Bacterial morphology, metabolism and growth

Dr Ömer Küçükbasmacı

Cell

• Fundemental unit of living things

(smallest bacterium-largest plants-animals)

Bacteria

• The smallest cells

• Visible only with the aid of a microscope

• The smallest bacteria: Chlamydia and Rickettsia-0.1-0.2 micrometer

• Larger bacteria: many microns in length

A newly described species

• Hundred of times larger than the average bacterial cell

• Is visible to the naked eye

Diversity!

Most bacterial cells

• Approximately 1 micrometer in diameter

• Visible by light microscope

• Resolution: 0.2 micrometer

Microscopes

• Light:– Bright-field– Dark-field (Treponema pallidum-

Syphilis_Frengi)– Fluorescence– Phase contrast (details of the living cell)

• Electron

Staining

• Simple• Differential: Gram and Acid-fast stain

Gram-stain: cell wallAcid fast stain: Mycobacterium

• Negative stain: Indian ink(capsule)

• Special staining

Animal and plant cells

• Much larger

• Ranging from 7micrometer (red blood cells)

• To several feet (certain nerve cell)

Each cell

• Genetic basis for reproduction (DNA genome)

• Biochemical machinery (genetic information is transcribed in mRNA and mRNA translated in proteins)

• The machinery for energy production and biosynthesis

• This is all packaged by a membrane.

Each cell

• Replicates by cell division.

Cells

• Eukaryotic (Greek for true nucleus)

• Prokaryotic (Greek for primitive nucleus)

Eucaryotes

• Animals

• Plants

• Fungi

Procaryotes

• Bacteria

• Blue-green algae

Major characteristics of Eucaryotic and prokaryotic cell

Eucaryote Prokaryote• Size >5 μm 0,5-3 μm• Nuclear

structure :

Nucleus classic membrane no membrane

Chromosomes

strands of DNA single circular DNA

diploid genome haploid genome

Major characteristics of Eucaryotic and prokaryotic cell

EucaryoteProkaryote

• CytoplasmicStructuresMitokondria + -Golgi bodies + -Endoplasmic reticulum + -Ribosomes 80S(60S+40S) 70S(50S+30S)Cytoplasmic membrane with sterols no sterol

Major characteristics of Eucaryotic and prokaryotic cell

Eucaryote Prokaryote

• Cell wall -/composed of kitin complex structure(protein, lipits and peptidoglycans)

• Reproduction sexual and asexual asexual (binaryfission)

• Movement complex flagellum simple flagellum(If present) (If present)

• Respiration via mitokondria via cytoplasmic membrane

Bacteria

• Lack nucleus membrane and membrane bound organelles

• A smaller ribosome• Peptidoglycan cell wall which protects it from

environtment with low osmotic pressure, at temperature extremes (both hot and cold), dryness and with very dilute and diverse energy sources.

• They have evolved their structures and functions to adopt these conditions.

Differences

• Between Eukaryotes and prokaryotes

Differences between Prokaryotes

• Bacteria differ:

-morphology (size, shape, stainig characteristics)

-metabolic

-antigenic and

-genetic characteristics

Size

• They are diffucult to differentiate by size

Shape

• Spherical: coccus (Staphyloccus)

• Rod-shaped: bacillus (Escherichia)

• Snakelike: spirillum (Treponema)

• Branched filamentous (Nocardia and Actinomyces)

( Clusters: diplococcus (Neisseria)

chains (Streptococcus)

grapelike (Staphylococcus) )

Bacterial shape

Sperical (coccus) Rod-shaped

Spiral

Spiral or spirillum Helix or spirochete

Bacterial arrangement

Chains: streptococcus

Diplococcus

Clusters: staphylococcus

Packets of eight:sarcina

Treponema by dark-field microscopy

Gram stain

• Two major classes of bacteria are distinguished:

• Gram-positive and • Gram-negative bacteria• Except: • Mycobacteria (waxy outer shell ,

distinguished by acid fast stain)• Mycoplasmas(no peptidoglycan)

