Molecular Bicrobiology Chapter 1-6

• Mobility apparatuses are complex machineries attached to the membrane
• Are powered either by Protons or ATP
• 3 Main mechanism: Flagellar-basses swimming, pili-based twitching (on surface) and gliding (also on surface)
• Some bacteria evolved more than one mechanism
• Advantages: move towards nutrients and away from toxins, repellants or general stress conditions

Import into the IM is done by permeases, three main groups:

• 1 Symporter/antiporter
  • use concentration gradients to move substrate across the membrane
  • example: Lactose permase LacY works with proton force to move lactose
  • have many TMs, that form a pore for the substrate and proton
• 2 Phosphotransferase system (PTS)
  • imports sugar like glucose, fructose, mannose, galactose etc…
  • uses the energy from PEP that is bound on the substrate (made by enzymes)
• 3 ABC transporter
  • Multiprotein complex: 1 protein binding substrate ind periplasm, 1 ind cytoplasm binding the ATP and 1 or 2 tmIMP
  • Can control the flux (abfluss) of toxic substances
  • Example Siderophore-iron complex is taken up trhough the OM  and then by Tol-coupeled system shuttled to the inner membrane by  a ABC transported, then in the inside the iron is reduced and released

• OM is not in contact with ATP (in contrast to the IM) and thus the OMP have no acess to ATP (no ATP in periplasm). Transport of specific solutes though specialized prions need ATP (example Iron is transported by siderophores)
• Siderophore goes through OM porin FhuA and this is energized by coupling protein TonB (energy transducer from IM to OM). The exact process in not fully understood

• Rotary Nano machine (consist out of F_O, F₁) that can produce ATP with letting Protons trough the membrane. If needed it can be reversed.
• Here only small differences between mitochondria, bacteria’s and chloroplast
• F_O Part consist out of three subunits (a, b, c; 1:2:9) translocates the proton across the membrane and induces a rotation of the c unit (very hydrophobic part), when Protons enter into it over the a part form the outside. B part holds the F₁
  F₁ consist of 5 subunits (a,-Delta). a and b part catalyce 1 ATP for each rotation

• Respiration: Transfer of electrons to an oxidizing substrate like O₂ or also NO₃^- (can be anaerobic)
• Alternative way to produce Energy is fermentation: Means transfer of electrons from one part of the substrate to another part of the same substrate. The electron are then eliminated by reduced metabolites (H₂, acids or alcohols)
• Many bacteria can both production ways, depending on the environment

• IM has many different proteins (20%≈ of total proteins ) compared to OM (≈2%), but in smaller and more regulated amounts
• Proteins are essential for Respiration (energy production, transport and motility)
• IMPs have 1-18 a-Helices, linked by hydrophilic loops and one helix is made out of 20-27 residues (length needed to span the bilayer) (can be perpendicular or tilted)
• Loops can easily be identified, but orientation (cytosol or Periplasm) more difficult, this can be done experimentally with the phosphatase fusion strategy
  • PhoA only gets active if it can make disulfide bonds between internal cysteine residues and this oxidation only happens in the periplasm
  • Gene sequence of PhoA can be inserted to artificial transponsons, which allows them to jump and fuse randomly in the gene if the IMP à PhoA will either be in the periplasm or cytoplasm (active or not active)
  • Active PhoA can be monitored
  • If many colonies are screened and insertion site later determined by DNA sequencing

It was found that generally positive charged residues are on the cytosol side (positive inside rule)

• Made of a bilayer of phospholipids and embedded proteins ( 1:1 amount)
• Three major phospholipid species depending on the species, strain (Belastung) and growth condition in different proportions: Phosphoatidylethonalamine (PE) (70-80%), phosphatidylgycerol (PG) (15-20%) and diphosphatidylglycerol (cardiolipin) (5-10%) (example E.Colu) but no cholesterol or sphingomyelin (typical in Eukaryots)
• To solve membranes and membrane proteins, detergents with long hydrophobic tails can be used. IM and OM (also Proteins) habve different solubility in detergents. Example Triton x-100 solves IM (protein) and not OM à possible to determine if it is from the OM or IM

