Biology 2EE3 – Intro to Micro & Biotech (1-3)

Biology 2EE3 – Introduction to Microbiology and Biotechnology

Lecture 1: What is Microbiology?

Microorganism:- Any living organism (or life form) we cannot see with naked eye

o Certain stages we can see easily (e.g. mushroom)- What is life?

o An object that avoids the decay into equilibrium (Shrodinger)o A self-sustained chemical system capable of Darwinian evolution (NASA)o An object with the capacity for metabolism, growth and reproduction, responsive to stimuli and

evolution (operational definition)- Animals are morphologically very diverse- Microorganisms are morphologically very similar (evolutionarily they are very diverse)- Virus = no cell structure (a-cellular, parasites)- Always be some bacteria on lettuce, but numbers are so small they don’t cause problems; if numbers of

E.coli are sufficiently high, can cause pandemic of infectious diseases (e.g. cucumbers in Germany)- Irregular shapes

Why study microbiology?- satisfy intrinsic interest (basic interest)

o gene expression (marker proteins), metabolic pathways, genetic engineering (TI plasmid, used to transfer DNA – rocket-like)

- Industrial microbiology o Enzymes, antibiotics, food additives (AAs, vits), bulk chemical (ethanol, methanol, acetic acid),

fine chemicals (stereo-specific synthesis, e.g. steroid production – find specific enzymes that has high activity in converting one step, switch to different enzyme with higher efficiency for next step)

o Leaching off metals (help recycle electrons, e.g. Ca2+ Ca0)o Pharmaceuticals (hormones, blood proteins, immune modulators, vaccines)

Insulin production from E.colio Microbial fuel cells (potentially replace fossil fuel-based economy)

- Food and drinko Fermented food (yoghurt, cheese, sausage, soy sauce, beer, wine)

- Agricultureo Forestry, fishery and animal husbandryo N2-fixing microbes – 3 million, soil fertility enrichment (free-living:

cyanobacteria, symbiotic: legumes) Why are all organisms not capable of fixing nitrogen? Too

energy costly.o Mycorrhizal fungi: all land plants contain them in their roots, help

plants obtain/extract minerals, water, phosphorous) Fungi evoluved earlier than plants, allowed them to move to

terrestrial environmento Rumen microbes: in livestock (sheep, cows) – completely rely on

microorganisms to digest celluloseo Plant pathogens: >$200 billion losseso Animal pathogens

A source of human pathogens and superbugs – great overuse on farms >80% of antibiotics produced are used in agricultural farms

- Ecoloy/environmento 5x1030 prokaryotic cells on Earth, microbial carbon – plant carbon >> animals carbon

increase microbial component in environment to capture/fix carbon, reduce CO2

o metabolic diversity (greater number of substrates, pH, T)o global cycling of N, S, Co biodiversity in the environmento sewage plants and bioremediation of xenobioticso bio-degradable plastics (poly-hydroxy butyric acid)

- Medical microbiology (infectious and chronic diseases)o Many of the major discoveries were based on human infectious diseases (caries, infections,

bioterrorism)o Normal flora vs. pathogens (organisms that can cause diseases, organisms that will cause

diseases and organisms that can/will cause disease In specific people) Normal flora = can invade host tissue (be pathogenic) if you have compromised immune

system HIV, chemotherapy or if you take in so much that your body cannot cope

o Vaccination – immunology Most research based around virulence mechanisms (what makes them infectious) What is the specific agent that causes disease? Develop cures/treatment strategies 50% of the time, microbiologists/epidemiologists awarded Nobel prize in

medicine/physiology (e.g. Marshall, Warren in stomach ulcers from Helicobaccter pylori) diagnostics and prevention of diseases can be used to monitor human migration since stomach bacterial is not in much

communication with outside world

Importance in Human Society- 10000 BC, fermentation, Chinese, Egypt- Antoni van Leeuwenhoek (1676) first to observe microbes under microscope (didn’t invent it)- Edward Jenner (1790) cowpox vaccination prevents smallpox- Louis Pasteur (1822-1895)

o Basic science: falsified the hypothesis of Spontaneous Origin of Live Introduced sterilization, then showed that food didn’t grow bacteria (cannot

automatically, spontaneously give rise to life) Scientific basis for fermentation (microbe is required for wine-making, beer brewing

o Practical medical applications: Anthrax vaccine Chicken cholera vaccine Rabies vaccine

- Robert Koch (1843-1910)o Not the first one to suggest that some human diseases are caused by infectious germs; but no one

scientifically proved that a specific bacteria causes a specific diseaseo Basic science:

Established the principles for identifying infectious disease agents, known as Koch’s postulates (used to identify all the infectious disease agents)

