Module 2: Bacteria (Part 2) Flashcards
What is the bacterial cell wall made of?
Cross-Linked Peptidoglycan
Peptidoglycan
A polymer consisting of alternating sugars linked together by short peptide chains
What is the structure of the glycan backbone of peptidoglycan?
Alternating NAM + NAG
(attached to one another via B-1,4-glycosidic linkage)
NAM = N-acetylmuramic acid
NAG = N-acetylglucosamine
How are NAM and NAG connected?
Beta-1,4-Glycosidic Linkage
Describe the entire structure of cross-linked peptidoglycan?
Glycan backbone strands (made up of B-1,4-linked NAM and NAG units) are cross linked together via short 3-5 AA peptide chains on NAMs
= Forms a mesh-like network!
B-1,4-Glycosidic Linkage
A COVALENT bond between two sugar molecules
Oxygen on C1 of one sugar (from OH grp) bonds to C4 of another sugar molecule (loses an OH)
B-1,4-Glycosidic Linkage Formation Rxn
Dehydration Reaction (releases H2O)
Oxygen from OH grp on C1 of one sugar attacks C4 of other sugar causing the C1 OH to lose the hydrogen and C4 of the other sugar to lose an OH
Bond forms between C1 oxygen and C4 of other sugar while the lost OH + H form water
In both gram + and gram - bacteria, what is the same about the NAM peptide chain involved in cross-linking?
1st AA (attached to NAM) = L-alanine
4th/5th AA in chain = D-alanine
What crosslinking-peptide AAs vary between gram - and gram +?
2nd AA
–> Gram (-) = D-glutamine
–> Gram (+) = D-isoglutamine (gluNH2)
The THIRD AA –> Varies amongst all bacteria (even within gram -/+ groups)
In peptidoglycan crosslinking, what AAs get linked?
4th AA of one chain connects to 3rd AA of another chain
What are the 2 methods of crosslinking?
1) Direct AA-Linking
2) Interbridge Linking
Crosslinking: Direct-AA-Linking
4th AA on one NAM covalently bonds to 3rd AA on second NAM (directly linking the two chains)
Crosslinking: Interbridges
Bridge of AAs connects the 3rd AA on one NAM to the 4th AA on a second NAM
–> Exact length and composition of interbridge can vary!!
What interbridge does staphylococcous aureus have?
A pentaglycine interbridge (5 glycines)
What crosslinking methods are most common for Gram (+) and Gram (-) bacteria?
Gram (-) = Direct AA Linking
Gram (+) = Interbridge Linking
Crosslinking
Connection formed between peptide chains attached to NAM subunits
–> Very important for the STRENGTH of the peptidoglycan network!!!
What is weird about peptidoglycan AAs?
Many of them are the D-isomer AAs
–> D-AAs are rarely ever found elsewhere
–> L-AAs are almost always used by ribosomes
D-AA + L-AA =
Stereoisomers! (same formula, different 3D arrangement)
Synthesis Pathway of Peptidoglycan
CYTOPLASM:
1) NAG (already made) reacts with UTP to form UDP-NAG
2) UDP-NAG converts to UDP-NAM
3) AAs are added to UDP NAM to form UDP-NAM-pentapeptide
4) Phosphate attached to bactoprenol attacks the UDP on UDP-NAM-pentapeptide = knocks of UMP and links bactoprenol to NAM-pentapeptide
5) NAG is bonded to the NAM attached to bactoprenol (B-1,4-glycosidic linkage forms)
6) Bactoprenol FLIPS in the PM, pulling the attached NAM-NAG complex to the ECF/periplasm
ECF/PERIPLASM:
7) TRANSGLYCOSYLATION = NAM/NAG complex gets added to a growing peptidoglycan strand (OH of NAG from the bactoprenol complex forms link with NAM on already existing strand)
–> By the Transglycosylase Enzyme
8) Crosslinking = Pentapeptide chain from the newly added NAM gets crosslinked to peptide chain of another NAM (5th AA breaks off in this process)
–> By the enzyme Transpeptidase
9) Bactoprenol detaches from the NAM-NAG complex and flips back over to the cytoplasm again
What are the enzymes transglycosylase and transpeptidase used for?
