Microbiology - Module 1

Bacteria, Eukaryotes, and Archaea

What Are Common Prokaryotic Cell Arrangements?

- Bacilli
- Spirochetes
- Cocci

They can be in:
- Single
- Diplo
- Tetrad
- Strepto- chains

Typical Structure of a Bactrium

- Have a cell membrane;
- Chromosomal DNA (nucleoid) and extrachromosomal DNA (plasmids)
- Ribosomes;
- Cell WALL

Can also possess :
- Flagella;
- Pili;
- Fimbriae;
- Capsules

Bacterial Membrane Structure

- Lipid bilayer
- Membrane proteins responsible for ATP synthesis;
- Integral or peripheral proteins;
- Transport proteins;
- Sensing proteins;
- Secretion proteins.

Functions of a Bacterial Cytoplasmic Membrane

Permeability barrier
- Prevents leakage and functions as a gateway for nutrients and wastes.
- Transport proteins accumulate solutes against concentration gradient (and move charged molecules).

Protein Anchor
- Sites of proteins that participate in transport, bioenergetics, and chemotaxis.

Energy Conservation
- Sites of generation and dissipation of the proton motive force.

Bacterial Membrane Lipids

- Cardiolipin;
- Palmitic and oleic acid;
- Cyclopropane fatty acid;
- Hopanoids

Definition of Cardiolipin (Diphosphatidylglycerol)

- Localizes to the cell poles
- Binds certain environmental stress proteins.

Ex. Protein that transports osmo-protectants when the cell is under osmotic stress.

Definition of Palmitic and Oleic acid

- Saturated or unsaturated
- Add fluidity to the membrane in cold temperatures

Definition of Cyclopropane Fatty Acid

- Stiffens the cell membrane;
- Conversion of unsaturated fatty acid to cyclopropane is important for Mycobacterium Tuberculosis pathogenesis.

Definition of Hopanoids

- Stabilizes membranes like cholesterol

Function of a Cell Wall

- Protects against changes in osmotic pressure;
- Maintains the shape of the bacteria.
- Confers shape and rigidity to the cell.

THE CELL MEMBRANE IS ATTACHED TO THE CELL WALL.

What is a bacterial cell wall made of?

- Peptidoglycan layer of single interlinked molecules.
- 100+ distinct peptidoglycans have been described.

Peptidoglycan Structure

Alternating modified glucose:
- N-acetylglucosamine (NAG);
- N-acetylmuramic acid (NAM) (Beta 1,4 linkages)

Amino acids:
- L-alanine;
- D-alanine;
- D-glutamic acid;
- L-lysine or diaminopimelic acid (DAP)

What can destroy Peptidoglycan?

Lysozyme - enzymes that cleave glycosidic bonds between sugars.
- Found in human secretions; major defence against bacterial infection.

Gram Negative Cell Wall Structure

THIN peptidoglycan; separated from membrane by PERIPLASMIC SPACE; OUTER MEMBRANE on top of the cell wall contains phospholipids, LIPOPOLYSACCHARIDES (trigger SEVERE immune response; more than LTAs)

Sugar backbones with a peptide cross link (bridge between two peptidoglycans).

Gram Positive Cell Wall Structure

Many layers of interlinking peptidoglycan crosslinks and teichoic acid.

Contains a PENTAGLYCINE interbridge.

How does Penicillin work?

Blocks the formation of peptide cross links in the peptidoglycan cell wall.

Blocks transpeptidase's active site so it cannot form crosslinks between peptides.

How does Vancomycin work?

Prevents crosslink formation by binding to the terminal D-Ala-D-Ala dipeptide.

How does growth of a peptidoglycan wall form?

Growth occurs via a synthesis complex that extends the chain of amino-sugars.

These include PENICILLIN-BINDING PROTEINS which catalyze the formation of the peptide cross-bridges.

DIRECTED by a protein complex including MreB (actin homologue).

What is MreB?

Maintains shape of a cell wall and its extension by positioning peptidoglycan synthesis machinery (like PBPs).

It is an ACTIN homologue.

How does peptidoglycan form in different species?