1. Microbe is found in all cases of disease, non from healthy individuals2. Microbe is isolated from diseased host, grown in pure culture3. When microe is introduced into healthy susceptible host the same disease occurs4. The same strain of microbe is obtained from the newly diseased host

- Now know that there has to be a specific number of cells to cause disease (quantitative disease, not just 1 or 0, lots of grey) (or host with suppressed immunity)

- Isolate grow pure culture: most microorganisms cannot be grown in pure culture; can never (no matter what the conditions) culture all the microbes in a natural sample

- Cannot infect healthy host humans; can use surrogate animals, but can differ- Scientific community has accepted many modifications to this postulate

Introduced solid agar medium for culturing pure microbes (idea came from an assistant’s wife)

o Disease Agents identified: Bacillus anthracis causes anthrax Mycobacterium tuberculosis causes tuberculosis Vibrio cholera caused cholera

Discovery of AntibioticsAlexander Fleming (1929)

- fungal contamination (Penicillium notatum) killed his cultures of the bacterium Staphlococccus aureus

Discovery of DNA as the genetic material- 1944 Oswald Avery et al. The genetic material responsible for transformation is DNA- 1953 Rosalind Franklin (& Maurice Wilkins) used x-ray diffraction analysis to show the structure of

DNA is a double helix- 1953 Watson and Crick – porposed that the double helix DNA consists of anti-parallel

- 1973 Stanley Cohen – recombinant DNA molecule is created in a test tube- 1977 Carl Woese: a third domain of life is discovered (bacteria, archaea, eukaria – super-kingdoms)

o Fungi and animals in same super-kingdomo DNA as a genetic marker

- 1981 Kary Mullis: invented PCR and the genomics evolution

Lecture 2 - Microbial Cell Biology 1:

Morphological diversity of microbesSize range of microbes (with RBC as reference)

- smallest: 0.4 microns prochlorococcus marinus (not capable of self-sustaining)- large: paramecium, 100 micron- not a good way to classify microbes; not a good indicator of evolutionary relationships between

microbes

Tools for visualizing microbes- light microscope- light as electromagnetic energy: the shorter the wavelengths, the more energy they have, the better

resolution for images they can provide, and the more damage they can do to cells- Magnification: product of magnification of objective and ocular lens- Resolution: a measure of light gathering ability of objective lens (0.2 microns-0.2nm) (0.5gamma

divided by numerical aperture)- Contrast: differential diffraction of light, enhanced through staining.

o Staining process (*look up)- The gram stain: gram positive (dark purple), negative (reddish) different cell wall (much thicker in

positive)o basic shapes and forms of bacteria

rods (bacilli) flagella inside cells (spirochetes) cocci in pairs cocci in chain

- Dark field microscopy: for examining small bacteria and extracellular structure- Phase-contrast microscopy for working with live eukaryotic microbes with transparent cytoplasms (esp.

protozoan)- Interference microscopy for defining the shape of cells- Fluorescence microscopy: for detecting cells or cell parts based on fluorophore binding- Transmission Electron microscopy: electron beams travel through the object where (want to look inside

the cello- Scanning electron microscopy (want to look at cell surface)- Laser confocal scanning microscopy: take a series of plane 2-D pictures for building 3D images of

microbial communities- Atomic force microscopy: used van der Waals forces between a probe and a cell surface to map 3D

topography of a cell for surface ultra-structures of live cells- Mollecular visualizations:

o X-ray crystallography detects interference patter of x-rays entering the crystal lattice of a molecule: for determining 3D structure of crystallized macromolecules at atomic resolution (ribosomal structure was determined using this method, has 20-30 different parts)

o Nuclear magnetic resonance: using magnetic properties of atomic nuclei to build structures of molecules in liquids

Microbes have a large surface-Volume ratio- allows more exchange

First impressions- FtsZ fused to GFP to determine the location of the gene when expressed- Typical bacterial cell is a mess when cut open (scanning electron microscope)

o Model of a bacteria cello Porins: about 10micron in size, etc. (do for all other enzymes, proteins)o Add all necessary cellular enzymes, components = 200 nmo 70% of cell weight is water, 16% is protein 2000 is upper limit of genes that are expressed.o Most RNA is mRNA (degrades within the cell very quickly), other RNA lasts longer because it

has so many modified bases (cell can’t digest it)- to break up cell: detergent, freezing/thawing, sonication, enzymatic digestion-

Cytoplasmic membranes- cell membrane

o functions: transport, energy generation, signaling, defense structural integrityo Hopanoid adds rigidityo ATPase (proton-driven) veryo Phospholipids provide basic framework for cell membrane