Transglycosylase = Enzyme that forms glycosidic linkage between new NAG and NAM from pre-existing peptidoglycan strand
Transpeptidase = Enzyme that conducts the crosslinking between NAM peptide chains
Lysozyme
An enzyme that degrades peptidoglycan by hydrolyzing the B-1,4-glycosidic linkage between NAM/NAG subunits
What bond does the lysozyme break in peptidoglycan?
Through HYDROLYSIS
–> Addition of water to break a bond
Breaks the B-1,4-linkage by adding water
Lysostaphin
An enzyme that breaks down cross-linked peptidoglycan by attacking and cleaving the interbridges
What type of enzyme is lysostaphin?
An ENDOPEPTIDASE –> Breaks peptide bonds between AAs
–> Breaks the peptide bonds between interbridge AAs!
Lysostaphin action on S. aureus
Lysostaphin breaks the peptide bonds between the glycine AAs in the pentaglycine interbridge
Why is breakdown of the peptidoglycan layer dangerous for bacteria?
It can leave them susceptible to lysis! (Due to osmotic effects!)
What are the two enzymes used to breakdown peptidoglycan?
1) Lysozymes
2) Lysostaphin
Protoplast
A bacterial cell WITHOUT its cell wall
–> Has ONE membrane –> Made from GRAM (+)
Spheroplast
A bacterial cell without its cell wall BUT with TWO membranes –> Made from GRAM (-)
Protoplast in Isotonic Solution vs Hypotonic Solution
Isotonic Solution = Protoplast survives
Hypotonic Solution = Protoplast LYSES (cell swells with H2O until it bursts)
How do most antibiotics work?
By targeting the bacterial cell wall!
B-Lactam Antibiotics
A family of drugs that contain a B-Lactam Ring
–> These drugs PREVENT the formation of bacterial cell wall
What does the B-Lactam Ring do?
The B-lactam ring MIMICS the structure of the 4th AA of peptidoglycan crosslinking peptide chains = D-alanine
–> Therefore, it “tricks” transpeptidase into binding to the ring rather than the peptide chain!
–> Upon binding the ring to its active site, transpeptidase is permanently destroyed
OVERALL = prevents peptidoglycan crosslinking and therefore cell wall formation
What is the limitation to B-Lactam action?
Only works on GROWING bacteria
–> It can’t destroy the cell wall once its formed, it can only PREVENT its formation in the first place
Examples of B-Lactam Drugs
1) Penicillin
2) Carbapenem
3) Monobactam
4) Cephalosporin
What is the structure of the B-Lactam Ring?
A 4-membered cyclic amide (square ring with 3 carbons and one NH)
The nitrogen in the ring is adjacent to a carbonyl (C=O) carbon
B-Lactamases
Enzymes that break open the B-lactam ring, rendering it inactive
–> Hydrolyze (add water to break) the C-N bond in the ring
(carbonyl grp becomes carboxylic acid by OH addition)
(NH grp becomes NH2 by H addition)
Why was amoxicillin made?
Why was it still not enough?
A new drug made that was more resistant to B-Lactamases
HOWEVER, bacteria quickly altered their B-lactamases to also attack the amoxicillin
Clavulanic Acid
A variant of the B-Lactam Ring that is used as a competitive inhibitor for B-lactamases
Why does clavulanic acid prevent B-Lactam drug inactivation?
Because calvulanic acid is a form of B-lactam ring with a higher affinity for B-lactamases!
–> The silly B-lactamases then bind to this clavulanic acid instead of the antibiotic B-lactam ring, allowing the antibiotic to stay intact and work!
How is clavulanic acid used now?
Used in COMBO with B-lactam drugs to protect the drugs from deactivation
Augmentin
A drug combo of Amoxicillin and Clavulanic Acid
All bacteria can be separated into one of two classes:
Gram (+) or Gram (-)
Who discovered the difference between Gram +/- bacteria?
Hans Christian Gram (1884) –> (via the Gram Stain)
The Gram Stain (original purpose)
Used as a method of detecting bacteria in living samples of individuals who died of pneumonia
What type of stain is the gram stain?
A “differential” stain = Bacterial species stain differently upon the structure of their cellular envelope
Gram Stain Steps
1) Stain cells with Crystal Violet (CV)
2) Treat cells with Iodine
3) Treat cells with Alcohol
4) Stain cells with Safranin
What is the purpose of each component of the gram stain?
