LSM3232 CA1 Preparation

Microbes

a large and diverse group of living organisms that are too small to be seen by naked eye

Size/Nature of Virus

0.01 - 0.25 um (Acellular)

Size/Nature of Bacteria

0.1 - 10 um (Prokaryote)

Size/Nature of Fungi

2mm - 1m (Eukaryote)

Size/Nature of Protozoa

2 - 1000 um (Eukaryote)

Size/Nature of Algae

1 um - several meters (Eukaryote)

Invention of Microscopes

Antonie van Leeuwenhoek

Who discovered Penicillin?

Alexander Fleming

Germ Theory

theory that certain diseases are caused by the invasion of the body by microorganisms, organisms too small to be seen except through a microscope.

• marked beginning on microbiology

• provided hints on good sterile tefchniques

Miasma Theory

the belief that diseases were caused by organisms arising spontaneously from bad air, swamps, and putrid matter.

Yersinia pestis

Gram negative, rod-shaped pathogen that causes black plague

3 manifestations: bubonic, pneumonic, septicemic

Identification of Antibiotics from Soil

Antibiotics found: chloramphenicol, tetracycline, erythromycin, macrolides, polymyhxin, vancomyxin

Are all microbes harmful?

Not all microbes are harmful, some are crucial for daily life such as microbes for fermentation and antibiotic production

Can you see microbes without a microscope?

Without a microscope, one can still grow the cell in agar or liquid broth to “visualize” microbes

Basic features of a microbial cell:

Cell wall, Plasma membrane, Capsule, Pili, Mesosome, Cytoplasm, Flagella, Chromosome

Bacterial Cell Wall

Made of Glycan strand and Peptide crosslinks.

Glycan strand: repeating units of N-acetylglucosamine and N-acetylmuramic acid

Peptide crosslinks: Usually 3-5 amino acids, containing D-alanine

Function of Flagella

For movement.

The flagellum moves by whirling about its long axis.

Flagella of motile bacteria differ in structure from eukaryotic flagella.

Function of Pili

Surface structure that are important in adhesion to host surfaces

Function of Capsules

• evade host immune responses

• escape mucus (majority negatively charged)

• shield underneath antigens — some pathogens can be difficult to develop a protein-based vaccine

Organelles in Bacterial Cells

Bacterial cytoplasm densely packed with 70S ribosomes.

Other granules represent metabolic reserves.

Endospores

Bacillus and Clostridium species can produce endospores.

Refer to heat-resistant, dehydrated resting cells that are formed intracellularly.

Contains a genome and all essential metabolic machinery, encased in a complex protective spore coat.

Visualized by endospore staining (malachite green and safranin)

Antibiotic Drug Targets

>50% target cell wall synthesis (e.g Vancomycin, Carbapenems)

Overdose of antibiotics targeting cell walls lead to kidney issues.

Last line antibiotics target cytoplasmic membrane structures (e.g Polymyxins and Daptomycin), have high toxicity

Antibiotics targeting cell wall synthesis

Vancomycin and Carbapenems

Last-line Antibiotics

Polymyxins and Daptomycin

Classification of Bacteria based on Cell Morphology

• Coccus (pl. cocci): spherical or ovoid

• Rod/Bacillus: cylindrical

• Spirillum: curved or spiral

• Spirochetes (tightly coiled)

• Grouped/clustered chains of Streptococcus, cubes of Sarcina, grapelike clusters of Staphylococcus.

Cell Shape in Bacteria

generally determined by the peptidoglycan cell wall

Peptidoglycan

made of glycan strands and peptide crosslinks.

Thickness range from 2.4 to 30nm.

Forms the basis of Gram’s staining.

Gram Staining

provide additional information that helps researchers to identify the species.

Preliminary identification of gram nature of bacteria - positive (purple) or negative (red)

Process of gram staining

1. Fixing (heat fix) - kill cells and adhere them to slide

2. Crystal violet (primary stain) - stain all cells

3. Iodine treatment (mordant) - forms a complex with crystal violet

4. Ethanol and acetone (decolorization) - disrupt outer membrane.
   - Gram +ve cells retain CV-Iodine complex and remain purple
   - Gram -ve cells lose CV-Iodine complex and are now colourless

5. Counter stain with Safranin

Gram positive and Gram Negative Cell Envelope

Gram Positive - thick peptidoglycan layer that interacts with the environment

Gram Negative - thinner peptidoglycan layer

Purified Cell Walls retain cell shapes

Cell wall confer the characteristic cell shape and provide mechanical protection.