Bacterial Ultrastructure

• Internal structure

• External structure

• Gram-positive and gram-negative bacteria have

-Similar internal structure

-But different external structure

Cytoplasm

• DNA chromosome

• mRNA

• Ribosomes

• Proteins

• Metabolites

Bacterial chromosome

• Unlike eukaryotes

• A single

• Double stranded circle

• Not in a membrane bound nucleus

• In a discrete area called nucleoid

Bacterial chromosome

• Unlike eukaryotes

• No histons

Plasmids

• Smaller• Circular• Extrachromosomal DNAs• Not usually essential for cellular survival• Most commonly found in gram-negative

bacteria• Often provide a selective advantage:

resistance to antibiotics

Lack of a nuclear membrane

• Simplifies the requirements and

• Control mechanisms for the synthesis of proteins

Ribosomes

• Unlike the eukaryotic 80S(40S+60S)ribosome

• Bacterial 70S chromosome (30+50S)

• Proteins and RNA of the ribosome are significantly different

• Major targets for antibacterial drugs

Cytoplasmic membrane

• Lipid bilayer

• Similar to eukaryotic membranes

• But no sterols (cholesterol)

Exception: Mycoplasmas

Cytoplasmic membrane

• Responsible for many functions

• Attributable to organelles in eukaryotes:

-electron transport

-energy production (mitokondria in eukaryotes)

Cytoplasmic membrane

• Transport proteins: uptake of metabolites release of other substances

• Ion pumps: to maintain a membrane potential

• Enzymes

Mesosome

• A coiled cytoplasmic membrane

• An anchor to bind and pull apart daughter chromosomes during cell division.

Cell wall

• Distinguishes gram-positive and gram-negative bacteria

The cytoplasmic membrane in most prokaryotes surrounded by • Rigid peptidoglycan (murein) layer• Except: Archaebacteria (pseudoglycan

and pseudomurein) and mycoplasmas (no cell wall)

• Peptidoglycan provides rigidity and determines the shape of a bacteria

• Gram-negative bacteria. + outer membranes

Gram positive bacteria

• Thick multilayered cell wall

• Consisting mainly of peptidoglycan

Gram positive bacteria

• Peptidoglycan

• Sufficiently porous(allows diffusion of metabolites to the plasma membrane)

• Essential for structure, replication, for survival

Peptidoglycan

• During infection

• İnterferes with phagocytosis

• Stimulates lymphoctes

• Pyrogenic activity (induces fever)

Peptidoglycan

• Degraded by lysozyme

• Enzyme in human tears, mucus (produced by bacteria and other organisms)

• Degrades the glycan backbone of the peptidoglycan which protects it from osmotic pressure changes

Protoplast

• Removal of cell wall with lysozyme

• Lysis unless it is osmotically stabilized

Gram-positive cell wall

• Peptidoglycan +

• Teicoic acid

• Lipoteichoic acid

• Complex polisaccarides (C polysaccharides)

• M protein of streptococci

• R protein of staphylococci

Gram-positive bacteria

• Teicoic acid : covalently linked to peptidoglycan• Lipoteichoic acid : anchored in the cytoplasmic

membrane• Common surface antigens• Distinguish bacterial serotypes• Promote attachment to other bacteria and to

spesific receptors on mammalian cell surfaces (adherence)

Gram positive-bacteria

• Teicoic acid: important virulance factors

• Lipoteicoic acid are shed into media and host

• Although weaker

• Can initiate endotoxic-like activities.

