Prokaryote Biology Module 8: Cell Wall and Capsule Biosynthesis

Saccharides

Simple sugars, combination sugars, polymerized sugars

Oligosaccharide

3-10 sugars

Glycogen

Polysaccharide with energy/carbon storage function

Examples of polysaccharides in cell structures

LPS, Capsules, Glycan strands in PG

Glycogen structure

Polymer of alpha-1,4 (glycan) and alpha 1,6 (cross-bridge) linked glucose units

All domains of life

____ synthesize(s) glycogen.

High energy environments where excess glucose is available

Glycogen is generated in…

It doesn’t generate osmotic stress

Glycogen can be stored in large amounts because…

UTP and ATP; PPi

Glycogen is activated by ______ and is driven by the hydrolysis of _____.

Glucose + ATP → G6P

First step of glycogen formation

G6P → Glucose-1-phosphate (mutase)

Second step of glycogen formation

G1P + UTP → UDP-glucose + PPi

Third step of glycogen formation

UDP-glucose

High energy molecule that can be attacked by nucleophile to drive forward polymerization

Peptidoglycan structure

Repeating glycan strands of NAG, beta 1,4 to NAM (linkages cleaved by lysozyme) connected via peptide cross bridges

Peptide chains of peptidoglycan

NAM lactyl group (D-lac) is covalently linked to pentapeptide chain w/ D and L amino acids

Third

The ____ amino acid in the pentapetide chain varies between organisms and used for bacterial classification (DAP in E. coli, multi-amino acid bridge in G+)

Small structures; major polymerization

During peptidoglycan synthesis, ______ are made inside the cytosol, while ______ occurs outside the cell.

ATP

During peptidoglycan synthesis, all _____ input occurs inside the cell.

F6P, Gln, Acetyl-CoA, PEP, NADPH, L/D-amino acids, ATP, UTP

Substrates for formation of NAG and NAM-pentapeptide UDP-precursors in cystosol

F6P gets aminated by glutamine, forming Glucosamine-1-P and glutamate byproduct

First step of cytosol PG synthesis

Acylation of Glucosamine-1-P into NAG-1-P

Second step of cytosol PG synthesis

Activation of NAG-1-P by UTP-dependent reaction, forming UTP-NAG (ready to receive nucleic attack) and PPi

Third step of cytosol PG synthesis

Phosphoenolpyruvate (PEP)

Lactyl group that will eventually form NAM w/ pentapeptide chain

Steps of NAM formation

Addition of lactyl group

Reduction

ATP-dependent alanine addition (first acid)

Mycobacteria have side pathways

Stepwise addition of amino acids onto NAM

ATP; DNA and the ribosome

The stepwise addition of amino acids onto NAM is ____ dependent but independent of ____.

Second; the inner leaflet of the cell membrane; membrane enzymes

Attachment of UDP-sugars to lipid carrier is the ______ overall step of PG synthesis and occurs on ______ and is catalyzed by ______.

Meditates enzyme catalyzed nucleophilic attack on UDP-activated NAM

Function of undecaprenyl lipid carrier in PG synthesis

UDP-NAM-pentapeptide attacked by P-lipid to form lipid I, releasing UMP

Formation of lipid I in UDP-sugar attachment to lipid carrier

-OH group on NAM attacks phoshoryl group of UDP-NAG, forming beta 1,4 glycositic linkage

Formation of lipid II in UDP-sugar attachment to lipid carrier

tRNA; high-GC G+ bacteria

Formation of PG amino acid bridge is ____ dependent in ____, but still does not involve the ribosome.

Other biochemical reactions involving aminoacyl-tRNAs (independent of ribosome)

Porphyrin formation

N-terminal protein modification

AA-phosphatidylglycerol

MurJ flippase (floppase)

Member of MOP superfamily that facilitates movement of UDP-sugar-lipid complex with hydrophilic AA chain to outside of cell membrane

Identification of related flippase/floppases in MOP superfamily for lipid-linked oligosaccharides (even in mammalian cells)

MurJ enables…

On the outer leaflet of the cell membrane.

Polymerization of peptidoglycan strand and formation of peptide crosslinks occurs…

The third amino acid of one pentapeptide stem and the fourth amino acid (D-alanine) of an adjacent pentapeptide stem.

In peptidoglycan cross-bridging, the cross-link forms between

Cleaved off by transpeptidase enzymes during the cross-bridging reaction.

The fifth amino acid of the pentapeptide chain (D-Alanine) is….

Also occurs on cell membrane outer leaflet

Polymerization of peptidoglycan strand and formation of peptide crosslinks

Class A Penicillin Binding Proteins

Polymerize peptidoglycan from disaccharide peptides (glycosyltransferase) and form muramyl peptide cross-links (transpeptidase)

Class B Penicillin Binding Proteins

Monofunctional d,d-transpeptidases that form only the peptide cross-links

Transglycosylation reaction

Extracellular polymerization of glycan chains

Independent

Transglycosylation reaction is ATP _____.

Lipid-PP-disaccharide transferred to growing PG via transglycosylase; C4 -OH on NAG attacks activated glycan strand, forming beta-1,4 linkage and displacing old lipid carrier

Transglycosylation reaction mechanism

Gets hydrolyzed as pyrophosphate to form lipid phosphate to serve as carrier for next round

Fate of old lipid carrier in transglycolsylation reaction

Hydrolysis; exergonic; energy; ATP

PG cross-bridging is driven by ______ of existing peptide bonds, which is ______, meaning ______ is released and no ____ is needed.

