Midterm 2 Flashcards
What is a methyl accepting chemotaxis protein?
- 2 transmembrane domains
- N-terminus sensing region binds
ligand directly or via binding protein - C-terminus signalling region and methylation regions (highly conserved)
What is the role of the N-terminus of a methyl accepting chemotaxis protein?
A sensing region binds ligand directly or via binding protein
What is the role of the C-terminus of a methyl accepting chemotaxis protein?
Signalling region and methylation regions (highly conserved)
What does CheW do?
Coupling protein, enhances CheA interactions with receptors.
What does CheA do?
sensor kinase, autophosphorylates,(de)phosphorylates CheB and CheY
What does CheY do?
Regulator, when phosphorylated interacts with Flagellar motor to reverse direction
What does CheZ do?
Phosphatase, dephosphorylates CheY-P
What does CheR do?
Adds methyl groups to MCPs
What does CheB do?
When phosphorylated demethylates MCPs
What does Tsr do?
Serine, repellents, (heat, oxygen)
What does Tar do?
Aspartate, maltose, repellents
What does Trg do?
Ribose, galactose (glucose at certain concentrations)
What are the different types of MCPS in E. coli
Tsr, Tar. Trg, Tap, Aer
What does Tap do?
Various dipeptides
What does Aer do?
So-called Aerotaxis receptor, has signalling domain, but not methyl accepting sites or transmembrane helices
What are the major receptors present in cells?
Tsr and Tar
What are the minor receptors present in cells?
Trg and Tap
What do Tsr and Tar have that Trg and Tap don’t?
NWET/SF site at the C terminus which seems to be involved in CheR binding (Implies interaction between receptors)
Where are MCP’s clustered in a cell?
At the pole(s) of the cell. In E. coli this is mostly at the pole opposite the flagellar bundle.
How can we prove the location of MCP’s?
Using antibodies, GFP fusions.
What is PTS taxis?
Links transport of sugars (eg. glucose, mannose, mannitol, galactitol, glucitol, glucosamineetc -21 different ones in E. coli) to Chemotaxis via phosphotransferase system
What does PTS taxis require?
CheA, CheW, CheY, and at least one major MCP
What does sugar transport inhibit?
CheA phosphorylation
What does AER
An “energy sensing” mechanism
Does not sense Oxygen directly, but seems
to be able to detect rate of electron transport.
What is Aer
Aerotaxis receptor; MCP like but lacks methylation or transmembrane domains
Where were the effects of Aer first noticed?
In taxis on glycerol
What does high electron flow equal?
Decreased CheA
phosphorylation. This will happen if Oxygen, Substrate, NADH etc. are high
What are some of the deviations from the E. coli model in other bacteria
Many more MCPs (up to 89; 42 in Vibrio), no PTS, multiple flagellin genes, multiple Chemotaxis
systems (as many as four; cytoplasmic vs. clustered at poles); Undirectional flagella, CW rotation = swimming
How is the chemotaxis system different in B. subtilis
Different: CheC, CheD, CheV, No CheZ methylation works backwards (sometimes).
Where does chemotaxis matter?
- In infection of Eukaryotes
- In colonization of roots and infection of plants by rhizobia
- In biofilm formation and spread ?
- In oxygen avoidance by anaerobes, optimal foraging by all microbes.
Where does swimming motility usually occur?
Surface phenomenon
Usually only happens on softer agar surfaces (0.5 to 0.7 %) or in viscous media.
Proteus swimming motility
one type of flagellin, cells elongate from 2-4 µm to 80 µm, more flagella. Works on 1.5 to 2.0 % agar.
Vibrio swimming motility
new lateral flagella (different flagellin, some elongation, works on 1.5 % agar)
E. coli swimming motility?
only on 0.5-0.7 % agar. Much less elongation and differentiation.
What does infection of P. aeruginosa by bacteriophage do?
Inhibits motility and induces repulsion of healthy swarms
What is swarming good for?
Possibly colonization of surfaces
Possibly movement Through viscous media
more resistant or at least tolerant to antibiotics
What is swarming motility?
A form of flagellar movement that cells exhibit on surface or in viscous media. Hallmarks of Swarming include coordinated multicellular movement, appearance of more, sometimes distinct, lateral flagella, and differentiation of cells to larger forms
What are the challenges of cell division?
Correct timing
Correct placement of septum; not breaking Chromosome
Assembly and disassembly of cell wall must be coordinated.
What happens in G1?
Binding of DnaA to oriC initiates replication
What happens in S?
Blocking of oriC regions by SeqA and cell elongation.
The second step in S phase is segregation of chromosomes
What happens in G2?
Z ring formation
How is cell division studied?
Microscopy
Mutants, often Temperature sensitive (Ts)
Characterisation of proteins
Localisation of proteins (immunogold Labelling, GFP labelling etc., Fluorescent
probes)
Nomenclature rules for gene
Lower case, italics
Nomenclature rules for proteins
First letter capitalized
Major players in cell division
Fts proteins, Min proteins, Zip, Zap, DivI, MipZ and other proteins like
YyaA;some of these variable between bacteria
What happens to ftsZ mutants?
They don’t divide; form elongated filamentous cells. ftsZ is essential.
What happens to MinC, MinD or MinCDE mutants in E coli?
divide normally at centre, but also at poles generating “anucleate” (no chromosome or nucleoid) “minicells
What are the uses of minicells?
Expression of genes and synthesis of proteins encoded/ cloned on plasmids
Could be used nowadays for proteomics of low abundance proteins
What is a minicell?