Withstand boiling in denaturing detergent (SDS), survives at high osmotic pressure.

Inhibition of Peptidoglycan synthesis

Can cause cell lysis (e.g in E. coli)

What makes peptidoglycan synthesis inhibitors so effective and valuable as antibiotics?

• Humans don’t make peptidoglycan

• PG surface is exposed, does not need to enter inner membrane

Acid-fast staining

used to visualize mycobacteria (e.g Mycobacterium and Nocardia species)

• stain cells with high lipid and wax-like surface known as mycolic acids

Process of Acid-Fast staining

1. Fixing (heat fix)

2. Carbol fuchsin (primary stain, red)
   - Trap carbol fuchsin dye in mycobacterial cell envelope, due to presence of mycolic acids

3. Heat treatment
   - Help stain to penetrate the cell wall
   - Steam helps to loosen the waxy layer and promotes entry of primary stain inside cell

4. Ethanol and acid (decolorization)
   - Strip stain from all non-acid-fast cells

5. Counter stain with methylene blue

Function of Cell Membrane

• Compartmentalization

• Selective permeable barrier for molecules

• Protein transport systems for nutrient uptake, waste excretion, protein secretion, and others

• House enzymes to produce energy (e.g photosynthesis, respiration, synthesis of lipid and cell wall)

Structure of Cardiolipins (specialized lipids)

Two phosphatidic acid moieties connect with a glycerol backbone in the center

Phosphatidylglycerol

consists of a L-glycerol 3-phosphate backbone ester-bonded to either saturated or unsaturated fatty acids on carbons 1 and 2.

SEC system

• In bacteria, it functions as a major pathway for moving proteins from the cytoplasm across the inner membrane to the periplasm or for insertion into the membrane itself.

• Required for growth in all bacteria, hydrophobic part of protein can be stabilized by chaperone binding, transmembrane protein complex SecYEG serves as a conduit for protein translocation, protein can be translocated while being synthesized

• Key components: SecA, SecYEG and Chaperones

• Requires energy for transport

Type VI Secretion system

• Delivers toxins to kill competitors in the vicinity - contact dependent killing

• Resembles an inverted contractile phage, delivers toxins to victim’s periplasm or cytoplasm

Outer membrane of Gram -ve Bacteria

Contains lipopolysaccharides (LPS) and O-antigen

Lipopolysaccharides (LPS)

Highly immunogenic (endotoxin)

Block many harmful molecules such as antibiotics

O-antigen

used to classify strains, stabilised by divalent cation

S-Layer in Bacterial Cells

• Outer protein coat that provides mechanical support

• Play roles in pathogenesis

• Protects against pH, ion fluctuation and osmotic stress

Bacterial cell envelope from inside out

Cell membrane, cell wall, outer membrane or mycolic acid (optional), and capsule/S-layer (optional)

Bacterial cytoplasm

• Molecular crowding present - high conc of macromolecules that occupy significant portion of cell volume

• Not just viscous, showed characteristics of colloidal glasses

• Bacterial ribosomes

  • about  15,000 ribosomes per E. coli cell, roughly 30nm in diameter

  • 30S and 50S subunits, vs 40S and 60S in eukaryotes

Types of Flagella

1. Monotrichous (single flagella)

2. Amphitrichous (2 flagella on opposite sides)

3. Peritrichous (many that are spread out)

4. Lophotrichous (many but only on one side)

Assembly of Flagella

Heavily regulated

Assembles from inside to outside, any misstep will lead to a stall in assembly

Chemotaxis

• Positive = attractant

• Negative = repellent

• Detected by chemoreceptors at the cell envelope (usually at cell pole)

Types of Bacterial Motility

i) Brownian motion, sliding motility (non-motile)

ii) Swimming motility

iii) Twitching motility 

iv) Gliding motility

v) Swarming motility

Nucleoid

• extremely packed DNA

• packing achieved by supercoiling and a protein called HU (histone-like) that bends DNA drastically

Cell division in bacteria

• Binary fission
  i) Cell elongation
  ii) Septum formation
  iii) Completion of septum, formation of walls and cell separation

Endospores formation

i) Late sporulation

ii) Mother cell lysis

iii) Germination

iv) Assymetric cell division

v) Engulfment

Bacterial Genome

• Double-stranded, supoercoiled, circular DNA

• Organized by histone-like proteins, similar to eukaryotic DNA packing

• Large range in sizes: Smallest genome of a living cell

Plasmids

• Small, circular double-stranded DNA

• Vary widely in size

• Origin of replication - determines the copy number

• May encode a variety of genes that lead to a better survival

• May contain transposable elements, can be integrated into the genome

Mutations

• Spontaneous mutation - random, undirected, alteration of the nucleotide sequence

• Caused by alternation in the nucleotide sequence at some point of DNA, which can occur due to insertion, deletion or substitution.