• IM is the diffusion barrier and the electrostatic isolator of the cell
• To Study the function it has to be separate from the OM
• OM can be labeled with ¹⁴C galactose in the LPS and with ³H labeled Glycerol both membranes (IM and OM) label the phospholipids
• The IM and OM can be collected by centrifugation of the derbies after ultrasonic disruption since the LPS makes the OM more dens than the IM
  • The separation process is done by centrifugation. The cell membranes are put into a 55% sucrose solution and then decreasing gradient (until 25%) is put on top and solution is centrifuged à outer membrane is lower, with the labeled Glycerol and Galactose it is possible to differentiate the,

• Only discovered in 1990 in chloroplast and since they derive from bacteria homologous genes were found in many gram – and + bacetia
• Tat genes are not found in Lactobacilli, which are fermentative bacteria, thy use no terminal electron acceptors and take energy from electron redistribution
• Mutation in path ways are not lethal but disable respiratory pathways, for example in E.coli tat pathway transports enzymes that are involved in electron transfer
• Those enzymes are folded in the cytosol and Tat has to secret fully folded pathways
• Tat pathway signal is similar to the Sec signal (charged N terminal, Hydrophobic core and polar C terminus) and also cleaved of after the transportation, but two difference
  • C region has basic residues and contains a conserved AXA sequence (also in Sec singal the AXA) and  of two arginine residues on the N side, that are almost invariable
• Structure and Function is not understood yet, only that its made from proteins lioke TatA, B,C and A forms the chanel
• It is guesed that A is highly dynamic and only assembles around the substrate during the trandfer
• Tat B and C associate at the beginngin of the process and bind the signal peptide (energy independend stepp) à tat Ais build and transfers the Substrat à signal is cut of and tat A depolymerizes
• No ATPase near the tat proteins which indicates H transfer is used as energy source

• Tat also exports proteins with co-factor or metal which can be shown with GFP, that is fold in the cytosol and with the signal then exported

• Secretion is after the translation but befor the actual folding
• After translation the folding is stopped by SecB chaperone à secA ATPase then mediates interaction with SecYEG chanel à SecA (Dimer)  uses ATP to make conformational changes and pushes about 25 residues through the channel (at once) (ADP/ATP exchange makes the push and ATP hydrolysis reopens SecA chanel and closes SecYpore) à  at the end the signal peptide is removed

• Composed of three domains: N,H,C
  • N terminal domain (N) is 1-10 residues long with 1-3 positive charged residues
  • H: central hydrophobic domain has 10-15 hydrophobic residues
  • C terminal domain is polar and has the recognition site for signal peptidase (separates the signal peptide after secretion

• Is in bacteria, eukaryotes and archaea and insert proteins into the IM (for bacteria)
• In gram – for periplasmic and OM proteins and gram +  cell wall-associated and hydrolytic enzymes
• Exported proteins are first synthesized as pre-protein with and N‘ signal sequence

• Two major pathways: Sec and Tat
• For gram –, proteins are brought to the periplasm, OM or across the OM
• For gram +, translocated proteins are either anchored to the peptidoglycan or released to the outside

• Can cross the OM by their own
• The Peptide consists of three parts: Signal peptide (for Sec), Passenger Domain and b-Domain (c terminal))
• b-Domain forms a b-Barrel in the OM (Bam-Dependent manner) and then the passenger domain can move through into the outside
• Then the the passenger part is either cleaved of (self-catalyzed or by other protease) or stays anchored in the OM
• Many proteins secreted by autotransport are virulence factors

• Membrane association is mediated by a diacylglycerol linked to an N-terminal cysteine
• Lipoproteins (LP) have a signal sequence with a cysteine at +1
• First they are transported through the IM by Sec à 2^nd a diacylglycerol is attached to the SH of the cysteine à 3^rd signal sequence is cleaved of behind the cys à 4^th NH₂ is acylated and anchors the IM
• Then if at the +2 position there is an Aspartate, the LPs stays in the IM
• Otherwise the +3 residue determines the final localization. LP for the OM are transported by an ABC transporter, LoLCDE
• Deletion of lol genes is lethal since it prevents the attachment of OM to Pep.gly.

