the sigma factor to the promoter

-Positive regulators assits in binding sigmal factors to the promoter

(regulatory proteins)

What is an activator?

What is the operator?

What are positive regulators?

Activator: The DNA sequences positive regulators bind to

-Negative Regulators inhibit the binding of the sigma factor to the promoter.

Operators: The DNA sequences negative regulators bind to

-Genes for regulators can be located nearby or far away from their targets.

 Positive Regulators: bind to an inducer ligand, followed by the activation sequence.

-Inhibit the binding of the sigma factor to the promoter.

1)induction: a negative regulator can be removed by an inudcer ligand?

2)depression: a negative regulator can form a complex with a corepressor ligand.

- a sensory relay protein(kinase) phosphorolates a response regulator in response to the enviorment. 

-The regulator binds and alters expression.

(Gene Inversion)

Phase variation?

Immune avoidance?

-certain components of the immune system are very specific for certain proteins.

Phase variatoin: alteratoin of proteins that compose surface structures.

Immune Avoidance: The use of phase variation to avoid antibodies the immune system.

What are steps of gene inversion?(Salmonella Enterica)

*Salmelle enterica alters the proteins in flagella

-usese recombination to flip flagellum gene over. Causing an inversion mutation turning it off. It can express H1 or H2 flagellin.

1) The promoter drives expression of FlijB(H2flagellum) and FlijA(reppressor)

2)Hin recombinase binds to the hix sequences and caues recombination

3)The promoter is inverted to the wrong orientation, and can no longer drive expression

4)H1 flaagellin is transcribed by fliC.

-Northern Blot(RNA)

-Western Blot(Protein)

-DNA Micro-array(RNA)

*Souther Blot is used to identify DNA

1) RNA is extracted from mutiple cells

2)It is seperated by size on a gel

3)RNA is transfered to membrane

4)The RNA o the membrane is hybridized with a DNA sequence for the gene.

5)RNA levels are determined based on intensity levels of bonds.

1)Proteins are extracted and run on a gel

2)They are transcribed to the membrane with electrical current.

3)The membrane is probed with antibodies

4)Protein levels can the be determined.

-It measures thousands of genes at once. Complimentary DNA is made from thousands of RNA's and printed on a chip. Each is pre attached to a fluorescent.

1) Make cDNA from RNA, or buy it.

2)Extract RNA and add it to the chip.

3) Fluorescence allows you to determine the relative expression levels.