Exam 4 Flashcards

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1
Q
  1. Draw the basic components of one and two-component signal transduction networks. Describe one
    example of a two-component system? Describe the input and output.
A
  • Caulobacter control of CtrA activity
  • Input: Cellular levels of cdG
    switches cckA from a phosphatase to a
    kinase
  • Output: Phosphorylated CtrA binds
    to the chromosome origin to repress DNA
    replication
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2
Q

Name one intracellular bacterial second messenger. What are some of the bacterial processes
controlled by this second messenger?

A

c-di-GMP: Biofilm formation, polysaccharide production, motility, cell cycle and cellular development,
virulence

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3
Q

Describe one reason why being small (like E. coli) is hard.

A

A bacterium as small as E. coli cannot coast since the mass of the cell is very small and the viscosity of
water in comparison is very large. If the flagellum stops rotating, the cell will only coast less the 0.1
angstroms. Also, cells are buffered about by Brownian motion, so they can’t sit still. They also cannot
swim in a straight line, due to the rotational Brownian motion. So, the cells will wander off about 90
degrees in 10s. This means the cells “forget” where they’ve been, so any measurement made about 10
seconds ago are meaningless… so the cell must respond within that time to play the chemotaxis game.

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4
Q

What is one way Caulobacter crescentus produces different levels of phosphorylated CtrA in the
swarmer cell and the stalked cell

A

a. In order to produce high levels of phosphorylated CtrA in the swarmer cell and low in the stalked
cell, C. crescentus controls the overall amount of CtrA by polarly localizes proteases that
degrade CtrA, to the pole of the stalked cell.
b. C. crescentus also polarly localizes the kinase and phosphatases. In the swarmer cell, the
kinase is localized to the pole to phosphorylate CtrA, while the phosphatase in the stalked cell

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5
Q

What are the major components of the biofilm? Why are each so important

A

Adhesins: Important for cellular attachment to surfaces and to other cells in the biofilm
Matrix: Important for biofilm maturation, production of 3D structuring of the biofilm, adhesion, andprotection
Polysaccharide degrading enzymes, proteases, DNases: All important for cellular detachment from the
biofilm

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6
Q

You want to determine how climate change is influencing the microbial communities in Terry
Trueblood. You’ve been collecting samples from the water over time. What are two questions
you have about these communities? What are some techniques you could use to interrogate
them?

A

Tons of possible questions! You could ask how the community composition changes with increasing in
water temperature by collecting water samples, isolating microbial DNA, and performing 16S
sequencing, this will give you genus level information and the relative abundance of the bacteria in your
sample. If you wanted to ask about the species or stain level diversity in the sample, then you could
perform metagenomics, which would also allow you to ask what the genetic potential of the population
is and also if the population is evolving with increasing water temperature

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7
Q

Describe ways that P. aeruginosa and V. cholerae type VI secretions systems differ.

A

V. cholerae expresses T6S constitutively, while P. aeruginosa senses attack from competitors and
increases T6S when attacked by another T6S, by sensing membrane perturbation. P. aeruginosa is
also thought to sense lysed kin cells and increase T6S in response.

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8
Q

What is the difference between one component and two component signal transduction?

A

A one component system has the input and output domains on a single protein. A two component system has one protein with an input domain that transfers the signal to a second protein with the output domain.

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9
Q

Example of a one componenent system

A

Arabinose utilization:
* Growth on two carbon sources
(here, glucose and arabinose)
* Bacteria will use the one that yields
the highest growth first – this is the
one it “prefers” (here, glucose)
* Once the preferred C source is
used it will adapt its metabolism to
use the next available C source
(here, arabinose)
* For optimal efficiency, bacteria only
express the genes needed to import
and utilize one C source at a time.

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10
Q

AraC regulation summary

A
  • Signal: Arabinose
  • Input: Dimerization and Arabinose Binding Domain
  • Output: DNA-binding to upstream of araBAD
  • Absence of arabinose, AraC binds O2 and I2
    – No recruitment of RNA Pol = No expression
  • Presence of arabinose, AraC binds I1 and I2
    – Recruitment of RNA Pol = Expression
  • Function: Regulates arabinose utilization genes (araBAD)
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11
Q

How to study signal transduction

A
  • Monitor OUTPUT = gene expression
    – Northern Blot
  • Isolate mRNA
  • Hybridize with labeled probe
    – Radioactive 32P
    – Biotin
    – Quantitative Real Time PCR
  • Isolate mRNA…convert to cDNA
  • qPCR amplify using specific primers
    – Reporter gene fusion
  • Gene expression correlates with reporter output
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12
Q

What is reporter gene fusion

A
  • Clone regulatory region of gene upstream of reporter gene lacking
    its own regulatory region
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13
Q

Example of reporter gene fusion

A
  • lux – luciferase
    – generates light
  • lacZ – beta-galactosidase
    – ONPG turns yellow
    – X-gal turns blue
    – pH Indicator plates
  • Color change with
    fermentation of sugar (lactose)
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14
Q