Crystal Violet = Primary Stain
Iodine = “Trapping Agent” (forms an insoluble complex with CV)
Alcohol = Decolorizer (for gram (-) = membrane dissolver and washes out CV-iodine complex )
Safranin = Counterstain (secondary stain to visualize decolorized bacteria)
Gram (+) Bacteria: Cell Envelope Order
Cytoplasm to ECF:
1) PM
2) NARROW periplasmic space
3) THICK peptidoglycan layer (~20-80nm)
4) LTAs + WTAs within the peptidolgycan layer
Think of it like being (+) for the gram test, CV stains it so the gram test is positive!
Gram (+) Bacteria: Cell Wall composition
Thick peptidoglycan layer (~20-80nm) that contains unique glycopolymers: TEICHOIC ACIDS (LTAs and WTAs)
Teichoic Acids
Glycopolymers unique to gram (+) cells
–> Polymers of repeating sugar + phosphate grps
–> Two types:
1) Lipoteichoic Acids
2) Wall teichoic acids
Lipoteichoic acids (LTAs) vs Wall teichoic acids (WTAs)
LTAs = Teichoic acid molecules anchored to the PM via a lipid tail
WTAs = Teichoic acid molecules anchored IN the cell wall by covalent bonds to peptidoglycan chains
Function of Teichoic Acids
Mainly to provide RIGIDITY and STABILITY to the cell wall
–> Due to the (-) charge of the molecules (from phosphate grps), they attract CATIONS (Mg2+, Na+)
What percentage of the dry weight of Gram (+) bacteria is peptidoglycan?
~90% of the dry weight
How wide is the peptidoglycan layer in Gram (+) cells?
~20-80nm
Gram Stain in Gram (+)
1) CV stains the cells purple
2) Iodine forms the CV-iodine complex
3) Alcohol cannot get fully through the cell wall, the CV-iodine complex persists/remains
4) Safranin counterstain is applied but does not show due to the darker CV stain dominating
= PURPLE COLOR
Gram (-) Bacteria: Cell Envelope Order
Cytoplasm to ECF:
1) PM
2) LARGE periplasmic space
3) THIN peptidoglycan layer (followed by a little more periplasm)
4) OUTER MEMBRANE
Gram (-) Bacteria: Outer Membrane
A lipid bilayer with two different leafs:
Inner Leaf (facing periplasm) = Phospholipids
Outer Leaf (Facing ECF) = Lipopolysaccharides (LPS)
Gram (-) Bacteria: Cell Wall Composition
(Thickness + % Dry weight)
Thin peptidoglycan layer that is ~7-8nm wide!
(Peptidoglycan accounts for ~10% of cellular dry weight)
Lipopolysaccharide Structure
3 main parts:
1) Lipid A
2) CORE polysaccharide
3) O-Side-Chain
LPS: Lipid A
Lipid A is hydrophobic and makes up the OUTER LEAF of the OM!
–> Holds the entire LPS unit to the cell envelope
LPS: Core Polysaccharide
Series of sugar monomers (roughly the same in all bacteria!)
LPS: O-side-chain
Shorter series of sugar monomers whose sequence and composition largely varies between species
What is the function of LPS?
Protection from external threats! Makes up a second lipid bilayer which adds another barrier to prevent many substances that could be harmful from entering the cell
What do LPS and LTA have in common?
Both trigger a strong immune inflammatory response in humans! = Responsible for many symptoms of infection
What part of LPS is specifically an immune trigger?
Lipid A!
Gram stain in Gram (-)
1) CV stains the cells
2) Iodine treatment creates the CV-Iodine complex
3) Alcohol treatment DISSOLVES the OM, making the CV-iodine complex able to wash OUT
–> CV-iodine complex is washed away!(NOT in cell anymore) = COLORLESS
4) Safranin counterstain stains the cells PINK
Outcome = PINK COLORED CELLS
What are two main mechanisms of transport through the Gram (-) OM?
1) PORINS
2) TON-B
Porins
3 subunit pores in the OM that allows for the DIFFUSION of polar molecules from the ECF to the periplasm
(once in the periplasm, some PM transport mechanism must get the material into the cytoplasm)
The largest known porin can fit molecules up to…
600 daltons
How does porin size influence antibiotic effectiveness?