Synthesis can occur as:
- Disperse zones (several columns growing at different locations).
- Septal zonal growth (from the centre of the bacteria outward)
- Polar growth (from one pole to the other)

Where can the S-Layer be found?

Gram Negative:
- Associated with the lipopolysaccharide layer

Gram Positive:
- On the surface of the peptidoglycan cell wall and teichoic acids

Teichoic Acid

A glyco-polymer with a negative charge in GRAM POSITIVE bacteria that have multiple functions:
1. Aids with proton motive force;
2. Maintains rigidity of the cell and its shape;
3. Protects against high temp and high salt conditions.
4. Protects against antibiotics like beta lactams.

Lipoteichoic acids

Teichoic acids covalently bound to membrane lipids.

Lipid A

The lipid component of lipopolysaccharide, which is released from dead Gram-negative bacterial cells and can trigger shock and other symptoms in human hosts.

Lipopolysaccharide

A component of Gram Negative cell wall structure. It is a phospholipid layer with sugars.
- O-polysaccharide and LIPID A.

E.coli O157:H7

O defines the bacterial serotype

H refers to the flagellar protein.

K is sometimes used for capsular polysaccharide

Braun Lipoprotein (Murein Lipoprotein)

Covalently linked to the thin peptidoglycan and binds the outer membrane to the cell wall in Gram Negative Bacteria.

Periplasm

The gel-like material that fills the region between the cytoplasmic membrane and the outer membrane of Gram-negative bacteria.

Gram Staining

A process by which components of bacterial cell walls are bound to Gram's stain. Depending on the amount of peptidoglycan in their cell walls, bacteria stain differently and are classified as Gram-negative or Gram-positive.

Thick cell wall like those in Gram Positive cells retain crystal violet.

Gram Negative do not retain crystal violet dye.

Acid-fast stain

A differential stain used to identify bacteria that are not decolourized by acid-alcohol.

Eg. Carbolfuchsin used to stain Mycobacterium species.

Spore stain

Malachite green used to detect spores of Bacillus and Clostridium.

Negative stain

Colors the background, which makes capsules more visible.

Acid-fast Bacteria

Retain the primary stain even when treated with acid alcohol. Cell walls contain a hydrophobic layer including mycolic acid (a lipid).

Looks light blue with Gram stain but bright pink with a special stain.

Includes Mycobacterium tuberculosis.

Glygocalyces

Not considered part of the cell wall.
- Contains polysaccharide layers (thick or thin, rigid or flexible);
- Capsules (tightly attached, tight matrix).
- Slime layer (easily deformed, assist in adherence, maintenance of biofirms, protects against phagocytosis, prevents dehydration/desiccation).

Cell Surface Structures: S-Layer

Additional protectively found in Gram-positive and Gram-negative bacteria and archaea.

Crystalline layer of thick subunits consisting of protein or glycoprotein.

May contribute to cell shape and protect the cell from osmotic stress.

Cell Surface Structures: Fimbriae and Pilli

Filamentous proteins about 2-10 nm wide.

FIMBRIAE
- Enable organisms to stick to surfaces or form pellicles (thin sheets of cells on a lipid surface).

PILI
- Longer and fewer found per cell.
- Allows conjugation (genetic exchange through sex pili)
- Allows twitching motility and adherence in Type IV pili.

Features of the Cytoplasm

- Nucleoid
- Extrachromosomal DNA
- Enzymes
- Regulatory factors
- Ribosomes
- Cell inclusions;
- Gas vesicles;
- Magnetosomes;
- Cytoskeleton.

Nucleoid

Made of DNA, RNA, or protein.
Stores genetic information and allows gene expression.

Extra chromosomal DNA

Made of DNA that is variable, and encodes non-chromosomal genes for a variety of functions.

Enzymes

Proteins which regulate replication, transcription, metabolism, and cell signalling.

Regulatory Factors

Protein and RNA which control replication, transcription, and translation.

Ribosomes

RNA and protein that allow protein synthesis, translation.

Cell Inclusions

Allow for storage and reserves of various polymers; function as energy reserves and carbon reservoirs.

