MOLECULAR MICROBIOLOGY
AB_470610
P3 2021
,Inhoud
MolMic HC 1: OMP biogenesis ................................................................................................................ 2
MolMic HC 2: Protein trafficking in bacteria: tool and target ............................................................... 14
MolMic HC 3: Secretion systems ........................................................................................................... 22
MolMic HC 4: The mycobacterial cell envelope and membrane transport .......................................... 34
MolMic HC 5: Biosynthesis and function of LPS .................................................................................... 42
MolMic HC 6: Flagella, pili and fimbriae................................................................................................ 53
MolMic HC 7: The Twin Arginine Translocation system ........................................................................ 67
MolMic HC 8: Host-pathogen interactions (bacterial virulence) .......................................................... 74
MolMic HC 9: Regulation of bacterial respiration ................................................................................. 84
MolMic HC 10: TLR-mediated innate immunity & pathogen escape mechanisms ............................... 95
MolMic HC 11: Reverse vaccinology ................................................................................................... 106
,MolMic HC 1: OMP biogenesis
Outer membrane proteins (OMPs) are essential for the surviving and proliferation of gram-negative
bacteria. If you try to mutate or delete the complex that is involved in OMP biogenesis, then the
bacteria are no longer viable.
First, let’s take a look at the membranes. Gram-negative bacteria have two
membranes (see picture): (i) an inner membrane (cytoplasmic membrane) that
protects the cytoplasm and compartmentalizes it. Then follows the periplasmic
space and (ii) the outer membrane shields and envelops this space.
The inner membrane (see picture) is a classical membrane like the ones found in
eukaryotes and consists of a phospholipid bilayer (two leaflets of phospholipids)
that creates a hydrophobic barrier which protects the cytoplasm. This membrane
is symmetric, because both leaflets consists of the same types of phospholipids
(same components). Due to the hydrophobicity of this barrier, transport is
facilitated across this barrier with the help of integral membrane
proteins. These proteins are embedded in the phospholipid bilayer,
in such a way that they make contact between the compartments
that are separated by this phospholipid bilayer. Therefore, they have
hydrophilic surfaces to interact with the compartments and
hydrophobic surfaces to not be expelled from the membrane itself.
Additionally, there are peripheral proteins that are anchored on one
side of the membrane. These proteins are often lipoproteins in
gram-negative bacteria when they are in the periplasm (peripheral
lipoproteins). These peripheral lipoproteins have a lipid tail that
anchors these proteins to the membrane, but the protein itself sticks
out into the periplasm.
The outer membrane (see picture) is not a phospholipid bilayer and
therefore it is asymmetric. The inner leaflet consists of
phospholipids, but the outer leaflet consists of (another type of
lipids) primarily lipopolysaccharides (LPS). These LPS molecules have
a lipid part that anchors them to the outer leaflet of the outer membrane, but the remainder of these
LPS molecules are sugars (polysaccharides) that extends from the cell surface into the bacterial
environment and serves as an extra protective layer for the bacterium. The outer membrane also
consists of integral membrane proteins and this summary will explain how these outer membrane
proteins look like and how they are inserted into the outer membrane. The outer membrane also has
peripheral lipoproteins. In the case of the outer membrane, these peripheral lipoproteins can be in
both the inner and outer leaflet and can therefore be sticking into the periplasm or the bacterial
environment, respectively. Of course, these peripheral lipoproteins are anchored to the membrane
leaflets with lipid moieties (parts/components). However, the peripheral lipoproteins will not be the
main topic of this summary, but are sometimes important.
The topics of this summary is as following:
1. What are integral outer membrane proteins: structure, function
2. Targeting of the outer membrane: periplasmic transit, chaperones
3. Assembly and insertion into the outer membrane: discovery of the assembly machinery (Bam-
complex), assembly complex and its mode of action, targeting signal
, To start off with, let’s take a look at the structure of the integral
membrane proteins (see picture). On the left you can see a
typical integral membrane protein of the cytoplasmic
membrane (inner membrane) (IMP: inner membrane protein)
and on the right you can see a typical integral membrane
protein of the outer membrane (OMP: outer membrane
protein). Judging from this picture, structural differences can be
observed. For example, there is a clear difference in secondary
structure element. The stripes of the IMP picture indicate the
position of the membrane (the phospholipid bilayer) and this membrane, of course, has a hydrophilic
head group (outer part of the bilayer) and a hydrophobic tail (inner part of the bilayer) that constitute
the barrier. In order to have an integral membrane protein inserted into this membrane, these integral
membrane proteins have hydrophobic transmembrane α-helices that serve as a membrane anchor.
Both IMP and OMP proteins have in common that the surface that makes contact with the hydrophobic
acyl chains need to be hydrophobic to interact with these acyl chains (of course, if they were
hydrophilic they would be expelled from the membrane otherwise). The integral membrane proteins
are characterized by these α-helices that form the membrane anchors (this can be one (completely
hydrophobic) α-helix or a bundle of α-helices) and at least the ones that are on the outside are
hydrophobic. If the integral membrane protein is a channel through which a hydrophilic molecule is
transported, it should have hydrophilic residues inside of the integral membrane protein for the
transport. If you look at the OMP, it is a β-barrel protein consisting of β-strands that form a β-sheet
and this β-sheet is shaped into a tube. This tubular shape contacts the acyl chains on the outside of
the entire OMP with the help of hydrophobic residues. All-in-all, this characteristic of a membrane
protein is an important requirement for these proteins to be integrated within such a membrane. In
the same picture of the OMP, the inside is hydrophilic. In the case of this OMP, there is an α-helix
within to plug this channel in a way to prevent that not any molecule diffuses through by chance.
The integral IMPs are α-helical and their transmembrane helices
(TM) acts as membrane anchors (there could be 1 TM (the only
anchor and completely hydrophobic) or 10 TMs (some can be
anchor, some can be hydrophilic). A nice thing about these TM is
that you can identify them very easily with database searches,
because you look for a stretch of amino acids that have the
capability to form α-helices. On top of that, the amino acid
sequence is often a length of around 20 amino acids and they are
hydrophobic.
The integral OMPs also have known characteristic features, but
are more difficult to predict when you do database searches.
OMPs do not have continuous stretches of hydrophobic residues,
because the strands that make up the β-sheet are amphipathic
(the surface of the tube is hydrophobic, the inner part of the tube
is hydrophilic). A feature (discovered by analyzing and comparing
many OMPs) of OMPs is that they tend to have an even number
of β-strands, with a minimal number of 8 and a maximum of 24
(in gram-negative bacteria). The strands can be from 10 to 40 amino acids in length (the minimal length
to cross the membrane is 10 amino acids). The β-strands of the OMPs are, of course, amphipathic,
meaning that they have alternating hydrophobic and hydrophilic residues. This has to do with the fact
that if you look at the structure of a β-strand in cartoon fashion (see picture), you can see that the side-