Missense mutation

DNA change that results in different amino acids being encoded at a particular position in the resulting protein - may/may not alter function

Silent mutation

Mutations that arise when a single DNA nucleotide alteration inside a protein-coding region of a gene does not affect the amino acid sequence that makes up the gene’s protein.

Nonsense mutation

occurs in DNA when a sequence change gives rise to a stop codon rather than a codon specifying an amino acid.

Frameshift mutation

refers to the insertion or deletion of nucleotide bases in numbers that are not multiples of three.

Prevalence of mutations

0.1% of cells will become a point mutant per replication

Horizontal gene transfer

transferring genes among the same/other organism(s)

3 types of gene transfer:

i) Transformation

ii) Transduction

iii) Conjugation

Transformation

• genes transferred from one bacterium to another as “naked” DNA

• aka direct uptake of foreign DNA, usually single strand

• results in bacteria with new traits (transformants)

Transduction

• DNA transferred from one bacteria to another through a virus

• “Sloppy” bacteriophages pack bacterial DNA at a low frequency

Conjugation

• plasmids transferred 1 bacteria to another via a pilus

Basic Characteristics of Fungi

• Cell wall often contains chitin. Other components include mannan and glucan.

• Morphology:
  i) Yeast-like single cells
  ii) Mycelium or hyphae — septated or non-septated hyphae

• Reproduced by budding, septation, or sporulation

Dimorphism of Histoplasma capsulatum

Different morphology in soil vs in lungs.

Temperature shift triggers this change in morphology.

Capsule in fungi

Huge capsule formed by C. neoformans makes the cells too big to be phagocytosed

Dikaryote and diploid

Dikaryotic mycelium of Crytococcus neoformans.

Not diploid, not haploid, two nuclei co-exist.

Interesting Archea

1. Pyrococcus furiosus
   - Source of Pfu DNA polymerase
   - Optimal growth temperature = 100 degrees celsius

2. Candidatus Prometheoarchaeum syntrophicum MK-D1 - Asgard Archaea

Growth of E. coli

Doubling time = 20 minutes

Mass of an E. coli cell = 2 × 10^-12 grams

Estimated time to gain mass of earth = 43 hours (<2 days)

Origin of eukaryotes

Asgard archaea as the common ancestor

Origin of mitochondria

• have circular chromosome

• sizes (2-8 um) and shape

• Lipid composition (cardiolipin)

Asgard archaea growth

• 60 days lag phase

• Doubling time - 14 to 25 days (>6 months to grow)

Typical Microbial Growth Curve

i) Lag phase - cells adapt to new environment

ii) Exponential phase - rapid, predictable doubling

iii) Stationary phase - growth stops as resources run out

iv) Death phase

General requirements for microbial growth

• Carbon source

• Nitrogen source

• Phosphate source

• Oxygen requirement

• Temperature and pH

• Other important growth conditions

Carbon Source

• Need to synthesize biomolecules and provide energy

• Autotroph/Heterotroph - use inorganic carbon like CO2 or organic carbon for growth

• Auxotroph/Prototroph:

  • Sugars: lactose/glucose/arabinose/fucose

  • Organic acids like acetate or glycerol

  • Alcohols (ethanol, methanol, etc)

  • Sole carbon source

  • Amino acids as carbon source

Autotroph (e.g Cyanobacterium)

Organisms that are capable of producing their own food by using various inorganic components like water, sunlight, air, and other chemical substances.

Heterotroph

Organisms that do not produce their food and depend on other organisms for their food and energy.

Auxotroph

Microorganisms that are unable to synthesize an essential nutrient because of a gene mutation.

• Met mutant = Met auxotroph (cannot grow without Met bc it cannot produce it)

Prototroph

Microbes capable of synthesizing all essential nutrients required for their growth and survival.