Enclosed by a thin membrane; reduces osmotic stress.

Contains polymers like:
- Glycogen (glucose polymer);
- Poly-beta-hydroxybutyric acid (PHB) (lipid polymer stored as lipid droplets)

Gas vesicles

Proteins that allow for buoyancy.

Magnetosomes

Contain iron, proteins, and lipids which orient the cell during movement across a magnetic gradient.

Cytoskeleton

Proteins which guide cell wall synthesis, cell division, and possibly the partitioning of chromosomes during replication.

Bacterial Cytoskeleton

Implicated in cell division and morphology.
- FtsZ
- MreB
- Crescentin

FtsZ

Tubulin-like protein needed for cell division.

MreB

Major shape-determining factor in prokaryotes, forms a coil in bacteria.

Aka Actin-like protein.

Crescentin

Shape-determining protein produced by vibrio-shaped cells of Caulobacter crescentus.

Filament-like protein.

Par system

The analogous system in some Bacteria to the mitotic spindles that are seen in eukaryotic cells.

Uses Actin-like protein (ParM) for the movement of molecules to the right location within the cell.

Segregates a dividing chromosomes and segregates extrachromosomal DNA.

Bacterial Cell Division by Septation

1. As DNA synthesis terminates, the cell divides by SEPTATION, the formation of a septum.

2. The Septum grows inward from the sides of the cell, constricting and sealing off two daughter cells.

3. FtsZ subunits assemble circles around the septum, directing septal growth.

How does FtsZ direct cell division?

- Septation requires rapid biosynthesis of envelope components (membrane and wall).

- the DIVISOME manages the overall process of division (a protein complex).

-FtsZ is part of the DIVISOME, it polymerizes to form a Z-ring.

- SEPTATION is coordinated with DNA replication.

Bacterial Cell Differentiation

Some bacteria generate two kinds of daughter cells: one stationary (sessile) and one mobile (warmer).

Eg. Flagellum-to-stalk transition of the bacterium Caulobacter crescentus.

Polar aging

The phenomenon where bacterial cell poles differ in their origin and age.

The poles of each daughter cell are chemically different from each other (POLAR AGING INCREASED BY STRESS).

A major form of asymmetry is Endospore formation.

Extracellular Membrane Vesicle Functions

1. Carry proteins and nucleic acids;
2. Attractors of partner heterotrophs;
3. Act as phage decoys;
4. Act as vehicles for DNA transfer.

Bacterial Nanotubes

Membrane extensions that merge directly with the membranes of neighboring organisms. These allow bacteria to directly share proteins and mRNA useful under hostile conditions, such as when exposed to antibiotics.

Specialized Bacterial Structures

- Thylakoids;
- Carboxysomes;
- Gas vesicles;
- Magnetosome;
- Cell inclusions;
- Pili;
- Stalks;

Thylakoids

A flattened membrane sac used to convert light energy to chemical energy.

Carboxysomes

Polyhedral bodies packed with the enzyme Rubisco for CO2 fixation

Gas Vesicles

Gas-filled cytoplasmic structures bounded by protein and conferring buoyancy to cells.

Pili

Straight filaments of pilin protein used in attachment of bacteria to tissues.

Gram-negative enteric bacteria use SEX PILI for conjugation.

Stalks

Membrane-embedded extensions of the cytoplasm whose tips secrete adhesion factors called HOLDFASTS.

Cell Motility

1. Flagella, archaella, and swimming motility.
2. Gliding Motility;
3. Chemotaxis.

Flagella and Swimming Motility

Occurs in bacteria with long, thin appendages (15-20 nm wide) with different arrangements:
- Polar;
- Lophotrichous;
- Amphitrichous;
- Peritrichous.

Flagella Rotate either Clockwise (CW) or counterclockwise (CCW) relative to the cell.

Flagella Structure

Helical in shape; composed of several components and filament composed of FLAGELLIN.

Has reversible rotating mechanisms which increase or decrease rotational speed relative to the strength of the PROTON MOTIVE FORCE.

