Exam 4 Flashcards
- 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.
- 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
Name one intracellular bacterial second messenger. What are some of the bacterial processes
controlled by this second messenger?
c-di-GMP: Biofilm formation, polysaccharide production, motility, cell cycle and cellular development,
virulence
Describe one reason why being small (like E. coli) is hard.
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.
What is one way Caulobacter crescentus produces different levels of phosphorylated CtrA in the
swarmer cell and the stalked cell
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
What are the major components of the biofilm? Why are each so important
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
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?
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
Describe ways that P. aeruginosa and V. cholerae type VI secretions systems differ.
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.
What is the difference between one component and two component signal transduction?
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.
Example of a one componenent system
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.
AraC regulation summary
- 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)
How to study signal transduction
- 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
What is reporter gene fusion
- Clone regulatory region of gene upstream of reporter gene lacking
its own regulatory region
Example of reporter gene fusion
- lux – luciferase
– generates light - lacZ – beta-galactosidase
– ONPG turns yellow
– X-gal turns blue
– pH Indicator plates - Color change with
fermentation of sugar (lactose)
Two component system mechanism
- 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
Do bacteria need to metabolize a chemoattractant to respond to it?
No
What is a biofilm?
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
Where do biofilms form?
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
Examples of places where biofilms form
-Medical devices
-Pond scum
-Rhizosphere
-Plaque and cavities
Biofilms and virulence
- Biofilms can be disseminated around the
body, either as single cells or clumps of
protected emboli
*Sporadic detachment can lead to cycles of
bacteremia
Assaying attachment bacteria to surfaces
- Incubate in glass or plastic vessel
- Wash off unattached bacteria
- Stain with crystal violet
- Wash off excess stain
- Solubilize stain
- Measure OD