A bacterial cell that is smaller than usual.
What is the Z-ring made of?
FtsZ and other proteins
What is minCDE important for?
Positioning of Z-ring
What does minCD inhibit?
Z ring formation
What does MinE inhibit
MinCD
What happens to Min proteins in E coli?
They oscillate back and forth between cell poles
In bacillus there is no _____
MinE
What does DivIVA do in bacillus?
sequester MinCD at poles
What organisms is FtsZ present in?
All bacteria and Archaea
What are spores?
Resistant structures made by bacteria, fungi, plants etc
What are spores used for?
Can be survival mechanisms, but also propagation mechanisms.
Primarily about surviving periods of environmental adversity.
Where are endospores formed?
In some low GC Gram positives (Firmicutes)
Where are myxospores formed?
In myxobacteria
Where are conidia formed?
Actinomycetes (Actinobacteria,High GC Gram positives)
Where are Akinetes formed?
Cyanobacteria
Where are cysts fomed
Azotobacteria
Where are UMBs fomed
most bacteria (see also persisters, viable but non-culturable state)
The _____ is most resistant to heat starvation, UV, radiation, desiccation etc.
Endospore
What are some low GC gram positives organisms?
Bacillus spp.
Paenibacillus spp.
Clostridium spp.
Desulfotomaculum spp.
Desulfitobacterium spp.
Sporosarcina spp. (cocci)
Why are endospores made?
Made in response to starvation and cell density
signals
What are endospores capable of doing?
capable of detecting signals in their environment, and in response to nutrients and water, will germinate to form a vegetative cell.
What signals do endospores respond to?
mainly sugars and amino acids (alanine)
What senses signals in endospores?
GerA, GerB and GerK
What is some evidence for extreme longevity?
Simulation studies
Isolation from samples 1000s of years old.
Isolation from within a bee entrapped in amber that was 25-40 million years old
Isolation from 250 Million year old salt crystals
T /F Endospores have ranging levels of elaticity
T
What are the conditions inside an endospore?
High concentration of Calcium and Dipicolinic Acid.
10-30 % of the water content of a vegetative cell
Lower pH, small acid soluble proteins (SASPs) which bind DNA protect from radiation and denaturation
What is dipicolinic acid?
A chemical compound which plays a role in the heat resistance of bacterial endospores
Stage 0 in formation of endospore
Vegetative cell
Stage 1 in formation of endospore
DNA becomes more dense
Stage 2 in formation of endospore
Cytoplasmic membrane invaginates to form spore septum
Stage 3 in formation of endospore
Spore septum grows around protoplast (engulfment)
Stage 4 in formation of endospore
Spore formation
Exosporium appears
Primordial cortex forms
Dehydration
Stage 5 in formation of endospore
Coat layers are formed
Stage 6 in formation of endospore
Maturation (development of resistance to heat and chemicals)
Stage 7 in formation of endospore
Lysis of cell and release of free spore
What is endospore formation controlled by?
Sigma factors
Sigma factor for early spore post-septation?
Sigma F
Sigma factor for late spore post-septation?
Sigma G
How is control of endospore formation also achieved?
a phosphorelay system (variant of the two-component type system)
What is the advantage of the phosphorelay system?
more checkpoints at which various Phosphatases can have input to the system - see RapA
What is Spo0A?
the master regulator for the whole process.
necessary for expression of sigma-E, Sigma-F genes (proteins made in inactive form)
How many sensor kinases are involved in phosphorelay system input in Bacillus Subtilis?
5 different sensor kinases labelled KinA,KinB, KinC, KinD, and KinE.
What are the disadvantages of endospores?
Non-motile
Heavy
Would be hard to make a similar structure in a Gram negative cell, or even a Gram positive with a complex cell envelope, like
Mycobacterium and some other high GCs .
What is myxobacteria?
Gram negative, rod shaped, large genomes, complex life cycles with multicellular behaviour. Belong to delta subgroup of the Proteobacteria
What are some examples of myxobacteria?
Species most studied:
Myxococcus xanthus
Stigmatella aurantiacum
Chondromyces apiculatus
What are the types of motility exhibited by myxobacteria?
Adventurous and Social
What is adventurous motility?
free living cells as individuals or groups, glide using the mechanism seen in Cyanobacteria and other gliders like Cytophaga.
What is social motility?
only large rafts of cells, using Type IV pili. Prelude to fruiting body development.
What type of motility is social and adventurous?
Gliding
What governs motility and differentiation in Mycococcus
All the Che gene sets
What is a fruiting body?
A fruiting body is the multicellular structure of an organism on which spore-producing structures, such as basidia or asci, are born.
Early stage of fruiting body formation
showing aggregation and mound formation
Initial stage of fruiting body formation
stalk formation
Slime formation in the head has not yet begun and so the cells that compose the head are still visible
Myxospores
Dormant, resistant to dessication, sonication, UV, detergents and heating to 50 degrees C.
Have thick spore walls containing novel proteins; exact structure appears
not to be well known. (14 % protein, 75 % polysaccharide with galactosamine and glucose)
Spores formed about 30 hours after starvation signal. (espA mutant 16 hours earlier, espB mutant 16 hours later)
Sporulation in Actinomyctes
High GC Gram positives such as Streptomyces spp., Frankia, Nocardia, Actinomyces etc.
Exhibit “hyphal” morphology and a multicellular mode of growth.
Spores produced on aerial hyphae
What is a Hyphae
spore bearing structure in Streptomyces
What is an Akinete
Spore-like survival structure
What are cysts for?
Starvation survival