Chemotaxis

The movement of bacteria in response to chemical gradients

Attractants cause CCW rotation:
- Flagella bundle together;
- Cell pushes forward into A RUN.

Repellents cause CW rotation
- Flagellar bundle falls apart;
- Cell "TUMBLES".
- Bacterium stops and changes direction.

Random Walk

The unpredictable movements of a bacterium trying to distinguish the location of an attractant.

Attractant concentration increases and prolongs a run.

Other Forms of Taxis

- Phototaxis (light);
- Aerotaxis (oxygen);
- Osmotaxis (ionic strength);
- Hydrotaxis (water).

CHEMORECEPTORS sense attractants and repellents.

Endospores

A thick-walled protective spore that forms inside a bacterial cell and resists harsh conditions once expelled. Occurs when environmental conditions are unfavorable.

Endospore Structure

- Exosporium;
- Spore coat;
- Cortex;
- Core wall.

Contains dipicolinic acid and is enriched in calcium both forming CALCIUM-DIPICOLINIC ACID (DPA) COMPLEX.

SASP (small acid-soluble spore proteins) that bind and protect DNA.

Cytoplasm, cytoplasmic membrane, ribosomes, and cell essentials.

Contents of a Bacterial Spore

- Dipicolinic acid enriched in calcium (DIPICOLINIC ACID COMPLEX - DPA).
- Small acid-soluble spore proteins (SASP) which bind and protect DNA and function as a carbon source for outgrowth.

Endosporulation

Endospore formation occurring when environmental conditions are not conducive for growth:
1. DNA replicates;
2. Membrane forms around DNA.
3. Forespore forms membranes;
4. Protective cortex forms around spore;
5. Protein coat forms around cortex;
6. Spore is released.

Can endospores form in gram-positive bacteria?

Yes, but only some (bacillus and clostridium).

NO SPORES form in archaea, suggesting the process evolved in bacteria after the split.

Bacterial Taxonomy

Bacteria utilize the binomial system of genus (closely related species) and species (organisms with common features).

Bacterial Strain

Distinct subtypes of species that differs genetically, and often phenotypically, from other subtypes.

Bacterial Serotypes

Strain identified by serotyping (identifying surface antigens particular to a species).

Bacteria Classification by Shared Characteristics

- Morphology (colony appearance);
- Gram colouring;
- Size and shape;
- Presence of structures;
- Metabolic traits;
- DNA sequence.

Archaea

Domain of unicellular prokaryotes that have cell walls that do not contain peptidoglycan. May or may not share phylogeny with eukaryotes. They are structurally and genetically different from bacteria.

Carl Woese

Discovered the existence of archaea through research into ribosomal RNA and their genes to distinguish archaea from bacteria and eukaryotes.

Common cellular ancestry means that certain molecules must be shared between all living organisms. Therefore, genetics can be used to compare the different organisms.

How many phyla of archaea can be grown in a lab and what are they?

5 and they are:
1. Euryarchaeota (methanogens, halophiles, thermophiles);
2. TACK (Crenarchaeota, thermophiles);
3. Asgard (ancestral eukaryotes?);
4. DPANN (symbionts-gene loss).

Genetic Properties of Archaea

- Possess singular, circular chromosomes without a nucleus.
- Contain DNA complexed with histones (like Eukaryotes).
- Similar transcription factors to Eukarya.

General Archaea Properties - CYTOPLASM

- Inclusion bodies (gas vacuoles and carbon storage);
- Protein content like bacteria (ribosomes, enzymes);
- Cytoskeleton homologues;
- NOT KNOWN TO FORM SPORES;
- Contain DNA within a nucleoid.

General Archaea Properties - CYTOSKELETON

Cytoskeletal homologs are found in both bacteria and archaea.

Archaea possess:
- TubZ (tubulin-homologue);
- Crenactin (actin-homologue)
- NO filamentous analogue.

Archaeal Cell Membranes and Walls

- Can have lipid bilayer OR MONOLAYER;
- Possess isoprene chains (INSTEAD OF FATTY ACIDS);
- Form Ether (C-O) linkages (INSTEAD OF ESTER COO linkages).
- NO peptidoglycan, have pseudomurein.
- Have an S-layer (protein or glycoprotein).