• Lol D provides the Energy from ATP hydrolysis for C and D which open the hydrophobic cavity of LolA à transports to LolB which then inserts LPs into the OM

• OMPs have a cleavable signal sequence and are exported do the periplasm by Sec
• Folding was thought to be spontaneous, until they realized that a mutated gene from Neisseria meningitis (OMP85 (also called BamA)) has a vital role (without it it died)
• Later it was tested, that when omP85 was knocked out and second gene was induced after a IPTG inducer, the PorB (one porin) was not inserted in the new built membranes when IPTG was in the medium
• b-Barrel assembly machine (Bam) consist out of 5 components (A-E) and form a complex in the OM and together they insert b-barrel proteins in the OM
• Protein for the OM are transportet trough the IM and then kept in the infolded state by Chaperones SurA
• BamA contains POTRA (polypeptide transport-associated) domains that fold and assemble b-barrels
• Two models of how it could work:
  • 1. Bam complex provides a template and initiates b-structure formation
  • 2. Hybrid-barrel formation: delivery factor shuttles unfolded OM b-barrel to TamA and b-strands are formed when the stands are led through the gate regions in TamA. At the end a fission mechanism releases the formed Protein into the OM

• Pili of gram + bacteria were only discovered recently
• They are assembled by covalently interlinking of different subunits through peptide bonds
• The polymerization proses and the anchoring in the peptidoglycan is carried out by pili-specific sortase
• SrtD does the polymerization and StrA the anchoring
• Pilus genes are organized and close to each other (exception SrtA is locates somewhere else)
• Minor pilin lacks the YPKN motive

Process in detail:

• First step: Sec-dependent secretion of pilin subunits, they have a C-terminal LPxTG and N-terminal Pillin motiv YPKN). After transport the subunits remain anchored in the cell membrane
• Second step: Sortase D cleaves the LPxTG motive (between T and G) à acyl-enzyme intermediate where thiol group of cysteine in catalytic pocket of the sorties and carboxyl group of LPXTG form a thioester bond
• Third Step: Oligomerization is done by repeated nucleophilic attacks of the thioester bond (bond between subunit and sortase). The nucleophile comes from the amino group of the lysine in the YPKN à pulus growths by subunit adition at the base of the pilus
• Forth step: Amino group of the pentapeptide from the pep.gly. Precursor lipid II makes the nucleophilic attack and this the pili is anchored in the cell wall.

·      Surface proteins are mostly anchored in the peptidoglycan

·      The proteins are synthesized in the cytoplasm having an N-terminal signal peptide and a C-terminal sorting signal

·      After the signal peptide is cleaved of the binds to the Sec chanel

·      Sortase recognizes the LPXTG sorting signal and cleaves it below it (chain is bond over a thioester to the sortase) (acyl-enzyme untermediate)

·      Free Amino group of the Penta peptide from the MurNac-pentapeptide makes a nucleophilic attack (cell wall crossbridges within lipid II)

·      At the end the surface protein is incorporated into the cell wall by transglycosylation reaction generate from matur pepglyc.

·      Different sortase recognize different sorting signals, which allows protein composition on the cell wall with responds to the environment

• Assembly of Type 1 pili happens at the OMP usher, which is exported by Sec pathway (as well as the pilus subunits)
• Subunits are bound by the chaperon, without the chaperon pilus canot bind to other pilus’s which prevents premature folding
• Usher forms a channel though which the subunits can pass through and dissociates after the last subunits
• Adhesin subunits is the first one and the terminator pilus the last

• One subunit consist out of 7 b-Strand, but for proper folding one extra strand is needed (from the chaperon) and so they can only fold around the chaperon which donates one of its b-strand. This is called donor strand complementation
• Formed subunits is then delivered to the growing chain and its N-terminal extension replaces the donor strand from the previous chaperon (donor strand exchange)
• Adhesin can only be at the beginning, since it has no N-terminal extension
• When PapH comes, the process stops (terminator) because no Nte fits there