Gram-negative bacteria

• Cell wall is more complex

• Both structurally and chemically

Gram-negative bacteria

• Two layers external to the cytoplasmic membrane:

• -Thin peptidoglycan layer• -Outer mebrane (unique to gram-negative

bacteria)

• No teicoic acid and lipoteicoic acid• +periplasmic space

Periplasmic space

• The area between the external surface of rhe cytoplasmic membrane and the internal surface of the outher membrane

Periplasmic space

• A variety of hydrolytic enzymes• Breakdown of large macromolecules for

metabolism• Lytic virulence factors ( collagenases,

hyalurodidases, proteases, beta-lactamases)• Components of sugar transport system• Binding proteins for the uptake of different

metabolites and of a chemotaxis system

Outer membranes

• Unique to gram-negative-bacteria• Maintains the bacterial structure• Permeability barrier to large molecules

and hydrophobic molecules• Provides protection from adverse

environmental conditions (For Enterobactericeae from digestive system of the host)

Outer membranes

• Asymetric bilayer

• The inner leaflet: phospholipits normally found in bacterial membranes

• Outer leaflet: amphipathic molecule( having both hydrophobic and hydrophilic ends) contains Lipopolysaccaride (LPS)

Outer membranes

• LPS molecules are only found in the outer leaflet

Lipopolysaccaride

• Endotoxin (Lipid A+polysaccharide-O antigen, antigenic variety is great)

• Powerful stimulator of immune responses• Causes fever and shock Shwartzman

reaction (disseminated intravascular coagulation) follows the release of large amounts of endotoxin.

Outer membrane proteins

• Porins: transmembrane proteins they form pores allow diffusion of hydrophilic molecules

• Structural proteins and receptor molecules for bacteriophages

Disruption of the outher membrane

• Weakens the bacteria

• +lysozyme

• Spheroplasts (osmotically sensitive) are formed.

External structures

• Capsule

• Pili

• Flagellum

• Proteins

Capsules

• Some bacteria (gram-positive and gram-negative)

• Loose polysaccaride or proteinlayer• Slime layer: loosely adherent and

nonuniform in density and thickness

• Glycocalyx: capsule and slime layer are also called.

Capsule

• Polypeptide capsule:

• Bacillus anthracis (polyglutamic acid)

Capsule

• Hard to be seen by microscopy

• Negative staining: Indian ink

Capsule

• Unnecessary for growth

• Very important for survival

Capsule

• Poorly antigenic

• Antiphagocytic and a major virulence factor (Streptococcus pneumoniae)

• Barrier to toxic hydrophobic molecules such as detergents

• Promote adherence (Streptococcus mutants: stick the tooth)

Quellung reaction

• For vizualizing the capsule

• Using antibodies

• The capsule is swollen

Flagella (Kirpik)

• Motility

• Protein (flagellin)

• Antigenic and strain determinants

• Anchored in membranes through a hook and basal body

• One or several

Flagella

• Monotrichous

• Polar: Pseudomonas aeruginosa

• Peritrichous :Escherichia coli

• Lophotrichous

Fimbriae (Pili)

• Protein(pilin)

• Different from flagella: smaller in diameter and not coiled in structure.

• Promote adherence to other bacteria or to th host (adhesins, lectins, evasins, aggresins)

Fimbriae (Pili)

• Fimbriae are important virulance factors as an adhesin in E. Coli (urinary tract), Neisseria gonorrhoeae

• The tips of fimbriae may contain proteins-lectins that bind to spesific sugars-mannose

Fimbriae (Pili)

• F pili (Sex)

• Promote transfer of large segments of bacterial chromosome between bacteria

• Encoded by a F plasmid.

Bacterial exceptions

• Mycobacteria

• Corynebacterium

• Nocardia

• Mycoplasmas

Mycobacteria

• Peptidoglycan (slightly different)• Waxlikelipit coat of mycolic acid• Cord factor• Wax D• Sulfolipids• Acid-fast staining• The coat responsible for virulence and

antiphagocytic.

• Corynebacterium

• Nocardia

Produce mycolic acid lipids.

Mycoplasmas

• No peptidoglycan cell wall

Structure of Bacterial Cell Wall

• The components are large structures

• They are made up of polymers of subunits

• Synthesis of it occurs outside the bacteria

Peptidoglycan(Mucopeptide, Murein)

• Linear polysaccaride chain:

-repeating disaccarides:

N-acetylglucosamine

N-acetylmuramic acid

• Cross-linked by peptides

Cell wall synthesis

• Cross-linking reaction is catalyzed by:

-membrane bound transpeptidases

-DD-carboxypeptidases-

penicillin-binding proteins (PBPs)

Cell wall synthesis

• Penicillin-binding proteins (PBPs):

-remove extra terminal D-alanines

-These terminal D-alanines limit the extent of cross-linking

-They are targets for penicillin and beta-lactam antibiotics

Cell wall

• Peptidoglycan is constantly being synthesized and degraded.

