BIMM 120 Final

What are the three interconnected functions of circadian clocks?

1. Timekeeping

2. Entrainment

3. Output signaling

What is timekeeping?

A cycle of slow biochemical processes that set the oscillation close to 24 hours (knowing differences between night and day)

What is entrainment?

The synchronization with their environment

What is output signaling?

Runs all the temporal information from the timekeeping apparatus to the rest of the cell

What is the transcription-translation feedback loops (TTFLs) in timekeeping?

Oscillation is achieved in cyanobacteria through delayed negative feedback, where a gene product represses its own expression when its levels become high enough, thus oscillating the expression level of the gene at crucial points in the oscillatory cycle

What are post-translational oscillators (PTOs) in cyanobacteria timekeeping?

Cyanobacteria can adapt to oscillations through conformational changes, protein-protein interactions, and or subcellular localization, these are going to change the gene products that are made

True or False: PTOs are able to link dynamic molecular processes to biological phenotypes they are able to operate a constant protein concentrations

True - PTOs are able to link dynamic molecular processes to biological phenotypes they are able to operate a constant protein concentrations

What are the three major protein gene products in timekeeping for cyanobacteria?

KaiA, KaiB, and KaiC

In the gene cluster, what happens if one stops functioning?

There will be no circadian clock

The core cyanobacterial clock is centered around the hexameric ATPase KaiC as it can ___

timekeep through phoshphorylation

During the day there is an accumulation of S/pT which is characterized by:

The binding of KaiA at CII promoting phosphorylation

During the night there is an accumulation of the pS/pT characterized by:

The formation of the KaiABC complex until phosphorylation reaches a minimum and KaiA is recruited back to CII

During the night, what does KaiB bind to?

KaiB binds to phosphorylated KaiC at the B-loops of the CI domain and the KaiA is recruited to the KaiB-CL complex becoming inactivated

What is KaiA?

The primary regulator of the CII domain of the KaiC (phosphorylation cycle)

What are the two ways KaiA can promote phosphorylation of CII?

1. Structural rearrangement of the CII catalytic cor

2. Exchange of active-site ADP molecules for ATP

What is KaiB?

The attenuator of KaiC phosphorylation

What are the three main enzymes of output signaling?

SasA, CikA, and RpaA

What’s an RR?

A response regulator

What’s an HK?

A sensor histidine kinase

What is RpaA?

An RR that governs transcription factors that mediate the global changes in gene expression associated with circadian rhythms

What is RpA regulated by?

Two cognate HK’s: SasA and CikA

How does SasA regulate RpA?

It recognizes and binds to phosphorylated KaiC, leading to some phosphorylation events to RpA leading the transcription factor ready for DNA binding

How does CikA regulate RpA?

It reverses the activation/phosphorylation of RpA done by SasA

What are the two ways cyanobacteria can have metabolic entrainment?

1. Through sensitivity of the phosphorylation cycle to ATP/ADP ratios within the cell. As the enzymatic sensitivity of the core oscillator is aligned to ATP/ADP ratios allowing for the alignment to the maximal photosynthetic period at midday (where the ratio is highest)

2. Through the presence of nighttime-associated photosynthetic metabolites. Quinone pools decrease during photosynthesis but the concentration of them increases rapidly but briefly when photosynthesis shuts down with the onset of darkness before other pathways restore the redox steady state leading to idea that the cell is in night mode

How do RR’s and HK’s work with each other?

HK may activate multiple RR’s or RR may be regulated by multiple HK’s

When does nitrogen fixation and photosynthesis occur?

Nitrogen fixation is during the night and phosphorylation during the day

What happens during the day with the KaiABC complex?

KaiA binds to KaiC in high concentrations - KaiA then phosphorylates KaiC

What happens during the night with the KaiABC complex?

KaiB inhibits KaiC through its dephosphorylation and antagonizes KaiA activities through competition of binding to KaiC

What is KaiC?

An auto-phophorylator, autokinase, and ATPase that works in a 24 hours cycle and has Ser/Thr residues

Do circadian rhythms respond to the environment?

No they are innate and endogenous

What is CikA?

An enzyme that is able to sense quinone.

What does CikA signal when quinone is high?

When quinone is high, CikA signals it is night time to other bacteria

How does quinone change in photosynthesis?

Quinone is high immediately after photosynthesis ends and it is low during photosynthesis

What are the other enzymes involved in entrainment?