Two component system mechanism

A
  • Has two components!
    1. Membrane-bound sensor
    kinase to sense the signal
    (ex: EnvZ)
    2. Response Regulator to bind
    DNA (ex: OmpR)
  • In response to stimuli, the sensor
    kinase is phosphorylated
  • The phosphoryl group is
    transferred to the response
    regulator
  • Phosphorylation of the response
    regulator triggers a
    conformational change allowing
    DNA binding
  • Phosphorylation of the sensor
    kinase occurs on a conserved
    histidine residue
  • Then the phosphoryl group is
    transferred to a conserved
    aspartate on the response
    regulator
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15
Q

Do bacteria need to metabolize a chemoattractant to respond to it?

A

No

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16
Q

What is a biofilm?

A

Defined as a community of microbes
Attached to a surface (biotic or abiotic) or each
other
Can be comprised of one or many species
The predominant lifestyle of bacteria in every
environment studied

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17
Q

Where do biofilms form?

A

On solid substratums in contact with moisture
On soft tissue surfaces in living organisms
At liquid-air interfaces
As aggregates of cells suspended in liquids

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18
Q

Examples of places where biofilms form

A

-Medical devices
-Pond scum
-Rhizosphere
-Plaque and cavities

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19
Q

Biofilms and virulence

A
  • Biofilms can be disseminated around the
    body, either as single cells or clumps of
    protected emboli
    *Sporadic detachment can lead to cycles of
    bacteremia
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20
Q

Assaying attachment bacteria to surfaces

A
  • Incubate in glass or plastic vessel
  • Wash off unattached bacteria
  • Stain with crystal violet
  • Wash off excess stain
  • Solubilize stain
  • Measure OD
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21
Q

Biofilm life cycle

A
  1. Reversible attachment
  2. Irreversible attachment
  3. Growth
  4. Maturation
  5. Detachment/Dispersal
22
Q

Important proteins fo biofilm attachment

A
  • Motility
  • Flagella
  • Type IV Pili
  • Adhesins
  • Type IV Pili
  • Ag43 (E. coli)
  • AtlE (Staph epi)
  • Ica (Staph aureus)
23
Q

Important proteins for biofilm maturation

A
  • Matrix
    – EPS
    – Protein
    – Extracellular DNA
24
Q

Important proteins for biofil dispersal

A
  • PS-degrading
    enzymes
  • Protease
  • DNAse
25
Q

ci-d-GMP and biofilms

A

High ci-d-GMP signals dispersal while low signals to aggregate

26
Q

quorum sensing

A

Quorum sensing allows cells to only perform certain
behaviors when the group is at a sufficiently high
density.
* Virulence factors
* Interspecies compeition

27
Q

Quorum sensing example

A

Hawaiian bobtail squid + Vibrio fischeri symbiosis
* Squid sleeps on the ocean floor during the day; swims at night
* Produces light (luciferase) at night to mask its shadow
* Luciferase is produced by Vibrio fischeri that lives in its light organ – BIOLUMINESCENCE
* Luciferase only produced at HIGH CELL DENSITY
* Each cell secretes a molecule called an
AUTOINDUCER
* Here: Homoserine lactone (HSL)
* Once the concentration of autoinducer
reaches a critical concentration, it binds the
transcriptional regulator, LuxR, which
activates the luciferase biosynthetic operon
(lux)
* When the bobtail squid is sleeping, it pumps V.
fischeri out of the light organ to reduce the cell
population and turn off the lights

28
Q

How do biofilms make cells less susceptible to antimicrobial treatment

A
  • Physical or chemical diffusion barriers
  • Bound by matrix
  • Slow growth, reduced metabolic activity
  • Altered microenvironment
  • pH, reduced O 2
  • Activation of stress response
  • Biofilm-specific state
  • Efflux pumps
  • Periplasmic glycans (P. aeruginosa)
29
Q

Biofilm prevention strategies

A

-Development of antimicrobial surfaces to prevent attachment

30
Q

Caulobacter crescentus

A

A model organism for studying asymmetric cellular differentiation
* Sporulation
* Yeast budding
Conceptually important too for:
* Cellular polarity
* Cell cycles

31
Q

Unequal cell division of caulobacter crescentus

A

Unequal cell division
– Swarmer cell
– Polar Flagellum and Pili
– Delayed replication
– Stalked cell is different
– No flagellum/pili
– Stalks used for attachment
and nutrient absorption (phosphate)
– Capable of replication soon
Stalked Cell Swarmer Cell

32
Q

Caulobacter crescentus types of cells

A

C. crescentus does not differentiate in response to
nutritional stress or environmental cues à
differentiation is a central part of the growth cycle
* Two very different cells are formed:
- Swarmer cells: motile and incapable
of replicating its DNA
- Stalked cells: nonmotile but capable
of DNA replication and give off daughter
swarmer cells

33
Q

caulobacter crescentus holdfast versus stalk

A

The stalk is an extension of the cell body. The center and cell surface are contigous with the cytoplsam and cell membrane respectively. Crossbands of peptidogylcan provide rigidity to the stalk.
Holdfast is located at the tip of the stalk. Polysaccharide adhesion. Mediates permanent surface attachment.