• Are appendages of enterobacertia (live typically in the gut) and essential to cause diarrhea and urinary tract infections
• They do not retract and are not involved in motility
• Two different types: Fim (low infections like bladder) and Pap (high infections like kidney)
• made out of a helical Pilus (FimA or PapA)
•  Ends with the fibrillum (lectin which binds sugar)
• Starts from the OM where it’s anchored by the OMP called usher (Pap C or Film D)

• About 4% of the proteins are found here
• Essential degradative enzymes like nuclease, alkaline phosphatase, beta lactamase, is like the lysosome of eukaryotic cells
• Has also proteins that are for the import of substrate
  • Example: TonB, anchored in the plasma membrane and acts as a reseptor with protein from the OM like FhuA à allows transport across the outer membrane
  • DsBA oxydo-reductase: catalyzes formation or S-S bonds ( no S-S bonds in the cytoplasm) or help folding of proteins that are secreted in unfolded form

• Phospholipids are similar to the ones in the inner membrane
• LPS give a hydrophobic character to the outside, it has more fatty acids than phospholipids (so more hydrophobic interaction) and the chains are saturated which increases the molecular packing à less fluidity à les permeability à more resistant to hydrophobic toxic compounds and antibiotics compared to Gram-positive
• Ca²⁺ and MG²⁺ neutralize the negative charge of the phospholipids, if they get into a chelate complex with EDTA the repulsion happens and phospholipids translocate form the inner to the outer sideà permeability increses
• There are also a lot of outer membrane proteins (OMP)
  • Most commonly general trimetric porins which allow of unspecific aqueous diffusion, they consist of anti-parallel beta strand forming a barrel, IM porins consist mostly out of alpha helixes
  • FhuA transports iron Fe 3+
  • LamB is a porin specialized on import of malrose

Global structure:

The lipopolysaccharide (LPS) is a hydrophilic polmer composed out of three parts:

• The anchor: Lipid A
• Polysaccharidic part: core polysaccharide
• Variable polysaccharidic part: repetition of tri, tetra or penta saccharides

Lipid A

• Phosphoglycolipid with beta(1-6)-linked hexosamine backbone with phosphoryl groups and fatty acids with ester an amide linkages with acyl resst
• Variation in nature occurs on the hexosamine type, phosphorylation tipe and chain length of the acyl group and its position
• It is one major microbe-associated molecular patter, which is recognized by the mammalian immunity system
• Toll like receptor 4 (TLR4) recognized them and triggers the process to be protected against infection
• If the response happened in the blood, the response can be to large and lead to a shockà organ failure
• TLR4 activation depends on the two acyloxyacyl chain and numbers of phosphate groups (can be more or less effective)

The polysaccharide

• The core is well conserved but varies from each species
• Variable part made of short repetitive units and usually tri-saccharides or tetra-saccharides, diversity is the basis of to “umgehen” the immune system (O-Antigens)

Assembly of the LPS

• Lipid A and the core are assembled at the cytoplasmic side of the plasma membrane and then flipped accros the bilayer by an ABC transporter
• The O-antigen repeat units by glycosyltransferase on a lipid anchor (C55) and then brought through the membrane by another ABC transporter
• On the periplasmic side the two parts are then added together by the WaaL Ligase
• At the end the hole complex is transportet to the outer membrane by a multiprotein system Lpt (lipopolysaccharide transport)

• Peptidoglycan layer is thinner than gram positive (5-10nm) but also has an outer membrane (lipid bilayer), which consist  of two different lipid layers
  • Inner layer is made of phospholipids
  • Outer layer out of phospholipids and lipopolysaccharide
• Outer layer contains many copies of a few proteins and is attached to the peptidoglycan via specialized lipoproteins, murein lipoprotein
  • In E.coli Braun’s Lipoprotein consist out of 58 AA with 5-7 AA repeats and it is attached to lipids at the N-Terminal Cys with a peptide bridge with the NH₂ group of a Lys
• Space in between is the periplasm