CikA, LdpA, Pex, rPaA

What is LdpA?

An enzyme that is able to sense redox

How does LdpA act?

During the day when photosynthesis is high, LdpA is going to sense it, leading KaiA to be more active and bind to KaiC leading to phosphorylation

What is Pex?

An enzyme that is able to sense day and night

How does Pex act?

When it is night, Pex will sense it, leading to the inhibition of KaiA binding to KaiC, allowing KaiB to bind to KaiC leading to dephosphorylation

What is rPaA?

A response regulator of KaiABC

What does rPaA do?

It controls the gene expression and transcription factors of the KaiABC complex

How are the morning genes (CII) activated?

The CII genes are activated by unphosphorylated rPaA.

• throughout the day, SasA phosphorylates rPaA (slow) but it is most active during the sun fall where SasA phosphorylates rPaA accumulating the most active rPaA

How are the night genes (CI) activated?

The CI genes are activated by phosphorylated rPaA

What happens before dawn?

• KaiC has ps/t

• CikA dephosphorylates RpaA

What happens at dawn?

• Unphosphorylated RpaA makes/expresses KaiA

• KaiA phosphorylates CII

• S/T (both unphosphorylated)

What happens during the day?

• Ser unphosphorylated, Thr is phosphorylated (S/pt)

What happens during dusk?

• SasA phosphorylates RpaA. The phosphorylated RpaA makes KaiB

• KaiB dephosphorylates KaiA, which undergoes conformational change making KaiA detach (stop phosphorylating) CII

• CII becomes inactive

• The phosphorylated RpaA activates CI by having KaiB phosphorylate CI

• CI becomes active

• pS/pT (both phosphorylated)

What happens at night?

• KaiB is bound to CI [phosphorylating it] while KaiA is bound to KaiB [which is dephosphorylated]

• CikA dephosphorylates RpaA (towards the end of night)

• ps/T (Serine phos, Threonine unphos)

What are the two advantages of supercoiling?

1. Makes DNA more compact, DNA gyrase/topoisomerase II is going to be able to supercoil the DNA by adding coils

2. Puts a strain on the DNA duplex, helping separation of the strands in DNA for DNA replication and initiation of transcription

Why is topoisomerase II important?

(Gyrase) Uses ATP to generate negative supercoils of DNA - required for growth

Why is topoisomerase I important?

Relaxes the supercoiled DNA - not required

Which topoisomerase is important for bacterial survival?

Topoisomerase II

What are bacterial nucleoids?

Sort of like a nucleus but its not membrane bound in bacteria

Is condensin activity required for bacteria?

It depends on the species

What are bacterial nucleoids composed of?

The bacterial nucleoid is composed of the circular chromosome, nucleoid-associated proteins (HU, H-NS, Fis, IHF, etc.), RNA molecules, topoisomerases, and supercoiled DNA loops. These components compact and organize the DNA inside the cell.

What is initiation? Is highly regulated or not?

Initiation is when two strands of DNA separate from each other with an OriC. It is highly regulated

What is elongation? Is highly regulated or not?

Making DNA longer. it is not highly regulated

Is termination highly regulated or not?

Termination is highly regulated

What is membrane invagination?

The process of the membrane folding inward to internalize substances

Why are plasma invaginations (ICM) important in bacteria?

It is believed that is how most bacterial organelles came to be. In E. coli and other bacteria, the overproduction of integral membrane proteins lead to the creation of intracellular membrane vesicles (ICM) that help create those invaginations of the plasma membrane - membrane-bound organelles were created

What are chromatophores?

Intracellular pigmented membrane structures

What function do chromatophores have?

They are capable of catalyzing light-droven reactions including pmf driven ATP synthesis/photophosphorylation

What are magnetosomes?

Magnetotactic bacteria

What function do magnetosomes have?

They allow organisms to align to Earth’s magnetic field

Where is magnetite found?

In aerobic bacteria

Where is gregite found?

In anaerobic bacteria

What are anammoxomes?

They are energy producing, mitochondrion-like organelles

What function do anammoxomes have?

They do energy metabolism of anaerobic ammonium oxidizing bacteria and conserve energy by generating a pmf

Why is the ladderane in anammoxosomes important?

Ladderanes are unusually rigid lipids and anammoxosomes have them because of their inflexible ladder-like structures. The ladderanes contribute to energy conservation by creating a more H+ leak-proof membrane and for the protection from the toxic hydrazine

What are acidocalcisomes?

