01/21- Lecture 3 Bacterial Cell Cycle

How is bacterial growth measured

growth of populations not individual cells

What are the phases of the lifecycle of a population

Lag, exponential, stationary, death

Lag phase

if a fresh culture is inoculated with cells from an older or stationary phase culture, the cells need time to resynthesize essential components before beginning growth again

What happens is cells are inoculated from exponential phase culture

there is no lag

Exponential phase

Population doubles in mass per unit time

How is exponential growth measured

measured by OD and plotted on a semi log scale

Stationary phase

the period during which growth slows dramatically, and cells are growth arrested

When does stationary phase occur

Cells run out of nutrients AND/OR waste product builds up

Death phase

The point at reach nutrients become so limited or toxins accumulate to a point where cells begin to die

Bacterial cell cycle

1. Initiation of replication

2. Elongation and origin separation

3. Assembly of FtsZ ring

4. Nucleoid segregation + termination

5. Cell division

Single fork replication

Replication begins at oriC and proceeds bidirectionally towards terminus

Multifork replication

New round of DNA replication starts before the previous one finishes

What is the main difference between single fork and multifork replication

multifork replication occurs in faster growing cells

What initiates DNA replication

Topoisomerases nick and unwind DNA near oriC

What happens after topoisomerase unwinds DNA

DnaA binds to origin and melts two DNA strands- rate limiting component for initiating DNA replication

What happens after DnaA activitiy

Open complex formation- helicase and single-strand binding protein (SBB)

What happens after open complex formation

RNA primer formation in replisome (complex of ~20 proteins one of which is primase- RNA polymerase)

What happens after the RNA primer forms in the replisome

Elongation- DNA polymerase III polymerizes nucleotides in the 5’-3’ direction

Gyrase/topoisomerase

Unwinds DNA, cuts and reseals it to relieve supercoils

Helicase

Unwinds DNA in front of the replication fork. Doesn’t cut DNA

SSB

Single-stranded binding protein. Keeps single stranded DNA at replication fork from reannealing with complementary DNA

Primase

An RNA polymerase that uses a DNA template to lay down RNA primer

DNA polymerase III

the replicative polymerase, has proofreading ability meaning it can “back up” and remove the incorrect nucleotide and then resume replication

DNA polymerase I

responsible for removing RNA primers and replacing them with DNA

DNA polymerase I exonuclease activity

5’ to 3’ exonuclease activity to remove RNA primers

DNA polymerase I polymerase activity

5’ to 3’ polymerase activity to fill gaps left by the removal of the primer

The sliding clamp

beta subunit of DNA Pol III that increases the rate and processivity of DNA replication:

with clamp: 1000 bp/sec

without clamp: 10 bp/sec

Processivity of DNA polymerase

Processivity of DNA polymerase enzymes refers to their ability to add many hundreds or thousands of nucleotides to a growing chain without dissociating from the template

Genetic approach to identifying replication proteins

Isolate conditional mutants that are temperature sensitive (Cells that grow at 30°C but not 42 °C)

Quick stop mutants

replication ceases immediately upon a shift to the restrictive temperature

Slow stop

after shifting to the restrictive temperature, replication continues but cells do not initiate another round of replication

Fractionate whole cell lysate

1. Grow E. coli

2. Break cells open to make lysate

3. Fractionate lysate to isolate different proteins from one another

4. Test fractions for DNA polymerase activity using biochemical assay: incorporate radioactive building blocks into DNA chains

Who discovered mechanisms in the biological synthesis of ribonucleic acid and deoxyribonucleic acid

Arthur Kornberg and Severo Ochoa

How GFP was used to find if DNA moved through polymerase

Fusion of pol III to a gene for the green fluorescent protein (GFP)- pol III with GFP fluoresces when 500 nm light and showed where Pol III localized during the cell cycle

Where was GFP isolated from

jellyfish Aequorea victoria

How do you prove that the foci of GFP represent active polymerase

Using slow growing cells you show how the polymerases don’t move relative to the DNA

Factory Model of DNA polymerase

DNA polymerase remains generally stationary and the DNA moves through the replication machinery

Steps in chromosome separation in bacteria

Origin separation and chromosome separation

Chromosome separation

1. Topoisomerases unlink chromosome dimers

2. SMC and HU proteins help keep DNA condensed and thus easier to separate

What do mutations in bacterial smc to do

Mutations in bacterial smc lead to decondensed chromosomes, and guillotining (when the septum bisects the nucleoid)

How did we visualize the origin of replication in live cells

tagged a region of the B. subtilis chromosome with a tandem array of lac operator cassettes and expressed a version of the Lac repressor (LacI) fused to GFP

What drives chromosome separation in bacteria if they don’t have any obvious cytoskeletal machinery

we dont know

Fts mutants

filamentous temperature sensitive mutatns- long mutants

FtsZ protein definition

essential cell division protein conserved in bacteria, archaea, and plants

FtsZ protein type

GTPase with similar crystal structure to tubulin

FtsZ protein function

Assembles into a ring at the future division site

FtsZ importance

establishes the location of the division site, required for localization of other Fts proteins to the septum

What is the one common requirement for all microbes to colonize an environment

water

What is a primary determinant of growth rate

Nutrient quality

Average vs single cell growth rate nutrient quality impact

Nutrient quality impacts average cell growth rate. Single cell growth rate is widely distributed

Nonhalophile

microbe that thrives in low-salt environments

Halotolerant

can survive and grow in high-salt environments but don't require salt for survival

Halophile

microbe that grows in saline conditions

Extreme Halophile

microbe that thrives in extremely salty environments

Psychrophile

like temps <15°C

Mesophile

like moderate temps (15-45°C)

Thermophile

like 60-80°C

Hyperthermophile

(>80°C) like high temperatures

Why are low temperatures challenging

Protein activity slows, and hydrophobic interactions weaken, causing conformational changes in proteins

Why are high temps challenging

Proteins and other macromolecules denature

How do microbes increase protein thermostability

Modifications to tertiary structure: hydrogen bonding, hydrophobic internal packing, salt bridges

Increase the number of charged amino acids, like glutamic acid and arginine

How do chaperone proteins help microbes survive high temperatures

help hold proteins 3D structutres together

Reverse gyrase

introduces positive supercoils to stabilize DNA

What are most thermophiles

Archaea

What do most thermophiles not have

phospholipid bilayers for cell membrane

Extreme environments

Temperature, pH, osmolarity, oxygen, and pressure