Bacterial Cell Structures

Stijn van der Veen

How do I know what bacterium makes my patient ill?

Bacterial species can be differentiated by:

Morphology (shape)

Composition (cell envelope and other structures)

Metabolism & growth characteristics

Genetics

Bacteria are tiny!!!!!

Comparing bacteria with a football is comparing a football with Mount Everest.

10-6 10-1 104 meter

So, what do we need?

Microscopes!!!!!!!

Optical Methods

The light microscope Magnification: 1000x (100x obj. +

10x oc.)

Phase contrast microscope Observe living cells.

Fluorescence microscope Observe fluorescent dyes or proteins

Optical Methods

Confocal microscope Provide three-dimensional images

in multiple layers (z-stack)

Electron microscopes Transmission electron microscope

(TEM) can resolve particles with 1

nm in size

Scanning electron microscope

(SEM) can provide three-

dimensional images

Differentiating bacterial species

Morphology (shape)

Composition (cell envelope and other structures)

Metabolism & growth characteristics

Genetics

Shape (morphology) of bacteria

Spherical (coccus)

Rod (bacillus)

Twisted (spiral)

Morphology

Bacteria are unicellular…

but they can stick together!!

Spherical-shaped bacteria

Cocci may remain attached after cell division. These group characteristics are often used to help identify certain cocci.

Cocci that remain in pairs after dividing are called diplococci.

Cocci that remain in chains after dividing are called streptococci.

Cocci that divide in two planes and remain in groups of four are called tetrads.

Cocci that divide in three planes and remain in groups cube like groups of eight are called sarcinae.

Cocci that divide in multiple planes and form grape like clusters or sheets are called staphylococci.

Rod-shaped bacteria

Bacilli only divide across their short axis.

Most bacilli appear as single rods.

Diplobacilli appear in pairs after division.

Streptobacilli appear in chains after division.

Some bacilli are so short and fat that they look like cocci and are referred to as coccobacilli.

Twisted bacteria

Spiral bacteria have one or more twists.

Vibrios look like curved rods.

Spirilla have a helical shape and fairly rigid bodies.

Spirochetes have a helical shape and flexible bodies.

Other shapes…

Some odd types…that you (as doctor) would generally never encounter!!

Differentiating bacterial species

Morphology (shape)

Composition (cell envelope and other structures)

Metabolism & growth characteristics

Genetics

Prokaryote vs. Eukaryote

Bacterial cell structures overview

Basic structures Cell membrane Cell wall Cytoplasm Chromosome Ribosome

Specific structures Plasmid Flagellum Pilus Capsule Inclusion Endospores (not shown)

Staining methods

Methods to study bacterial morphology and composition:

Common differential staining methods

Gram stain （ Gram-positive vs. Gram-negative)

Acid-fast stain (Mycobacteria)

Special staining methods

The spore staining methods

The flagella staining methods

The capsule staining methods

DNA staining methods

Bacterial cell structures overview

Basic structures Cell membrane Cell wall Cytoplasm Chromosome Ribosome

Specific structures Plasmid Flagellum Pilus Capsule Inclusion Endospores (not shown)

Cell envelope

Cell envelope

The cell envelope consists of the cell membrane, cell wall, and associated structures

Bacterial cell envelopes fall into two major categories

Gram positive & Gram negative

This is based on Gram staining characteristics that reflect

major structural differences between the two bacterial

groups.

1884: Christian Gram: First publication for the Gram stain method

Gram-positive cocci Gram-negative bacilli

Gram stain

Gramm staining procedure

Crystal violet (1 min) => rinse Iodine (1 min) => rinse Acetone / Alcohol (10–15 sec) => rinse Safrinin (1 min) => rinse & dry

Simplified diagram and electronic microscopy

pictures of the cell envelope of G+ and G- bacteria

(murein = peptidoglycan)

Cell envelope

Gram-positive cell envelope

Gram-negative cell envelope

Peptidoglycan

The peptidoglycan is a single bag-shaped, highly cross-linked macromolecule that surrounds the bacterial cytoplasmic membrane and provides rigidity (which decides the shape of a bacterium) .

It is huge (billions in molecular weight).

Peptidoglycan is found in all eubacteria except Chlamydia and Mycoplasma.

Peptidoglycan structure

Glycan (polysaccharide) backbone of alternating residues of N-acetyl muramic acid and N-acetyl glucosamine connected by -1,4 linkage.

Tetrapeptide side chains usually containing D- and L- amino acid residues, and in some instances diaminopimelic acid (DAP) residues.

The side chains are cross-linked by peptide bridges. These peptide bridges vary in structure among bacterial species (gram-negative bacteria have no peptide bridges).