• Have members that cause sever human infections (tuberculosis, leprosy (Lepra), Buruli ulcures)
• Based on their 16s rRNA they are grouped to the Gramm Positive, but they don’t led mild staining into their peptidoglycan layers
• They also have glycolipids, which makes mycobacteria specially impermeable to dyes and other chemical (also antibiotic)
• Only in 2008 with cryo EM it was possible to analyze them in their native state and since then they are trying to find the exact structure, it is known so far, that myocilin is required

• 20-80 nm thick
• Teichoic acid are linear polymers, alternated substituted polyols (glycerol or ribitol) and are covalently bounded to the peptidoglycan
• Lipoteichoic acids have a lipid substituent that anchor into the plasma membrane
• Different proteins are also anchored in the peptidoglycan (eg deradative enzymes)
  they can also be abundant for defense purpose and protect bacteria from complete deposition

• Exoskeleton is made out of peptidoglycan, proteins and Lipids, which hold from 5-20 bar)
• Gramm Test: Bakterias fixed with methanol, stained with crystal violet and Iodine à washed with ethanol à counter stained with eosin (pink) à two main types
• Gramm Positive:
  • Stay violet in the Gram test (the Iodine is “trapped” under all the peptidoglycan layers)
  • Thick cell wall, multiple layers of peptidoglycan (Teichoic acids hold them to the cell membrane
• Gramm Negative:
  • Stay collarless after the Gram test (loosely trapped Iodine is easily washed out, since there is only one pept. layer
  • Single layer of peptidoglycan and inner and outer membrane
  • Lipoproteins connect Pept. With outer membrane
  • Outer membrane is more porous
  • Better protected against harsh environment (eg. Toxic compounds)
• Both may have a s-layer on the outside and carbohydrates that form the capsule

• Consists of phospholipid bilayer, with hydrophobic fatty acids directed inward and phosphoryl (negative chargerd) outwards
• Hopanoids act as a stiffening agents (like cholesterol in eukaryotes)
• 50% of the volume consists of membrane protein

• Membrane lipid composition varies depending on the environment or region of the cell
  • Positive charged head groups are found often in membranes interacting with DNA
  • Cardiolipid increase under starvation
  • Cis unsaturated fatty acids increase membrane fluidity (for low temperature)

• Membrane is semipermeable, soluble protein, small, uncharged molecules (O2, CO2, H2O) go through, polar molecules, charged ions, large compounds do not
• Weak acid and bases can go through in their uncharged state
• Water permeability increases with specific channels (aquaporins)

• Range size is from one to a few um
• Cyto plasma is sourounded by a membrane, cell wall with a rigid layer of peptidoglycan and some have a capsule
• Pili are used to make contact with other cells
• Flagella and rotary motors allow mobility
• Genome is on circular DS DNA
• Chemical composition varies with the species and the environmental conditions
• E.Coli consist out of 70% water, 16 % Proteins, 6% RNA, 3%Lipids 1 %DNA, rest other molecules. Essential ions like Ka, Mg, Phospahte

• Peptide antibiotics like bacitracin interfere with the C55-cycle
• C55 cycle shuttels the disaccharide-pentapeptide from the cytoplasm into the periplasm

• Structural analog of D-alanine and inhibits alanine racemas (L-Ala into D-Ala) and also inhibits D-Ala-ligase, which dimerizes D-Ala

• Cyclic gylcopeptide active against Gramm-positive (cannot diffuse across the OM of -)
• Inhibits as well the PG synthesis but complexes terminal D-Ala-D-Ala from lipid 2 àtranspeptidase is not possible
• Coupling the drug to fluorescent dye it was possible to study, where and how peptidoglycan assembles
• Last active frontline drug against some staphyslococcy that have resistance against penicillins and other antibioticas à causes problems in hospitals
• Enterococci replaced Acyl-D-Ala-D-Ala by Acly-D-Ala-D-latcice acid, this modification only needed two enzymes:¨à resistant to vancomycin
  • VanH pyruvate, produces D-Lactate
  • VanA ligase, couples D-lactate to D-Ala
• High-level resistance uses VanX, which stops the normal pathway
• Terminal group of the pentapeptide does not play any role in the crosslinking à function of crosslinking is still normal strength
• Vancomycin is 1000 lower binding affinity to the Lac
• The Van… enzymes are encoded by the van… genes

