Exam 3 Flashcards
Fill in the blanks
In B. subtilis a spo0A mutant is blocked at stage ___________ of sporulation.
spo0A mutants fail to ______ and _____.
Stage 0, express early-stage genes, form polar septa
Describe an example of how cell-cell signaling controls sigma factor activation during sporulation.
One example is activation of SigmaE in the mothercell. When SigmaF is activated in the
forespore it induces expression of spoIIR which is secreted into the intramembrane
space where it activates the protease SpoIIGA in the mothercell. SpoIIGA cleaves pro-
sigmaE to activate
You identified a new spore-forming organism from the intestinal microbiota. You find that spores can be germinated in response to Lysine. Describe assays to monitor germination of the spores.
You can purify spores then add Lysine and see if there is a decrease in OD600 due to loss of phase-bright or (germination)
Describe why disruption of the intestinal microbiota allows C. difficile to cause disease.
One reason is disruption of the intestinal microbiota changes the bile acid profile of the intestinal tract. In a healthy gut the bile acids are mostly Secondary bile acids. However,disruption of the microbiome results in increased primary bile acids which can trigger
germination of C. difficile spores and 1o bile acids are less toxic to C. difficile than Secondary bile.
Describe the two iron paradoxes. Why are they important for understanding how bacteria have adapted to life on Earth?
The first iron paradox is that despite iron being the most abundant transition metal on Earth, it has low bioavailability for organisms to use. This is because the more soluble oxidation state Fe2+ (Ferrous Iron) is not very stable in the presence of oxygen, as it is oxidized to Fe3+, which is less soluble.
The second iron paradox is that even though iron is essential for life, it can also be toxic. Fe2+ can react to H2O2 or Fe3+ with H2O to for the hydroxy free radical, which is very toxic to cells. This is called the Fenton Reaction.
Understanding these paradoxes yields insight into why all cells have evolved so many strategies to acquire, store, and protect themselves from iron.
You discover a new bacterial species that grows extremely well under iron limited conditions. What phenotypes would you hypothesize the species possesses?
This species would likely have evolved strategies to scavenge iron from its surroundings, store intracellular iron, and tightly regulate the consumption of iron.
What are siderophores? How are they made?
Secreted proteins with high specificity and affinity for ferric iron. Siderophores are made from non-ribosomal peptide synthesis. This system synthesizes secondary metabolites independent of the ribosome, performed by multimodular mega enzyme called nonribosomal peptide synthetase.
You add hydrogen peroxide to a bacterial culture….
a. What cell components would you expect to be damaged?
Proteins, lipids, carbohydrates, DNA
You add hydrogen peroxide to a bacterial culture….
b. How would the cells need to respond to the stress in order to survive?
- Produce enzymes, like catalase and superoxide dismutase, to catalyze the destruction of reactive oxygen species
- Sequester iron or turn off iron transport to limit the Fenton Reaction
- Import manganese, since it is a cofactor for some SOD and can protect cells from ROS.
Describe how a Gram negative organism folds exported proteins with cysteine residues?
As proteins are secreted across the inner membrane through the Sec system, thioredoxin-like enzymes, DsbA and B catalyze the formation of disulfide bonds. DsbA is in the periplasm and donates a reactive sulfide bond to the reduced peptide. The DsbA active site is then reoxidized by inner membrane bound DsbB, which shuttles electrons to the electron transport chain. If mistakes are made in folding, they’re repaired by DsbC and D.
Describe the differences between bacterial swarming and swimming (2-3 sentences). Describe an experimental technique to monitor each and one gene deletion you would use as a negative control to confirm the motility you observe is dependent on the flagellum.
Swarming is a form of coordinated group motility for movement on surfaces using lateral flagella and secreted surfactants to wet the surface and reduce friction between the cell body and the surface. Swimming is a single-cell movement through liquid, where the flagella can be arranged at the poles or all around the cell body (peritrichous). Both swarming and swimming can be monitored macroscopically on agar plates. For swarming, cells are inoculated on the top of semi-soft agar (~0.6% agar) and swimming are inoculated into the center of soft agar (0.3% agar). Some examples of a mutant you could use to confirm dependence on flagella are in the gene encoding the filament (fliC) or the hook (flgK).
The bacterial flagellum is composed of three major structures:
(1) the Filament functions like a helical propellor and is composed of a single protein called FliC, (2) hook is a flexible linker that connect to the (3) basal body, which consists of a rotor, composed of a central rod and three ring sets the: L ring, P ring, and MS ring. The motor proteins surround and are anchored to the third set of rings and generate torque to rotate the flagellum. The energy for flagellar rotation is supplied by proton gradients and sometimes Na+ .
3 types of bacterial development
Cellular differentiation in which a cell acquires
phenotypic properties that clearly differentiate it
from a precursor cell;
* Cellular differentiation in which a cell divides to
produce 2 daughter cells that can be
distinguished morphologically and/or
physiologically; or
* Multicellular development to form specialized
structures – fruiting bodies and biofilms
Who first isolated B. subtillis
Ferdinand Cohn 1872
B. subtillis traits
- Gram positive
- soil bacterium
- motile
- competent
- unicellular differentiation
- multicellular behaviors
Alternative starvation responses (3)
Motility (find more nutrients)
Competence (Find new and potentially beneficial genetic material)
Sporulation (enter dormancy)
Sporulation traits
developmental program
* conversion of vegetative cell
into a dormant spore
* triggered by nutrient depletion
* Bacillus endospores are:
1) metabolically dormant
2) heat resistant
3) desiccation resistant
4) radiation resistant
Functions of TFP
Motility, chemotaxis, signaling, phototaxis, self organization, microcolonies, DNA uptake, twitching
Describe a twitching motility assay
Subsurface (stab below agar), dump out agar and stain biomass with crystal violet
How else can we identify twitching motility?
Introduce fluorescence and record under microscope
PilQ Secretin
outer membrane pore for secretion of pili filament
PilB
ATPase for assembly of pili
– Converts chemical energy from ATP hydrolysis into
mechanical engergy
– Polymerizes 1000s of PilA units/secon
PilT and U
-ATPase for disassembly of pilus and retraction
-Mutants are hyperpiliated
