Bacterial Cell Structure

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eukaryotic

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Compartmentalized; > 5.0 µm; cell membrane; nuclear membrane; golgi; SER; RER; lysosome; mitochondrion (site of cellular respiration); algae, fungi, protozoa, plants, animals

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prokaryotic

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NOT COMPARTMENTALIZED; size 0.5 to 3.0 µm; 10x smaller; cell wall, cell membrane (site of cellular respiration); plasmid; cytoplasm rich in ribosomes; single, circular chromosome; bacteria

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cytosol

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BACTERIA: Site of many metabolic reactions: proteins, metabolites; reserve granules or inclusions; ribosomes- number depend on growth rate; mRNA- transcription and translation are coupled; no organelles in bacteria; contains nucleoid and plasmids

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ribosome

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BACTERIA: Structure: 70S (30S subunit + 50S subunits); three types of rRNA: 5S, 16S, 23S; target for antibacterial drugs

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nucleoid

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BACTERIA: Bacterial chromosome is large single, double-stranded circular DNA, supercoiled, size depends on bacteria; Cell contains 2-4 nucleoids depending on growth rate; Tightly packed – DNA-binding proteins; No nuclear membrane; size of chromosome will change depending on how many proteins are needed to be made; chromosome divides before the cell

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plasmid

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BACTERIA:
• Small circular extra-chromosomal DNA – not essential
• Double-stranded molecule
• Replicates separately from chromosome
• Generally several copies of same plasmid
• Multiple types of plasmid may be present
• Often codes for virulence factors, antibiotic resistance or/and self-transmission

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Cytoplasmic membrane

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BACTERIA:
• Lipid bilayer structure
• Does not contain sterols (e.g. cholesterol); exception: mycoplasma
• Electron transport and energy production
• Transport proteins – uptake of metabolites and release of other substances
• Ion pumps – maintain membrane potential
• A lot more proteins in cell membrane of bacteria because there is more activity; everything that happens in organelles happens in membrane

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Cell wall

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BACTERIA:
• Physiological function
o protection from osmotic lysis
o gives shape
• Important in diagnosis and pathogenesis
o classification of bacteria according to their cell wall – Gram stain
• Target for antibiotics
• Mycoplasma spp. don’t have a cell wall (e.g. Mycoplasma pneumoniae)

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Gram (+)

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• Thick murein layer
• High susceptibility to penicillin and lysozyme
• Teichoic acid and lipteichoic acid in the thick peptidoglycan layer; also contains small amount of polysaccharides and proteins; teichoic acid and lipteichoic acid are common surface antigens and play a role in adherence to host cells or other bacteria

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Gram (-)

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• Thin murein layer
• Outer membrane present: made of lipopolysaccharide, phospholidid, lipoprotein connected to murein
• Periplasm present
• Low susceptibility to penicillin and lysozyme

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Murein

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Peptidoglycan layer found only in prokaryotes; responsible for the gram stain; peptidoglycan’s NAG and NAM are crosslinked by peptides
Examples:
• Fosfomycin – inhibits the first step in formation of N- acetylmuramic acid
• Cycloserine – prevents incorporation of D -alanine into peptide bridge
• Vancomycin – prevents elongation; breaks the glycosidic bond
• Penicillin – prevents cross-linking; breaks peptide bond

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GRAM (+) cell wall

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Thick peptidoglycan layer components and functions that contains teichoic and lipoteichoic acids, other polysacs and proteins
• protection from environment
• resistant to many mammalian enzymes; exception: can be degraded by lysozyme – breaks glycan backbone
• murein synthesis enzymes; targets of b-lactam antibiotics
• loss of murein Æ cell lysis; exception: formation of protoplast in isotonic medium

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Teichoic acid and Lipoteichoic acid

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• GRAM (+) CELL WALL
• Common surface antigens
• Role in adherence to host cells or other bacteria
1. Teichoic acid
o poly -ribitol or -glycerol phosphates
o covalently linked to peptidoglycan
o essential for cell viability
o important virulence factor
2. Lipoteichoic acid
o contain fatty acid
o anchored in cytoplasmic membrane
o when shed into host Æ host response similar to endotoxin