Porins of the OM only allow materials <600da however, many antibiotic agents are > 600da SO the OM prevents them from getting into the cell!
Ton-B System
Has 3 main components:
1) Ton-B Dependent Receptor (in the OM)
2) Ton-B (in periplasm/PM)
3) ExB/ExD (in the PM)
–> TonB and ExB/ExD form a complex together
Ton-B Transport Process
1) ECF substrate interacts with the Ton-B Dependent Receptor (TBDR)
2) TonB-ExB/D complex uses the PMF to interact with the TBDR triggering a conformational change in the TBDR
3) Conformational change of TBDR allows the substrate to pass through and into the periplasmic space
(once in the periplasm, the substrate is available to other PM transport systems to get into the cytoplasm!)
What is the energy source of TonB transport?
PMF from the plasma membrane!
What are the main functions of cell surface molecules? (4)
1) Allowing/Facilitating Movement
2) Sticking to surfaces
3) Sensing the environment
4) Acquiring nutrients
Flagella
Spiral filaments that extend from the surface of the cell and rotate in order to propel the cell
What are flagella used for?
1) ACTIVE movement of bacterial cells
2) Sensory structures
Monotrichuous
Bacteria with ONE polar flagellum
Lophotrichuous
Bacteria with MORE than one flagella at either ends
Amphitrichuous
Bacteria with one flagellum at either end
Peritrichuous
Bacteria with flagella all over the cell surface
What is the structure of a flagellum?
3 main structural units:
1) Flagellar filament
2) Hook
3) Basal Body
Flagellar Filament: Composition + Purpose
Purpose = Acts as a helical propeller; propels bacteria forward by rotating and pushing against surroundings
Composition = Made of multiple copies of FLAGELLIN
Length of Flagellar Filament
Typically longer than the cell body itself!
Usually around ~5-10nm long
Flagellar Hook
A flexible “rotating joint” that connects the flagellar motor (basal body) to the filament
Flagellar Hook Purpose
By connecting the motor to the filament, it allows for the rotational torque from the motor to be transmitted to the filament
Flagellar Basal Body
A structure made up of a central rod and multiple discs/rings that extends through the cell envelope (OM to PM)
–> Anchors the flagellum to the cell AND holds/interfaces with the MOTOR
What are the parts of the flagellar motor and their respective functions?
1) Stator = Harnesses the energy from the PMF (protons flow through it causing the rotor to turn)
–> Stationary component
2) Rotor = Rotating component that creates the rotational source for flagellar movement
Where is the flagellar motor found?
Within the PM of the cell (inner membrane)
How does flagellar movement occur in monotrichuous bacteria?
NO RUN + TUMBLE
–> The direction of the singular flagellar rotation simply pushes or pulls the cell
How does flagellar movement occur in peritrichuous bacteria?
Exhibit a RUN + TUMBLE method of movement
Run + Tumble
RUN = All flagella rotating in one uniform direction (counter-clockwise usually) that causes the flagella to twist together and propel the bacteria FORWARD
TUMBLE = All flagella reverse their rotation (goes to clockwise) which causes the flagella to fly apart = the cell spins in place (new direction is chosen)
RUN = All flagella go back to rotating in their original direction (counter-clockwise usually), bacteria starts moving forward again in a new direction
Chemotaxis
The use of chemical signals from environment to direct movement
How does chemotaxis occur in bacteria?
Chemical receptors on the bacteria’s surface detect ATTRACTANTS and REPELLANTS
–> Bacteria will try to move TOWARDS areas of higher concentrations of detected attractants (or areas of lower conc. of repellants)
–> Bacteria will try to move AWAY from areas of higher concentrations of detected repellants (or areas of lower conc. of attractants)
What occurs if a bacterium detects an increasing concentration of an attractant?
The bacterium will continue a RUN in that direction (flagellar motor will maintain its rotation direction; no signal to change rotation direction)
How do chemoreceptors communicate what they have sensed to direct flagellar movement?
Via cytoplasmic intermediary proteins
What occurs if a bacterium detects an increasing concentration of a repellant?
The bacterium motor will be signaled to change rotation direction initiating a TUMBLE to try and change movement direction away from the area of higher repellant concentration
What occurs if a bacterium detects a decreasing concentration of an attractant?