Function of the cell wall

Maintain the bacterial characteristic shape.

Provide resistance to osmotic changes.

Provide anchoring for surface appendages such as

flagella and pili.

Assist in cell division

The effect of lysozyme on the cell wall

Lysozyme can cut the -1,4 linkage.

So lysozyme can kill G+ and G- bacteria by destroying their glycan backbone .

Effect of penicillin on the cell wall

Penicillin can block the linkage of tetrapeptide side chains and peptide bridges.

So penicillin can kill bacteria by inhibiting their peptidoglycan synthesis.

But…only replicating/growing bacteria are killed.

Characteristics of gram-positive cell wall

Gram-positive cell wall is typically 20-80 nm thick.

It contains 15-50 peptidoglycan layers.

It may contain additional components such as teichoic acids and proteins

Teichoic acids are water-soluble polymers of polyphosphates.

Wall teichoic acids are linked to the

peptidoglyacan.

Lipoteichoic acids are anchored in

the cytoplasmic membrane.

Gram-positive cell wall is thin: 10-15 nm.

It only contains 1-2 peptidoglycan layers

No teichoic acids.

Characteristics of gram-negative cell wall

phospholipids

Gram negative outer membrane

Outer membrane consists of lipopolysaccharide (LPS) and phospholipids.

It contains lipoproteins such as porins. Porins form channels to allow passage of small hydrophilic

nutrients (such as sugars, amino acids and certain ions) through the outer membrane.

Lipopolysaccharide (LPS)

LPS is an endotoxin because it is poisonous to mammalian cells.

LPS consists of 3 regions O antigen: highly variable

polysaccharide region composed of repeating units of specific monosaccharides.

Core polysaccharide: conserved within a genus.

Lipid A: contains β-hydroxy fatty acids (bacteria specific), which display endotoxin activity.

Free lipid A may trigger fever, inflammation, and septic shock

Summary

Property Gram positive Gram negative

Peptidoglycan layers 15-50 1-2

Peptidoglycan content >50% 10-20%

Teichoic acids + -

Outer membrane - +

lipopolysaccharide - +

Sensitive to penicillin yes Less sensitive

Digested by lysozyme yes weakly

Mycobacterial cell envelope

1. Outer lipids 2. Mycolic acid3. Polysaccharides

The Mycobacterial cell envelope is waxy.

This enables Mycobacteria to survive exposure to:

acids alkalis detergents oxidative bursts lysis by immune

system many antibiotics

5. Molecules involved in evading host immune cells & function.

4. Peptidoglycan5. Plasma membrane 6. 6 &

Acid-fast (Mycobacterial) staining procedure Ziehls carbol fuchsin (3 – 5 min heat) => rinse Acid Alcohol (10 – 15 sec) => rinse Crystal violet (1 min) => rinse & dry

Cell membrane

Separates the cell from its environment

Consists of a phospholipid bilayer

Semipermeable (important for osmosis)

Flexible

Cell membrane and osmosis

Osmosis is the diffusion of water across a semi-permeable membrane.

Changes in the bacterial environment such as the amount of dissolved molecules results in changes of the osmotic pressure.

Water will move in or out of the cell.

Cells need water, which is important for many metabolic reactions.

The cell wall will protect the bacteria from exploding when too much water moves into the cell, but bacteria are sensitive to conditions when to much water moves out of the cell (dehydration/desiccation).

Cell membrane proteins

Transmembrane proteins, porins, membrane anchored proteins, etc.

Important for many processes Sensing the environment Provide active transport across the cell membrane

Proteins Solutes Lipids Cell wall polymers

Generation of energy

Bacterial cell structures overview

Basic structures Cell membrane Cell wall Cytoplasm Chromosome Ribosome

Specific structures Plasmid Flagellum Pilus Capsule Inclusion Endospores (not shown)

Cytoplasm

Jelly intracellular environment composed largely of water (80%), proteins, nucleic acids, lipids, salts, sugars, and various low molecular weight molecules.

The cytoplasm also harbors: Chromosome Ribosomes Inclusions Plasmids

Bacterial cell structures overview

Basic structures Cell membrane Cell wall Cytoplasm Chromosome Ribosome

Specific structures Plasmid Flagellum Pilus Capsule Inclusion Endospores (not shown)

Chromosome

Bacterial chromosome (nucleoid)

Freely floating double stranded DNA (not covered by membrane)

Haploid More efficient => grows quicker Mutations allow adaptation to environment

Circular

Bacterial cell structures overview

Basic structures Cell membrane Cell wall Cytoplasm Chromosome Ribosome

Specific structures Plasmid Flagellum Pilus Capsule Inclusion Endospores (not shown)

Ribosome

Translates messenger RNA’s (mRNA) into proteins

Bacterial cell contains multiple copies (usually thousands).