34
Q

caulobacter crescentus life cycle

A
  1. In response to an as-yet unknown intracellular signal, the swarmer cell
    sheds its flagellum and pili.
  2. The cell produces the holdfast and attaches to a surface
  3. A stalk is produced at the same pole while DNA replication is initiated
  4. The stalked cell is reproductively mature and gives off daughterswarmer cells, marking the completion of the dimorphic life cycle
35
Q

CtrA

A
  • Response regulator of 2-component Serine-Threonine (phosphorelay)
  • CtrA~P is active form
    1. Binds to the chromosomal origin to repress DNA replication initiation
    2. Directly regulates 95 genes
  • polar morphogenesis
  • cell-cycle progression
  • regulatory processes
  • CtrA activity controlled on three levels: transcription, proteolysis &
    phosphorylation
  • Essential for viability
36
Q

ClpP and ClpX

A

responsible for proteolysis of CtrA

37
Q

What controls CtrA proteolysis?

A

ci-d-GMP

38
Q

PopZ

A

Polar membranous organelle: Disordered protein
that creates liquid-liquid phase separated MLO
Selectively sequesters signaling proteins at the pole
reinforcing subcellular organization

39
Q

What do bacteria compete for?

A
  • Carbon Sources
  • Trace Elements – Fe, Cu, Mg, S
  • Oxygen
  • Space
40
Q

What do bacteria cooperate to obtain?

A
  • Carbon Sources
  • Trace Elements – Fe, Cu, Mg, S
  • Oxygen
  • Space
  • Protection
  • From the host
  • From the environment
  • From other microbes
41
Q

How do bacteria compete?

A

Contact-independent: bacteriocins and fratricins
Contact-dependent: molecular syringe (type 4 secretion) and contact dependent growth inhibition (CDI)(Vb secretion system)

42
Q

bacteriocins

A

Large and varied types of peptide and protein antibiotics
produced by bacteria

43
Q

Colicins

A

Type of bacteriocin mostly made by Gram negatives (E.
coli best characterized)
* Bind to receptors in the outer membrane, using them to translocate into the cytoplasmic membrane where they exert their cytotoxic effects (depolarization of the cytoplasmic membrane, Dnase and Rnase activity, or inhibition of PG synthesis)
Contact Independent

44
Q

Secreted metabolites

A

Siderophores
* 2-Heptyl-4-hydroxyquinoline n-oxide (HQNO)
* Binds electron transport chain and
inhibit aerobics respiration

45
Q

T6SS

A
  • Molecular Syringe or crossbow
  • Multiprotein complex in the cells’ periplasm
    that forms a needle-like structure similar to
    the bacteriophage T4.
  • The outer sheath functions as a piston that
    contracts and pushed the “needle” out
    puncturing the membrane of a neighboring
    cell and releasing toxic effectors into the
    cytoplasm
46
Q

T6SS composition

A
  • Gene are encoded in tightly clustered groups; 13
    conserved genes are the core components
  • P. aeruginosa contains 3 distinct gene
    clusters – HCP- I, II, III
  • Grouped into 3 categories:
  • Membrane associated proteins: (TssL, M, and
    J): Interact to form a complex that crosses
    the cell envelope
  • Proteins related to bacteriophage tail (more
    detail next page)
  • Proteins of unknown function
47
Q

T6SS proteins

A
  1. Syringe: Hcp and Vgr
    * Hcp forms a tube as a hexamer (~ λ phage tail protein,
    gpV)
    * VgrG forms a homotrimeric complex at the tip
    (bacteriophage T4 spike protein)
  2. Sheath: TssB/TssC (VipA/B in V. cholerae) (~gp18)
  3. Baseplate: TssE
  4. ATPase: ClpV
    * Contracted TssB/C is recognized by ClpV and is
    disintegrated by a process dependent on ClpV-
    mediated ATP hydrolysis
  5. Membrane associated protein IcmF
    * ATPase activity required structural component
    T6SS composition
48
Q

T6SS effectors

A
  • Most known to-date target the bacterial cell envelope and nucleic acids
  • Some examples:
  • Tse1 and 3: peptidase that degrade peptidoglycan
  • Tse2: Cytoplasmic toxin that inhibits bacterial growth
49
Q

T6SS immunity and defense

A

A. Spatially separate
B. Protect the cell envelope
C. Produce immunity proteins
* Kin cells are protected from toxins through the
production of a cognate immunity protein
D. Respond to attack

50
Q

Contact dependent growth inhibition T5SS

A
  • Different from T6SS in that it
    requires a specific receptor on
    the target cell surface for the
    CDI toxin to bind