• are evolutionary related to the transpeptidase and have a bit larger active site, allowing water to hydrolys the penicilloyl-enzyme complex
• β lactamase have lost their original transpeptidase activity

• β lactamase help to protect enzymes outside the cell à in Gramm-positive thy are secreted in negative they are localized in the periplasm (more cos-efficient, no losses by diffusion)

• Their expression in carefully controlled
• Inducer is a muropeptide (is a product from PG from uncontrolled hydrolysis from β-lactam inhibition, this mechanism differs from classical systems where the substate (e.g Lactose) induces the sysnthesis of the enzyme involved in its breakdown. This regulation makes sense since the targets of β lactam are in the periplasm and transcription machinery in the cell, if β lactam would regulate it, then the reaction would take to long and the bacteria would dye before
• AmpG transpotts the muropeptide into the cytoplasm

• β-lactams antibiotics function as structural analogs of the acyl-D-Ala-D-Ala but its reaction is irreversibly
• Transpeptidase reacts irreversibly with the β-lactam antibiotics and produce a stable penicilloyl-enzyme complex which cannot be hydrolyzed with water (Water cannot penetrated into the active site)

• Using ³H-Penicillin they can be visualized
• Β-Lactam are also bacteriocidal, not only stop bacterial growth but also kill them
• The cell wall is weakened an no longer to able to hold the pressure
• the peptidoglycan is under constant remodeling (for cell growth), Model proposes that a multi-enzyme complex goes along the peptidoglycan structure and replaces one strand with tree new strands

• β lactamase destroys the lactam ring of those antibiotic -->  no antimicrobial activity

• Largest family of antibiotics used, several hundreds of β lactam have been synthesized
• 4 main families, differ in activity, sensitivity to β lactamase

• Peptidoglycan is in almost all bacteria and only found theirà ideal target for antimicrobial chemotherapy
• Different compounds attacking different targets of the peptidoglycan assembly have been developed

• Three major steps occur in the three different places, cytoplasm, membrane and periplasm
• First step(plasma): UDP-Glc-Nac and UPD-Mue-NAc-pentapetide
  • UPD-Glc-Nac is a common metabolite
  • UPD-Mue-NAc-pentapetide has to be made from UPD-Glc-Nac by the six enzymes MurABCDEF
    • A: PEP ist attached and then the phosphate group removed
    • B: DB (where phosphate was removed) is reduced
    • C: L-Alanine is added
    • D: D-Glu is added
    • E: diaminated residue is added
    • F: D-ala D-ala is added

• Second step (plasma membrane):
  • Membraneintegral lipid carrier binds to the Mur-NAc-pentapeptide (lipid 1) then Glc-NAc is added (lipid 2)
  • MurJ flipase flips over the disaccharide-pentapeptide across the membrane
• Third step (Periplasm):
  • Transglycosylase (TGs) brings and binds the disaccharide-pentapeptide to a growing peptidoglycan chain
  • Trasnpeptidation reaction cross-links a “donor” D-Ala (4) with the amine of the meso-DAP(acceptor) via two steps:
    • 1. Donor acyl-D-Ala-D-ala forms an Acyl-Enzyme complex and on D-Ala (5) is released
    • 2- Amino group from acceptor binds to it and the enzyme is released
    • Carboxypeptidase controls the process

• Synthesis through polymerization of disaccharide peptide units (M and G) linked over an betha 1-4 bound and form the glycan chain.
• Each M saccharide has e chain with the peptide (L-Ala-D-Glu-Meso-Dap (L-Lys)-D-Ala-D-Ala) (the D-Glu iscoupled over its gamma and nod alpha carboxyl group)
• Two side bridges are linked together and the energy needed for this comes from de D-Ala-D-Ala hydrolysis, the linking varies form species, for E-colie the Carboxyl group from D-Ala (4) binds to the NH₂ group from the diaminated residue (3)