The bacterium motor will be signaled to change the rotation direction, initiating a TUMBLE to try and change the movement direction away from the area of lower concentration in attempts to find where higher concentrations of attractants exist
Bacterium move _______ attractant gradient and move _________ repellant gradient
1) Move UP attractant gradient (towards higher conc)
2) Move DOWN repellent gradient (towards lower conc)
What bacteria have axial flagella?
Spirochetes
Axial Flagella
Flagella WITHIN the periplasm whose filaments wrap around the cell body
Polar Flagella
Flagella at the ENDS of cells
How do spirochetes move?
When the axial flagella rotate, it causes the entire cell to spin like a corkscrew!
Pili (general term)
Proteinaceous, hair-like fibers that protrude from the cell surface
Types of pili
2 main types of pili:
1) Fimbriae (adherance)
2) Pilus (sex)
Fimbriae
Pili that serve to allow bacteria to attach to surfaces (including other cells!)
What are pili made of?
PILLIN proteins
What gives fimbriae the adhesive characteristics?
Tips of the fimbriae contain distinct proteins that act as ADHESINS designed to bind to specific molecules on target surfaces
Importance of Fimbriae (2)
1) Pathogenic bacteria: fimbriae facilitates the adherence of bacteria to target cell in a host (often the first step of infection)
2) Bacteria in environment with fluid flow: Allows the bacteria to adhere to a surface, allowing them to persist in the environment and not get washed away
Conjugal Pilus
AKA Sex Pilus
A structure used for CONJUGATION in bacteria
Conjugation
Process by which one bacterium transfers genetic material (usually a plasmid) to another bacterium through direct contact
Conjugation Process
1) Donor cell produces a pilus
2) Pilus of donor cell attaches to a recipient cell, bringing the two cells together
3) The plasmid getting donated is cut to release a single strand that gets passed to the recipient cell
4) The donor and recipient detach from one another and both synthesize a complementary strand to re-produce the original ds plasmid
5) Recipient cell can now go on to be a donor cell
Capsule
A thick polysaccharide layer surrounding some bacterial cells –> Uniform in composition (made up of the same sugars over and over again but the sugars can vary between bacteria)
Capsular Polysaccharides are also referred to as…
K-Antigens
Main functions of bacterial capsule (3)
1) Immune evasion
2) Dessication protection
3) Biofilm formation and stabilization
How do capsules contribute to virulence of bacteria?
Capsule polysaccharides COVER the bacterial antigens found on their cell surfaces (which get detected by the immune system), prevents detection
AND
Capsule polysaccharides are often NOT recognized by host immune system and so bacterial detection does not occur! = no immune response possible
How does the bacterial capsule protect against dessication?
Capsule building-blocks (saccharides) re hydrophilic!
–> The capsule helps to retain water!
Biofilm
A microbial community adhered to a surface with a matrix of capsular polysaccharides holding the cells together
Why are biofilms beneficial?
Provides protection and enhanced survivability in harsh environments!
S-Layer
AKA. Surface Arrays
A MONO-molecular, crystalline-like layer of identical proteins or glycoproteins on a bacterium’s surface
Where is the S-layer found?
Above the outer membrane (gram -) or peptidoglycan layer (gram +) but BELOW the capsule!
How is the S-layer attached to Gram (+) cells?
S layer is bound to the peptidoglycan layer via SECONDARY CELL WALL POLYMERS
How is the S-layer attached to Gram (-) cells?
S-layer is bound to the OM via the Lipopolysaccharides
Functions of the S-Layer
1) Prevents bacteriophage infection = prevents phages from directly attaching to bacterial membrane receptors needed for infection
2) Prevents host immune attack = acts as “camoflauge” to the immune system (hides key molecular patterns used by the immune system to recognize bacteria as foreign (Ex: LPS)
What is the hierarchical taxonomical system (by groupings)?
1) Phylum
2) Class
3) Order
4) Family
5) Genus
6) Species
6A) Strains
How are bacteria named?
Using the binomial system:
GENUS-SPECIES
Bacterial Species
Grouping of strains that share common features while differing considerably from other strains
Genus
Group of closely related species
Taxon
Each named grouping in the hierarchical taxonomic system