Exergonic; transglycosylation; forward; ATP

The _____ nature of transpeptidation is coupled to _____ and also drives the reaction _____ without requiring _____.

Transpeptidase mechanism

Nucleophile from one chain attacks peptide bond, releasing terminal (fifth) alanine after bond hydrolysis, forming new peptide bond.

covalent.

Peptide bonds are…

LPS

Forms outer leaflet of G- outer membrane

Provides structural support for bacteria

Can trigger immune system

LPS components

Lipid A (A), Core Polysaccharide (C), O-Antigen (O)

Lipid A

N-Glucosamine in beta 1,6 linkage esterified to fatty acid chains (formation is UDP dependent)

Covalently linked

Lipid A and the Core polysaccharide are _____.

O-Antigen

Hexose sugar units that repeat up to 30 times

Lipooligosaccharide (LOS) biosynthesis

First step of LPS synthesis—same as inner-membrane steps of PG; product has dissacharide linkage

Lipid carriers for LOS biosynthesis

Acyl chains of lipid A serves as lipid carrier for core antigen and lipid A group

Flops LOS to outer membrane in ATP-dependent reaction in LPS biosynthesis

MsbA flippase/floppase of ABC-type family mechanism

O-Antigen synthesis

Happens on undecaprenol lipid carriers (same as PG synthesis); flipped by O-antigen floppase (Wzx).

A member of MOP superfamily (related to MurJ)

Wzx floppase is…

O-antigen; outer membrane

Like transglycosylation in PG, extension of the _____ also occurs on the ______.

Transglycosylation (LPS)

Extension of the O-antigen also occurs through…

Lpt system

Transports LPS from outer leaflet of inner membrane to outer leaflet of outer membrane.

Lpt system structure and function

Hydrophobic channel allows hydrophobic fatty acid chains to traverse hydrophilic periplasmic space

Bacterial capsules structure

98% water, 2% polysaccharide, glycoprotein, polypeptide, or both

Protects cell (against dessication, mechanical injury, temperature shifts, phagocytosis)

Virulence

Biofilm attachment and chemical repulsion (function of charge)

Nutrition

Functions of bacterial capsules

Polysaccharide layers attached to the cell wall, usually with lipophilic anchors (eg. LPS and grp II K)

Compared to slime layers, bacterial capsules have…

Loosely associated polysaccharide layer without membrane anchor that can be easily removed (levans or dextrans).

Compared to bacterial capsules, slime layers have…

Variable

The saccharide units making up most of the bacterial capsule are highly _____, even in a single species like E. coli.

Capsule formation in pathogenic E. coli

Similar transglycosylation mechanism to O-antigen, using an undecaprenal PPi carrier and Wzx floppase.

Nucleotide diphosphates

Like in LPS and PG biosynthesis, sugar units in capsule synthesis are charged with _____.

Wza channel

Moves oligosaccharide chain through outer membrane in capsule synthesis

Wzc and Wzb enzymes

Bacterial capsule assembly is regulated by…

Wzb

Phosphatase that removes phosphoryl groups from the enzyme complex to activate it

Wzc

Bacterial tyrosine autokinase (BY-kinase)

Dephosphorylated form

Wzc/Wzb is active in its _____, where its channel functions and allows capsule to move to the outside of the cell.

inhibits; activates

Wzc dominance _____ capsule transport while Wzb dominance _____ it.

not phosphorylated (Wzb phosphatase > Wzc kinase)

Capsule transport can only occur when Wzc/Wzb is ________.

Penicillin structure

Cyclic analogs of D-Ala-D-Ala

D-Ala-D-Ala

Terminal two amino acids in pentapeptide chain before getting cleaved and forming peptide cross bridges

Transpeptidase

Catalyze isopeptide bond formation which cleaves terminal alanine off of the pentapeptide and forms cross bridges.

active site of Penicillin Binding Proteins (D,D-transpeptidases (PBPs))

Beta lactams bind to _____.

Beta lactamases

Evolutionarily related to penicillin binding proteins but they are more hydrophilic; can accommodate and cleave antibiotics

Polypeptide antibiotics

prevent PG synthesis on inner-membrane; includes bacitracin, teixobactin, and clovibactin

Bacitracin antibiotics and mode of action

Binds to divalent (II) metal ions; complexes bind to lipid-PP, preventing hydrolysis to lipid-P

Hydrolysis of lipid-PP to lipid P (required for PG, LPS); binds and oxidatively cleaves DNA

Bacitracin blocks ______; auxiliary function: ______.

Teixobactin antibiotic and mode of action

Two-pronged attack on cell envelope

First prong of teixobactin attack

Binds lipid II, which has both NAG and NAM w/ pentapeptide attached, sticking it into the membrane preventing use in biosynthesis

Second prong of teixobactin attack

Attachment of lipid II to membrane purterbs it and compromises integrity, making it leakier

Clovibactin antibiotic and mode of action

Binds to lipid II, lipid I, and lipid carrier (C55 PP), attaching them all to the membrane to inhibit PG synthesis and presumably make the membrane leakier.