bisc exam 3

binary fission

-Most bacteria and archaea divide by binary fission.

A parent cell divides into two daughter cells of equal size

-growth and DNA replication precede formation of a division septum, followed by cell wall synthesis and cell separation (septation).

Cells typically grow by elongation Chromosomal DNA is replicated

Cells replicate DNA while they grow. Division is typically triggered once the cell hits a critical size

FtsZ

-a cytoskeletal protein that forms a structure called the Z ring

-Z ring is anchored to the plasma membrane, at the division septum.

- The Z ring contracts, pinches the cell envelope inward

FtsZ protein –

is a tubulin homologue – GTPase – breaks down GTP – polymerization forms Z ring – orients peptidoglycan synthesis

Cell division requires

breaking down the existing cell wall, and synthesizing new cell wall material.

autolysin

enzymes break down existing peptidoglycan

penicillin binding proteins

crosslink strands during synthesis and can break existing

crosslink

Penicillin Mechanism:

• Penicillin inhibits PBPs and prevents peptidoglycan crosslinking; actively growing bacteria are especially susceptible because their expanding walls weaken and may lyse.

transpeptidase

– links peptidoglycan strands during peptidoglycan synthesis

Generation Time / Doubling Time:

• The time for a bacterial population to double; under optimal conditions growth is exponential (e.g., E. coli ~20 minutes; rates vary greatly by species).

Exponential growth

N = N₀ × 2ⁿ, where N₀ is starting cell number and n is number of generations (n = elapsed time ÷ doubling time).

Growth phase-log/exponential phase

FASTEST DIVISION

Abundant nutrients, little competition. Cells grow at maximum rate.

Growth phase-lag phase

ADAPTATION

cells sensing nutrients, preparing to grow.

Growth phase-stationary phase

NUTRIENT DEPLETED/WASTE BUILDUP

Nutrients depleted, toxins accumulate. Culture may become acidic and hypoxic.

Growth phase-decline phase

Cells die off due to starvation or toxicity.

• Growth Graphs:

• Exponential population growth looks curved on a linear Y-axis but forms a straight line on a logarithmic Y-axis.

Bacterial growth in an open system = chemostat

An open system = growth environment where matter is continuously exchanged with the outside.

Viability stains

• Membrane-permeant green stain labels both live and dead cells; red stain enters cells with damaged membranes, identifying dead cells.

• differentiate between viable and dead cells

Fluorescence staining

• The primary green stain - penetrates intact cytoplasmic membranes, staining both live and dead cells.

• The secondary red stain - can stain only if the cytoplasmic membrane is damaged; detects dead cells

• Live cells are green since they only absorb the green stain, dead cells are red since the red stain displaces the green stain on their nucleic acids

Most Probable Number (MPN)

• Statistical estimate of viable organisms when samples are too dilute for reliable plate counts (e.g., coliform contamination in water).

• Turbidity / Optical Density:

• A spectrophotometer measures culture cloudiness (often OD₆₀₀) as an indirect proxy for cell density; quick, but it cannot distinguish live from dead cells.

BIOFILM lifecycle

Attachment → irreversible colonization → growth → EPS matrix and water channels → dispersal

Cells in biofilm

produce a sticky material aka the matrix containing Extracellular Polymeric Substances (proteins, DNA, lipids)

Cells in the biofilm are less susceptible to antibiotics:

The EPS slows the diffusion of antibiotics and antiseptics, preventing them from reaching cells

-biofilms are commonly associated with

medical implants

planktonic

Cells that float freely in liquid culture

-When they attach to a surface they become sessile

• Sessile bacteria can form complex biofilm structures

Sessile Growth

• attached cells are sessile and may form a biofilm in a self-produced extracellular polymeric substance (EPS) matrix.

How Microbes Talk: Quorum Sensing

-Microbes use chemical signals to communicate with one another

-Communication that senses population density

-cells secrete autoinducers (small signaling molecules) trigger coordinated gene expression

Obligate aerobes

– absolutely require abundant oxygen

Obligate anaerobes

– cannot survive in the presence of oxygen (o2 harmful)

-bottom of tube only

Facultative anaerobes

prefer O₂ but grow without it

-mostly at top but some dispersed down

Aerotolerant anaerobes

– tolerate but do not use O2

-dispersed everywhere in tube

Microaerophiles

– require low oxygen levels

-at top of tube only

• pH Preferences:

• Acidophiles grow best at low pH,

• neutrophiles near neutral pH,

• alkaliphiles at high pH

temperature Preferences:

psychrophiles-10 c

mesophiles-40c

thermophiles-70c

hyperthermophiles-95c

DNA serves two essential functions

DNA stores hereditary information and directs protein synthesis

Central dogma states that information flows:

• DNA → messenger RNA → protein

genotype

full collection of genes of an organism

phenotype

= observable characteristics resulting from those genes

Meselson–Stahl experiment

• ¹⁵N-labeled E. coli DNA shifted to ¹⁴N medium was separated by density

• the findings support semiconservative replication: each daughter duplex contains one parental strand and one new strand.

DNA Replication

-replicated by a semiconservative mechanism.

-The cell uses each parental strand as a template for a new strand.

-Base pairing between nucleotides allows one strand to serve as a template.

DNA polymerases

synthesize the complementary DNA strand, using the existing strand as a template – guided by base pairing

• DNA pol III is the major replication enzyme

• DNA pol I and DNA pol II participate in repair, and pol I also replaces RNA primers with DNA during replication.