Calcium/polyphosphate-rich acidic membrane-enclosed organelles found in all three domains of living organisms

What is the function of acidocalcisomes?

Functions include storage of cation and polyphosphates, osmo, pH, and Ca2+ homeostasis, and energy metabolism

What are OMVs?

Outer membrane vesicles

Why do bacteria have OMVs?

OMVs allow for:

• molecular communication

• Delivery of toxins to eukaryotic cells

• Protein and DNA transfer between bacterial cells

• Trafficking of cell to cell signals to other bacteria and hosts (quorum sensing)

• Delivery of proteases and antibiotics to host cells

• Removal of harmful incorrectly folded proteins

• Matrix vesicle production can be for biofilm production

What happens when bacteria are highly stressed and what does this correlate with OMVs?

Bacteria get stressed when protein folding is impaired. The release of OMVs is then directly correlated with the amount of accumulated stress.

What is quorum sensing?

Its a means of communication between bacteria

Why is quorum sensing important for bacterial communities?

It provides bacterial communication that can lead to differences in gene expression that help the bacterial community usually the production of energetically costly public goods

What processes can QS drive?

Bet-hedging, division of labor, phenotypic heterogeneity

What are the differences between bet-hedging and division of labor?

• Bet-hedging: Allows clonal bacterial population to optimally adapt to consecutive, rapid and frequency fluctuations in environmental conditions without the need to respond to external signals

• Division of labor: Individuals in a clonal population interact with each other and simultaneously express distinct, often complementary, traits

What are bet-hedging and division of labor caused by?

Phenotypic variation

What are indoles?

A QS sensing molecule to decrease motility, biofilm formation, adherence to host cells, and expression virulence related genes

How do indoles alter the bacterial community?

By helping coordinate behaviors within the community

How do indoles affect biofilm formation?

Adding indoles decreases biofilm formation

What QS signals do gram-negative bacteria release?

Acyl-homoserine lactones (Acyl-HSLs)

What QS signals do gram-positive bacteria release?

AI2

What are ways bacteria can degrade QS signals?

With chemical hydrolysis:

• use lactonases to degrade the lactone ring in AHL making QS defective

• use acylases to remove the acyl group of AHL, making QS defective)

What two morphologies do Caulobacter crescentus have?

1. Sessile (non-moving) stalk cell

2. Motile swarm cell

What are stalk cells?

Cells that have uncondensed DNA making them able to replicate, remains at the site of replication

What are swarm cells?

Cells that have condensed DNA making them unable to replicate

What is TipN and its importance?

• TipN is the tip of the new pole.The protein that localizes to the cell division site and is the morphogenetic protein

• Allows for morphogenesis (changes in morphology)

• Without TipN, polarity is lost

What is TipF and its importance?

• TipF is a catalytic protein that interacts with TipN

• Mutants without TipF cannot make flagelli

What happens with high concentrations of TipN?

An increased number of new poles occurs

What are actin nucleating proteins?

Proteins that direct motility through an animal cell or across cell barriers

How do actin nucleating proteins mediate movement of the bacterial cells?

By being displayed on the bacteria cell surface and inducing actin assembly. This then allows to build acting tails at the bacterial pole which propels the bacteria across cell boundaries

What are actin nucleating proteins dependent on?

The energy of the cell not the bacterium

What is sporulation?

The process by which certain bacteria form an endospore — a tough, dormant, and highly resistant cell type that allows survival during extreme stress.

Why do cells undergo the process of sporulation?

Endospores are utilized by bacteria as a means of survival especially in isogenetic communities

What are the stages of sporulation?

0. Vegetative growth

1. Chromosome condensation (axial filament formation)

2. Asymmetric septation

3. Engulfment

4. Cortex Formation (deposited inside membrane)

5. Coat formation (deposited outside membrane)

6. Maturation (resistance to environmental stresses develops to completion)

7. Lysis (mother cell death): release of the mature spore

What occurs in the stages of sporulation: Stages 0 and 1

Pre-septation

What occurs in the stages of sporulation: Stage 2

Asymmetric septation - spore divides

What occurs in the stages of sporulation: Stage 3

When sporulation occurs, naked forespores are present, mother cell engulfs forespore to protect it/provide nutrients

What occurs in the stages of sporulation: Stage 5-6

Coat formation/maturation - Mother cell turns into a coat which matures into a proper coat