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Summary Molecular Microbiology VUB
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Summary of the courses and higly possible questions with their answers for Partim 2 of the course Molecular Microbiology at the VUB.
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Describe the buildup and function of the cell envelope in classical Gram-positive bacteria.
Briefly indicate the nature of the cell wall components.
General role of cell envelope: It is a selective barrier of what you allow to enter and leave
the cell with a lipid bilayer as minimal boundary. A bilayer is very hydrophobic so if you want
to import things in and out of the cell, you’ll have to have active pathways. The cell
envelope in a way will help dictate and support the cell size and shape. In eukaryotes the
shape is mostly dictated by the exoskeleton, in bacteria it is the outer armor or shell. It is
also a scaffold for attachment proteins or appendages if it benefits the bacterial virulence.
Enzymes can be secreted in the environment or attached or injected when attached to a
host cell etc. It is cell supporting property and prevents rupture due to osmosis f.ex. Or
prevent water may leak out in a more hypertonic environment. Cell envelope also contains
the function for growth, the cell surface dictates if the cell divides or not and the envelope
pinches of or not for division. Cell envelope if everything above cellular membrane so above
the first lipid bilayer as cell membrane. Cell envelope is semi porous and allows larger
molecules to flow freely.
General role of cell membrane: First and minimal boundary of the cell. Is a very firm
permeability barrier, don’t allow many solutes to flow freely. Almost anything you want to
get across you’ll need to do it by active pathways. Also, the inner cell membrane has a very
important role for energy conversion, ATP synthesis protein motive force and electron
transport all happens at cytoplasm barrier. Even the smallest proton atom doesn’t flow
through the membrane passively and the cell uses that to create a gradient of protons
across the barrier with high proton concentrations outside the membrane and when you
allow protons to enter through aquaporins you go from high energy states to low energy
states which is a way to generate energy to power pathways as flagellar motion (not using
ATP hydrolyzation but by just letting protons flow in). Some bacteria choose to build sodium
gradients and let sodium enter to drive energy forces. So, anything you are letting in you
must do so in a very controlled way to not compromise that proton motive gradient and
force.
Bacteria has 3 main architecture: Gram-positive, Gram-negative and Mycobacteria.
Mycobacteria has Gram-positive staining but is also a diderm bacteria it has cytoplasmic
membrane; it has peptidoglycan like structures and has something that looks like a second
bilayer but its not a bilayer it is lipid nature only found in mycobacteria.
Gram-staining: a protocol where u expose bacterium to crystal violet and it diffuses into the
cell, iodine makes it precipitate and crystalize. You add ethanol to make it soluble again and
you can extract it again then you add Safranin as counter staining. Gram-positive means the
crystal violet remains inside the cell wall and wasn’t washed out by the ethanol due to the
different structure properties. Gram negative will be rose and positive dark violet.
Gram-positive cell envelope architecture: They just have a cytoplasmic inner membrane
and then different polymers on top of it. It is also called a monoderm or single skin. The
peptidoglycan cell wall is a very thick dens layer (variable thickness and density) and mostly
composed of peptidoglycan and also polymers.
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,Name: Class:
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Extracellular environment is immediately outside the cytoplasmic membrane and that is
where you have the cell envelope. Here cell envelope is the integrity of the wrap around the
cell and cell wall is more the peptidoglycan. It is a very thick (20-80 nm) peptidoglycan layer
of multiple layers of peptidoglycan polymer and is a crosslinked layer and major supporter
of the cell and the main part that gives the shape and shape retention and give physical
strength and protects against turgor pressure. No outer membrane. The peptidoglycan layer
is 50% of the cell wall and the other half are proteins imbedded or covalently linked to the
peptidoglycan and also secondary cell wall polymers (not primary) and are teichoic acids
and lipoteichoic acids as chemical derivative of the teichoic acids either covalently bond to
the peptidoglycan layer (teichoic acids) or linked on a lipid (lipoteichoic) and are anchored
onto a phospholipid of the cytoplasmatic layer and they are there not as a support but more
alter property, charge and permeability of the cell envelope and whether the envelope is
lipophilic or not and are projected and stick out beyond the envelope that can also be
involved in immune evasion or pathogenicity. Peptidoglycan in itself is very crosslinked and
very important as support and against turgor pressure it is very permeable for proteins and
large nm scaled molecules like a mesh.