• Autolysins such as lysozyme are important for determining the shape of bacteria.

Cell wall

• During starvation:

-New peptidoglycan synthesis does not occur

-Peptidoglycan is weakened

-Gram-staining property changes

(old cultures)

Biosynthesis of peptidoglycan

• Unique to bacterial cells

• İnhibited with no adverse effect of human cells

• An important target for antibiotics

(selective toxicity)

Lipopolysaccaride

• Lipid A

• Core polysaccaride

• O antigen

Lipoppolysaccaride

• Lipid A:

basic component

essential for bacterial viability

endotoxin activity

• O antigen: antigenic part (serotypes)

Inclusion granules

• Storage of energy

• Poly-beta-hydroxybutyric acid (PHB)

• Glycogen

• Polyphosphate

Inclusion granules

• Polymetaphosphate: Corynebacterium

-Babes-Ernst bodies

Spores

• Resistant to harsh conditions

• (loss of nutritional requirement, dessication, intense heat, radiation and attack by most enzymes and chemical agents)

Spores

• Exist for centuries

• Diffucult to decontaminate with standart disinfectants

Spores formers:

• Some gram-positive

• Never gram-negative

Spore formers

• Bacillus

• Clostridium

Kinds of spores

• Endospore: Bacillus subtilis

• Terminal endospore: Clostridium tetani

‘drumstick’

• Subterminal: Clostridium botulinum

Under harsh conditions

• Vegetative state is converted to dormant state (spore)

Localisation of the spore within a cell

• Characteristic of bacteria

• Can assist in identification of the bacterium.

Spore

• Dehydrated

• Multishelled structure

• A complete copy of chromosome

• Minimum concentration of proteins and

Ribosomes + High concentration of calcium bound dipicolinic acid

Spore

• Outside the core:

-inner membrane

-spore wall

-cortex

-outher membrane

-keratin-like protein coat

-exosporium

Sporulation

• 6-8 hours

Germination

• Spor__vegetative state:

disruption of the outher coat

by mechanical stress, pH, heat or

another stressor

It takes about 90 minutes

Bacterial metabolism

• Bacterial growth:

-a source of energy

-raw materials

*To build the proteins, structures and membranes

*That make up the structure and biochemical machines of the cell

Bacterial metabolism

• Bacteria should obtain or synthesize:

-aminoacids

-carbohydrates

-lipids as building blocks of the cell

The minimum requirement for growth

• Carbon

• Nitrogen

• Energy source

• Water

• Various ions

• Chemotrophs:

derive energy from inorganic material

• Chemoorganotrophs:

Most bacterial

Heat

• Cardinal temperatures:

-minimum temperature

-optimum temperature

-maximum temperature

Temperature

• Psychrophiles

• Mesophiles

• Thermophiles

• Hyperthermophiles

ph

• Acidophiles: Below pH 5 (many fungi)

• Alkaliphiles: Above pH 9 (Vibrio)

• Neutral pH: most pathogens

Oxygen requirement

• Obligate anaerobes :

Clostridium perfringens

• Obligate aerobes

• Facultative anaerobes

• Microaerophilic

Incubation for growth

• Aerobic

• Anaerobic

• Capneic (%5 Carbon dioxide)

Methabolism

• Catabolism: substrate breakdown and conversation into usable energy

• Anabolism: synthesis of cellular constituents (cell wall, proteins,fatty acids, nucleic acids

Bacterial growth

• Bacterial replication

• Two equivalent daughter cells

• Binary fission

(Escherichia coli: 20 minutes

Mycobacterium much slower: 12-24h)

Bacterial growth curve

• Lag phase

• Exponential phase

• Stationary phase

• Death phase