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GRAM (-) Cell Wall

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1. Thin peptidoglycan layer: inside periplasmic space; shape an integrity; loss of murein Æ spheroplast
2. Periplasmic space/gel - murein ; enzymes: hydrolytic and cell wall synthesis proteins; transport proteins; oligosaccharides
3. Outer membrane- permeability barrier to large molecule; asymmetric bilayer structure; phospholipids inner layer; lipopolysaccharide outer layer; transmembrane proteins & porins ; joined to the cytoplasmic membrane at adhesion points and is attached to the peptidoglycan by lipoprotein links
4. Lipopolisaccharide (LPS)- Specific to gram(-) bacteria; Also called endotoxin; strong stimulator of immune response; toxic to humans and animals; LipidA: lucosamine phospholipid; toxic part; Core: unusual carbohydrate residues; Antigen: polysaccharide chains; major surface antigen

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The Gram reaction with each step of the Gram stain

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Step 1: Crystal Violet
Step 2: Gram’s Iodine
Step 3: Decolorizer (Alcohol/Acetone)
Step 4: Safranin Red – stains gram negative pink

Gram (+): Stain goes thru cell wall, cell membrane and into cytoplasm to stain crystal violet precipitated by iodine and trapped in the thick peptidoglycan layer
Gram (-): decolorizer disperses the outer membrane and washes the crystal violet from the thin layer of peptidoglycan; bacteria visualized by the red counterstain
 Spirochetes – too thin to be seen
 Mycoplasma – no cell wall
 Mycobacterium – waxy cell wall; acid fast stain

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Mycobacterium cell wall

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• Unique among procaryotes (waxy, hydrophobic and high lipid content)
• Major determinant of virulence
• Peptidoglycan + complex lipids
• Lipoarabinomannan, free lipids, mycolic acid, polysaccharide, peptidoglycan, lipid bylayer
• Responsible for
o acid fastness & slow growth
o resistance to
 drying and chemical disinfectants
 common antibiotics
 osmotic lysis via complement deposition
 lethal oxidations Æ survival inside of macrophages
 traditional stains & dyes
o antigenicity
note: sensitive to heat and UV light

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acid-fast staining

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1. heat + fuchsin = acid-fast & non-AF stain pink
2. acid alcohol = acid fast stains pink & non-AF washes white
3. methylene blue = acid fast stains pink & non-AF stains blue

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One example for each of the following groups: Gram positive bacteria, Gram negative bacteria, acid fast bacteria and bacteria without cell wall

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1. Gram (+): Staphylococci (clusters); Streptococci (chains)
2. Gram (-): Enterobacteriaceae
3. acid fast: Mycobacterium
4. bacteria w/out a cell wall: Mycoplasma

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Pili (fimbriae)

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• Not essential for cell viability
• Found in many Gram(+) and Gram(-)
• Hair-like projections of the cell
• Composed of pilin
• 2 classes of pili Functions
o common pili – many around the cell
 adhesion to host cell surface
 important virulence factors – role in colonization
o sex pilus – one per cell
 in “male bacterium”
 sexual conjugation: genetic transfer between bacteria

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flagella

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• Not essential for cell viability
• Found in many Gram(+) and Gram(-)
• Function: motility
• Distribution
A. Monotrichous or polar – single polar flagellum
B. Lophotrichous – multiple flagella at the same spot
C. Amphitrichous – a single flagellum at both ends
D. Peritrichous – around the cell
• Rotating helical protein structures Æpropel cell by rotation
• Basal body: proteins organized as rings [4 rings in Gram(-); 2 rings in Gram(+)]
• Hook
• Filament composed of flagellin
• Motility and chemotaxis are important for colonization

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Spirochetes

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• Flexible peptidoglycan cell wall around which several axial endoflagella are wound
1. Motility
• from movements of endoflagellar filaments
rotation and flexion
2. Microscopy
• Gram(-) but stain poorly and too thin to be seen
• Darkfield microscopy, immunofluorescence or special staining techniques are effective
• Borrelia is the exception: large cells, visible in routine blood smears