DNA polymerase requires:

-a template – guides synthesis of complementary strand

-a primer – short starting DNA or RNA strand

-dNTPs (dATP, dTTP, dCTP, dGTP)

All DNA polymerases synthesize DNA

in the 5’ to 3’ direction

Replication in Bacteria

Bacterial DNA is circular and supercoiled.

Begins at an origin of replication and finishes at a termination region.

Replication in bacteria is

bidirectional

• proceeds in both directions from origin

DNA is actively unwound at a

replication fork

• two forks: one in each direction

Replication Fork Machinery:

Helicase unwinds DNA

single-strand binding proteins stabilize open strands

sliding clamp keeps DNA pol III attached.

Replication ends at the

termination site.

• Leading vs. Lagging Strand:

• Leading-strand synthesis is continuous in the direction of fork movement

• lagging-strand synthesis is discontinuous, forming Okazaki fragments.

Transcription involves

RNA polymerase copies DNA information into RNA (mRNA, tRNA, rRNA, and other RNAs). mRNA carries information later used for protein synthesis.

The sigma factor

-forms a sort of bridge between the RNA polymerase and DNA.

-Sigma factors are a critical layer of transcriptional regulation

-Conserved −35 and −10 promoter sequences are recognized by a sigma factor, which guides the RNA polymerase core enzyme;

During transcription, RNA polymerase reads the template strand

in 3’ → 5’ direction, and synthesizes a complimentary mRNA from 5’ → 3’

genetic code

• represents a sequence of amino acids as a string of nucleotides

• codons are three-nucleotides in length

• each codon encodes an amino acid or stop signal

how many codons are there

64 codons (= 43 )

61 codons encode amino acids – sense codons

AUG encodes

methionine

– also usually the first codon in the coding sequence (the initiator)

• Degeneracy vs. Wobble:

• Degeneracy means several codons encode the same amino acid

• wobble allows one tRNA to recognize multiple codons, often differing at the third codon position.

Ribosome

-huge complex of rRNA and protein

-distinct large and small subunits

-Bacterial 70S = 30S + 50S

Transfer RNA

ribosome works together with tRNA to translate mRNA

-have a folded 3-dimensional structure

• anticodon at one end,

• opposite end of tRNA binds amino acid

-delivers amino acids

Translation

Translation has three stages:

• initiation

• elongation

• termination

• A, P, and E Sites:

• A accepts aminoacyl-tRNA

• P holds the growing peptide

• E is the exit site.

• tRNAs cycle through the sites A → P → E.

In prokaryotes, transcription and translation occur

simultaneously in the cytoplasm.

In eukaryotes, transcription occurs in the

nucleus and translation occurs in the cytoplasm,

Mutations

-Mutations in the DNA sequence can alter proteins or expression patterns.

-Mutations can occur spontaneously or induced by mutagens

Types of Mutations-Point mutation

- one base is substituted or replaced by another

Types of Mutations-insertion

- addition of one or more bases

Types of Mutations-Deletion

- removal of one or more bases

Missense mutation

results in a different amino acid incorporated into the polypeptide.

Silent mutation

(same amino acid),

Conditional mutation

- a missense mutation may only be apparent under certain environmental conditions; eg, temperature sensitive mutations.

Nonsense mutation

– changes a codon encoding an amino acid (a sense codon) into a stop codon

-results in a truncated, non-functional protein.

Frameshift mutation

an insertion or deletion of base pairs that change the coding frame

• Replica Plating & Auxotrophs:

• Colonies transferred onto media with different nutrients reveal auxotrophs that cannot synthesize a required compound (e.g., an amino acid).

The Ames Test

- Measure the rate of mutations to test if compounds cause mutations - carcinogenic potential

• Uses a histidine-requiring Salmonella mutant; an increased number of revertant colonies growing without histidine indicates that a test compound is mutagenic.

ways that genetic variation can develop

Point mutations and vertical gene transfer

 Vertical gene transfer

passes DNA to daughter cells

Horizontal Gene Transfer

• genes transfer from one organism to another

• may transfer between same or different species

• involves acquisition of foreign DNA

Three mechanisms of HGT:Transformation

naked DNA is taken up from the environment

Three mechanisms of HGT: Transduction

bacteriophage-mediated transfer

Three mechanisms of HGT: Conjugation

• direct cell-to-cell DNA transfer.

-Closest analog to bacterial sex.

generalized transduction

within the lytic cycle

• During phage assembly, a phage head may accidentally package any random fragment of bacterial chromosomal DNA and deliver it to another host.

Specialized Transduction

- is tied to the lysogenic cycle

- It is biased towards transferring host DNA flanking the phage DNA due to imprecise excision.

Operon Theory

Prokaryotic genes are often arranged into operons

• multiple functionally related structural genes

• driven by a single promoter

• translated as one mRNA

• polycistronic gene & transcript

Repressible Operon: trp operon

Genes allow synthesis of the amino acid tryptophan and it binds/activates the repressor

Inducible Operon: lac operon

-Inducible operons are only expressed when induced.

-The lac operon is only expressed when: glucose is low and lactose is high

•  Lactose-use genes are OFF without lactose because the repressor binds DNA; when lactose is present, a lactose derivative inactivates the repressor, permitting transcription.