• can grow on the minimal media containing only inorganic salts a carbon source and water

Lac operon

Decides preferences of sugar to be metabolised

Key components:

i) LacI repressor - binds DNA when no lactose present

ii) Catabolite activator protein (CAP)

iii) Signaling molecule cyclic AMP (cAMP)

Both glucose and lactose present

• Glucose inhibits adenylate cyclase, decreases cAMP signal

• Lactose inhibits LacI repressor

• cAMP combines with CAP to inactivate it, hence lactose genes stay off

When no glucose and lactose

• No glucose to block inhibit adenylate cyclase, increase of cAMP signal = CAP active

• But LacI repressor blocks genes, hence lactose genes stay off (lac gene expression blocked)

Lactose only present

• No glucose to block cAMP signal, CAP active and can act on lac gene expression

• Lactose removes LacI repressor

• Hence lactose gene turns on

Diauxic Growth (Biphasic Growth Curve)

Diphasic growth represented by two growth curves intervened by a short lag phase produced by an organism utilizing two different substrates, one of which is glucose.

e.g When E. coli grows on a medium with lactose and glucose, it uses glucose preferentially until the glucose is exhausted. Then after a short lag phase during which bacterium synthesizes the enzymes needed for lactose use, growth resumes with lactose as a carbon source.

Water quality indicator

number of “coliform” bacteria rod-shaped bacteria that can ferment lactose —> contamination with fecal matter

Source of Nitrogen

• Common nitrogen sources in nature:
  i) Amino acids
  ii) Ammonia
  iii) Nitrate

• Not all bacteria can synthesize all amino acids. (e.g E. coli can synthesize all AA from nitrate)

• Cyanobacteria use heterocyst to fix nitrogen (oxygen sensitive)

Source of Phosphate

• absolutely required for life - synthesis of nucleotide phosphates, DNA, RNA, cofactors for various biochemical reactions, polyphosphate, etc

Can cells grow with arsenate instead of phosphate?

• Halomonadaceae bacterium GFAJ-1 can tolerate a high concentration of arsenate and low amounts of phosphate

Oxygen requirement

• dependent on its classification as an aerobe, anaerobe, facultative anaerobe

• Oxygen generates toxic products

• E. coli (facultative anaerobe)
  - aerobic respiration = 38 ATP per glucose, but anaerobic respiration = 2 ATP per glucose

Why is oxygen toxic?

• generates superoxide that can form peroxynitrite (ROS)

• Superoxide can also turn into peroxide (H2O2) that can form hydroxyl radicals (ROS)

• Reactive oxygen species can cause cellular damage, hence need both superoxide dismutase and catalase to turn superoxide/H2O2 into water and oxygen.

Temperature Requirements of Microbes

1. Mesophilic - grows best between 25-40 degrees

2. Psychrophilic - grows best below 20 degrees

3. Thermophilic - grows best at high temp, 55 to 80 degrees

pH range for Growth

• Neutral pH - most pathogenic bacteria

• Acidic = Lactobacilli or Helicobacter pylori

• Alkaline = Vibrio cholerae

Other requirements for Growth

• Metal ions (K, Ca, Mg, Fe, S, Mn..)

  • Competition between host and pathogens as biologically available Fe2+ is scarce

  • Siderophores: small, high-affinity iron chelating compounds secreted by microorganisms, examples such as pyoverdine and pyochelin from Pseudomonas aeruginosa remove iron from Lactoferrin from host

• Growth factors - hence why many viable but not culturable bacteria (<20% of soil bacteria can grow in rich medium)

VBNC bacteria

• stands for viable but not culturable bacteria

• can be overcome by a microchip (can grow ~80% of bacteria)

Concept of Pure Culture

• Originate from single cell 

• Homogenous and single species

Biofilm Microbes

• Can withstand higher antibiotic doses (20-50x higher)

• Consist of cells that are tiny but numerous and exhibit group behaviour that amplifies their impact on people and the environment

Types of Growth Medium

• From simple:
  - Minimal medium (e.g Salts as nitrogen source, glucose, phosphate)
  - Chemically defined medium
  - Semi-defined medium (where we know most of the components)
  - Rich medium (enriched)

• To complex

Selective Media

used for the growth of only selected microorganisms.

e.g Antibiotic selective media, MacConkey’s Agar (gram +ve bacteria cannot grow due to bile salts)