Free floating or attached to cell membrane.

Composed of ribosomal RNA (rRNA) and proteins.

Typically composed of two subunits (large and small)

Bacterial cell structures overview

Basic structures Cell membrane Cell wall Cytoplasm Chromosome Ribosome

Specific structures Plasmid Flagellum Pilus Capsule Inclusion Endospores (not shown)

Plasmids

Small extra-chromosomal double stranded DNA.

Generally circular (in few instances linear).

Usually present in multiple copies.

Capable of self-replication.

Often encode antibiotic resistance markers and virulence factor.

Generally not essential for survival.

Bacterial cell structures overview

Basic structures Cell membrane Cell wall Cytoplasm Chromosome Ribosome

Specific structures Plasmid Flagellum Pilus Capsule Inclusion Endospores (not shown)

Flagellum

Hair-like appendage on the bacterial surface that is responsible for movement (motility).

Consist of various different proteins

The main protein of the filament (flagellin) can be used for identification (H-antigen).

Flagellum-dependent motility is important for virulence and chemotaxis (movement towards food and away from toxics).

Only visible with light microscopy after specific flagellum staining

Flagellum structure

Flagellum arrangement

Monotrichous: single polar flagellum

Lophotrichous: multiple flagella at single pole

Amphitrichous: flagella at both poles

Peritrichous: flagella distributed all around

Flagellum-dependent movement

Bacterial cell structures overview

Basic structures Cell membrane Cell wall Cytoplasm Chromosome Ribosome

Specific structures Plasmid Flagellum Pilus Capsule Inclusion Endospores (not shown)

Pilus (plural pili)

Hair-like appendage on the bacterial surface that is involved in adhesion to host cells, surfaces, and other bacteria.

Composed of several different proteins and the structural protein of the filament is pilin.

Important for virulence.

Two major types can be distinguished: Common pilus (frequently referred to as fimbria).

Shorter, thinner, and numerous present per bacterium. Major role in adhering to host cells.

Sex (F) pilus Longer, broader, and only 1-4 per bacterium Important for bacterial conjugation (sex).

Conjugation

Donor

Recipient

Bacterial cell structures overview

Basic structures Cell membrane Cell wall Cytoplasm Chromosome Ribosome

Specific structures Plasmid Flagellum Pilus Capsule Inclusion Endospores (not shown)

Capsule and slime layer (glycocalyx)

Layer surrounding the outside of the cell envelope.

Usually composed of polysaccharides, and less frequently of polypeptides, glycoproteins, or glycolipids

Not present in all bacteria and even variable within capsule containing species.

Capsule contributes to virulence of pathogens and protects against phagocytosis and antimicrobial compounds secreted by host cells.

Capsule and slime layer

Helps in surface attachment and nutrient absorption, and prevents dehydration.

Not essential for viability.

Capsule: firmly attached and structured layer surrounding cells.

Slime layer: loosely attached unorganized layer surrounding cells

Bacterial cell structures overview

Basic structures Cell membrane Cell wall Cytoplasm Chromosome Ribosome

Specific structures Plasmid Flagellum Pilus Capsule Inclusion Endospores (not shown)

Inclusion

Aggregates of storage molecules, found as small bodies in the cytoplasm.

Consist of organic molecules (such as glycogen or PHB), or inorganic molecules (such as sulfur or polyphosphate).

Inclusions accumulate in conditions of excess nutrients.

Bacterial cell structures overview

Basic structures Cell membrane Cell wall Cytoplasm Chromosome Ribosome

Specific structures Plasmid Flagellum Pilus Capsule Inclusion Endospores (not shown)

Endospore

Highly specialized bacterial cell that is very resistant to extreme conditions such as heat, cold, desiccation, radiation, starvation, etc.

It is produced under unfavorable conditions and enables survival of the species.

Dormant endospores can survive for many years.

Endospores are unable to replicate in this form.

Under favorable conditions, endospores germinate (change) into vegetative (standard) cells again and are able to replicate.

Commonly found in the soil.

Sporulation (endospore formation)

Endospore structure

Serological identification

Serological detection and identification of bacterial cell surface antigens.

Binding of specific antibodies to the cell surface antigens.

Agglutination assays (clumping of bacteria due to antibody binding.

Serotyping (capsule, O/H antigens) Lancefield grouping (Streptococcus) Etc.

Next lecture

Bacterial Metabolism & Growth Characteristics