Teichoic acids:
Are covalently linked onto the peptidoglycan. The basic scaffold of peptidoglycan is the
alternation of 2 glycans: MurNAc and GlucNAc and on the MurNAc you can have via
Phosphate sugar linkage units and then you have Glycerolphophate as a linker. It can be
associated to virulence because it can be involved in escaping immune detection and in
biofilm formation.
Lipoteichoic acids: Sort of a variation to Teichoic acids. But here covalently linked to a
phospholipid so on a phospholipid background or diacyl glycerol and sugar units and in
repition of glycerolphophate or ribitolphosphate: two polycarbohydrates with a phosphate
linkage in between them and heavily negatively charge and altering it like that.
Mycobacteria: Have “Gram-positive” cell envelopes but are diderm:
Myco/Actinobacteria
They have a second membrane (not a bilayer) but a layer of cholic acids that build up and
act as a very strong diffusion barrier for, for example lipophilic proteins and is a resistant
important problem bacterium. Also, a semi permeable second layer and they can alter the
level of permeability in response to antibiotic or other pressure.
Secondary cell wall components: Capsules: large overarching term: thick slimey (mucoid)
layer usually linear polysaccharides deposited outside and don’t have a structure supporting
function but more a protective function from outside changes or events. Protect from drying
temporarely because of the polysachs and slime that attract water or take longer to lose it.
Often in pathogenic strains with capsules protecting them from the immune system and
phagocytosis. Very variable in changing the chemical composition of capsules and expressed
enzymes on it so not much immune memory. Immune escape.
ECM: also large overarching term: deposited outside and more loosely attached: often
polysachs as cellulose for example for E.coli in biofilm, it can be extracellular DNA or nucleic
acid polymers. Amyloids (Alzheimer) purposely formed for example curli forming rigid fibers.
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,Name: Class:
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Mostly the cement or matrix for bacterial communities for biofilms and linked to
persistence. You have mono- or interspecies biofilms.
S-layers are monolayers of proteins noncovalent glycopolymers and form a layer wrapping
the microbial cell. They crystalize and are compared as an exoskeleton found in almost all
Archea and 1/3 bacterial environmental isolates. Addition support system and for Archea
more important for extreme environments. Can be a molecular “zeef” for permeability and
known to be involved in pathogenesis. Requires lots of metabolic ability and production of
S-layers is lost in the lab.
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, Name: Class:
Date:
This EM image was obtained from pelleted bacteria (E. coli and Caulobacter crescentus)
that were heat-threated in 6% SDS. Please describe composition and function of the
observed structure.
Caulobacter crescentus: is not a pathogen it is an environmental species that lives in water,
but the unusual thing is its asymmetry. It has one planktonic cell and its neighboring cell as
daughter cell which is a sedimentary cell. The stick coming out of it is the holdfast and lives
on the sediment in waterous environment and is a model organism to study different
pathways of cell localization and how is the splitting daughter cell able to behave completely
differently.
TEM: on top E.coli and bottom Caulobacter both gram negatives. The structures were obtain
by resuspending bacterial cultures and heat in 6% sodium dodesil suffate: SDS is a detergent
and dissolves lipids and denatures in heat so lipids and proteins are washed out and
ultracentrifuged. What remains in the image is retaining the shape of the “about to devide
E.coli” and Caulobacter: What remains is the covalent polymer and is the peptidoglycan and
retains the shape.
Peptidoglycan nature:
Is a polymer of peptides and glycan: Glycans polymers are polymers of alternating N-Acetyl
Muranic acid (MurNAc) and N-acetyl Glucose amine (GlcNAc). The peptide nature at the
very minimal start as pentapeptides: 5 amino acids and the peculiar thing is they are D-
enantiomers (most are L) so it means they are not made by the ribosomes so not genetically
encoded but added on one at a time. Some gram-positives have more peptides up to 5
peptides more. The pentapeptides are two leveled cross linked: The minimal one is the
disaccharide units are covalently bond to one another forming long linear ribbon chains and
these chains are laterally covalently crosslinked by the peptides and you create the 2-
dimensional mesh.
How do you crosslink outside the cytoplasm? (make a covalent bond meaning going from
low energy to high energy state ?) Meaning hydrolising ATP or from other energy source:
The crosslinking of the building bonds are formed outside the cells without ATP: By
transpeptidations and transglycolisations:
You brake or replace the already established peptide bond with an incoming nucleofile
which is your other amino acid: Are important targets of big families of antibiotics.
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