Pseudomurein

Found in the cell walls of certain METHOGENIC archaea.
- Polysaccharide similar to peptidoglycan;
- Immune to lysozymes and penicillin;
- Composed of NAT and NAG;
- Possess Beta-1,3 glycosidic bonds;
- AMINO ACIDS ARE ALL L-stereoisomers.

Archaeal Cell Wall (S-layer)

The most common cell wall type.
- Consists of protein or glycoprotein;
- Paracrystalline structure of various symmetry (hexagonal, tetragonal, or trimeric);
- S-layers present in addition to other cell wall components (polysaccharides);
- ALWAYS THE OUTERMOST LAYER;
- Resists osmotic pressure in extreme environments.

Glycoproteins possible:
- Methanochondroitin;
- Protein shealth.

Methanochondroitin

Glycoprotein layer of archaeal cell wall that causes cell-cell adhesion. Looks like chondroitin found in connective tissues in animals.

Archaeal Protein Sheath

A protein sheath in archaea found covering multiple cells forming a chain instead of a single cell.

Archaeal Cell Surface Structures

- Hami;
- Rotating archaella;
- ATP usage and generation;
- Bacteriorhodopsin.

Hami

Fimbriae-like structures that radiate out from archaea, like barbed wire, function to securely attached archaea to biological and inanimate surfaces.

FORMS BIOFILMS.

Archaea Flagella Structure

- Simpler than bacterial or eukaryotic flagella.
- Utilizes ATP rather than PMF.
- Flagella ROTATE, not whip-like.

Methanogenesis

An energy-yielding metabolic process that produces methane. It is unique to archaea.

ARCHAEA ARE THE ONLY KNOWN ORGANISMS CAPABLE OF THE PROCESS:
- Chemolithotrophy (combine hydrogen and CO2 to methane);
- Chemoorganotrophy (reduce organic compounds - methanol, acetate - to methane).

Bacteriorodopsin

A method for which Archaea use light to power ATP production WITHOUT doing photosynthesis.
- Similar to light-gathering pigment in retinas;
- Cytoplasmic membrane proteins absorb light energy and pump protons across a membrane (ATPase produces ATP).

How does Bacteriorodopsin work?

1. Bacteriorhodopsin absorbs light (replaces ETC);
2. Retinal (PURPLE) is excited by rhodopsin);
3. Retinal changes conformation from TRANS to CIS.
4. Retinal transformation is coupled to one proton being pumped outside of the cell.
5. ATP synthase uses the proton gradient to generate ATP.

Archaea Gene Structure and Regulation

The genome of archaea resemble bacteria in gene size and density.

BUT
- Contain tRNA genes interrupted by INTRONS;
- Contain DNA and RNA polymerases and transcription factors like EUKARYOTES.
- Contain histone homologs.

UNIQUE TO ARCHAEA:
- Reverse gyrase in thermophiles introduce POSITIVE SUPERCOILS in chromosomal DNA (protection from high temperatures).

Archaeal Chromatin Structure

- Histones form TETRAMERS around DNA (not eukaryotic octamers).
- Nucleosome structure allows stacking of units (superhelix).
- Histone tails may allow for some epigenetic modifications.

Archaeal Gene Structure

Promoters resemble that of eukaryotic cells:
- Possess TATA box;
- Presence of BRE (beta-recognition element);
- Formation of pre-initiation complex (RNA pol 2 needs to be recruited by general transcription factors - TBP (TATA-binding protein) and TFBs (Archaeal transcription factor B))
- RNA pol 2 structure similar to eukaryotes.
- Multiple regulatory elements and binding sites for direct gene expression.
- Activators and repressors are more similar to bacterial DNA binding proteins.

UNIQUE TO ARCHAEA:
- Domain arrangements and sensing domains in response to environment;
- Modified bases in their tRNA molecules (archaeosine - guanosine analog).

Do archaea cause disease?

No known species of archaea causes disease.

Virus

A noncellular particle that infects a host cell and directs it to produce progeny particles (more virus).