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Capsule and slime layer

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• Also called glycocalyx
• Not essential for cell viability
• Found in some Gram(+) and Gram(-)
• Outside cell wall
• When well defined Æ capsule
• When less defined Æ slime layer
• Usually composed of polysaccharides (exception: polypeptide in Bacillus)
 Major virulence factor
 attachment to surfaces
 protection against phagocytic engulfment
 reserve of nutrients
 protection against desiccation
 The slimy or mucoid appearance of a bacterial colony is usually evidence of capsule production Complex aggregation of microorganisms marked by the excretion of a protective and adhesive matrix

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Discuss biofilm

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• Complex aggregation of microorganisms marked by the excretion of a protective and adhesive matrix
• Forms on household surfaces, but also on catheter lines and contact lenses.
• Grows on pacemakers, heart valve replacements, artificial joints and other surgical implants.
• ~ 65% of nosocomial (hospital-acquired) infections are caused by biofilms.
• Bacteria growing in a biofilm are highly resistant to antibiotics (up to 1,000 times more resistant than the same bacteria not growing in a biofilm).

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Functions of bacterial envelope

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1. Structure
• rigidity & packaging of internal contents
2. Physiological functions
• permeability barrier Æ outer membrane or plasma membrane
• metabolic uptake Æ membranes and periplasmic transport proteins, porins, permeases
• energy production Æ plasma membrane
• motility Æ flagella
• mating Æ pili
3. Host interaction
• adhesion to host cells Æ pili, proteins, teichoic acid
• immune recognition by host Æ all outer structures
• escape from host immune recognition Æ capsule
4. Medical relevance
• antibiotic sensitivity Æ peptidoglycan synthetic enzyme
• antibiotic resistance Æ outer membrane

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Spore/Endospore

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1. Dormant form of a bacterial cell
• non-metabolizing & non-reproducing state
• produced when starved Æ sporulation
• return to growing state (vegetative form) when nutrients/water available Æ germination
2. Resistant to environmental stress
• heat, dehydration, UV and gamma radiations, organic solvents…
3. Found only in gram positive bacteria
4. Important virulence factor
• e.g. Bacillus and Clostridium (know!!)
• B. anthracis spores can live in the soil for many years
• Respiratory anthrax: humans become infected by inhaling anthrax spores

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Describe the events of sporulation

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1. Spore septum invaginates newly replicated DNA
2. PM start to surrounds DNA, cytoplasm, and membrane
3. spore septum surrounds isolated portion, forming forespore (two membranes)
4. Peptidoglycan layer forms between membranes
5. Spore coat forms
6. Endospore is freed from cell

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Endospore resistance is due to:

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1. Various layers of spore coat
 coat – keratin-like protein
 cortex – peptidoglycan-like
2. Core
 Low water content
 DNA stabilization
• calcium dipicolinate
• acid soluble proteins
3. DNA repair during germination

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Endospore germination

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1. Spore
2. Activation
3. Germination
4. Outgrowth
5. Vegetative form- can have central spores and terminal spores

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Describe taxonomic classification

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• Family: a group of related genera.
• Genus: a group of related species.
• Species: a group of related strains.
• Type: sets of strain within a species (e.g. biotypes, serotypes).
• Strain: one line or a single isolate of a particular species.
• The most commonly used term is the species name
• e.g. Streptococcus pyogenes - abbreviation S.pyogenes
• 2 parts to the name, one defining the genus in this case ("Streptococcus“) and the other the species ("pyogenes").
• The genus name is always capitalized but the species name is not. Both species and genus are underlined or in italics.

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What are the phenotypic classifications for bacteria

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Phenotype = the expression of a specific trait based on genetic and environmental influences
1. Microscopic morphology
o e.g. gram stain, shape, organization…
2. Macroscopic morphology
o e.g. colony size and shape, hemolytic properties on blood agar, pigmentation…
3. Biochemical properties
o e.g. ability to ferment specific carbohydrates, presence of specific enzymes
4. Serotyping
o antigen identification
5. Antibiogram patterns
o susceptibility to various antibiotics
6. Phage typing
o susceptibility to specific bacteriophages

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Major groups of bacteria able to form spores

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• B. anthracis spores can live in the soil for many years. Will be breathed in and reside in lung bronchiole
• Respiratory anthrax: humans become infected by inhaling anthrax spores