Structure can be very diverse among microbial groups Head groups: phosphatidylglycerol, cardiolipin Side chainsL palmitic acid, oleic acid (cis or trans), cyclopropane F.A. (loops,

extra carbon-carbon chain, become much more hydrophobic, thus interactions would be much more stable than typical straight sidechains)

Bacteria Archaea EukaryaMembrane-strengthening lipids Hopanoids None sterolsHydrophobic side chain Fatty acid Isoprene FAChemical bonds linking glycerol and hydrophobic side chain

Ester Ether Ester

o Synthesis and structure of the domain specific lipids Hopane (sp?) very, very stable - Thermophilic organisms (many archaea, some bacteria) –

Significant modification of cell membrane – diglycerol tetraether, cyclopentane rings (upper limit 130-140 degrees, ADP is problem- need alternative energy)

19 September 2011Lecture 3 - Microbial Cell Biology II - Internal Complexities and External Augmentations :

1. Bacterial cell wall structure, synthesis, and comparisons (Gram+ and Gram-)

- Bacterial cell wall (peptidoglycan) - single molecule resembling a geodesic dome- Gram +: up to 40 layers, Gram -: one layer- chemical structure of peptidoglycan:

- alternating G-M-G-M (n-acteylglucosamine, n-acetylmuramic acid)- l-form of a.a.s is dominant (since multiple d-forms, suggested that peptidoglycan is ancient molecule)

- penicillin block enzyme that creates bond between d-alanine

- Comparisons of the cell walls and cell envelopes of G+ and G- bacteria:o S-layer in gram +; composed of proteins and glycoproteins

- Teichoic acids reinforce (Strengthen) the structure of Gram+ cell wall- Teichoic acid: phosphodiester-linked glycerol or ribitol (C5 monosaccharide) with sugars or amino acids linked to the middle OH groups

- Negatively charge (stains are positively charged)- Helps retain the Gram stain

Envelope, Cell Wall and Lipopolysaccharides in Gram- Bacteria - Antigenic properties vary with LPS structures - LPS as endotoxin, released after cell lysis

e.g. after killed by antibiotics, => over-stimulates host defense and results in toxic shock syndrome

- O-polysaccharide, repeating units of 40+ monosaccharides- lipopolysaccharide itself is endotoxin (not when cell is alive)- if you have gram- bacteria in blood stream, it is not recommended

to take antibiotics as it releases endotoxin into blood

- sugar is generally most efficient form of energy for bacteria

2. Cell division: DNA replication, septation

- The Bacterial Nucleoid - DNA is distributed throughout much of the bacterial cytoplasm organized into domains (~50) that are all connected to the origin of replication. Ori is typically- DNA is longer than the size of the bacteria, folding accomplished by DNA binding protein- Transcription and translation are tightly coupled in bacteria- SRP: signal recognition particle for proteins targeting membrane- soon after transcription, ribosome begins translation (unlike modifications before leaving nucleus)- polysome (mRNA get degraded very quickly)-

- how genomes are duplicated – Ori is usually right in the middle part of the cell- replisome (DNA through zipper) consists of helicase (unwind to single strands), leading strand replication

is very straight forward (5’ 3’ end)o lagging strand primase, ssDNA-binding proteins to protect, replicates in the reverse direction

- cell wall and cell envelope start expanding- average cell usually has 2 copies of its genome

-FTSZ formation of ___; universally conserved

3. Specialized internal structures: thylakoid, gas vesicle, magnetite - most bacteria do not have membrane-enclosed structures. Some exceptions exist:

- Thylakoids in photosynthetic bacteria- so many layers so max amount of internal surface area for reactions- carboxysome (O2 CO2)

- Gas vesicles: these vacuoles are enclosed by proteins that trap and collect gases - e.g. H2 and CO2- Function? Aquatic microorganisms use the gas vesicles to adjust their positions in water, mostly in

response to light intensity and temperature- difference between gas vesicle and thylakois and carboxysomes - enclosed by proteins not phospholipid

bilayer membrane- Magnetosomes: function to use earth’s magnetic field to help orient the direction of cellular movement

- use in sewage treatment can take up large amounts of metals from waste, life at bottom of ponds/tanks makes it easy to collect (magnet)

- Magnetite: Fe304

4. External modifications: pili, flagella- Pili

- attachment to surfaces- genetic material exchange- can be on both gram+/- bacteria

- Flagellar types:- named by location on bacterial cell surface- much bigger than pili, composed of multiple

proteins

Flagellar motor:- l, p rings are single protein- p ring in peptidoglycan layer- MS. C rings - driven by the proton gradient from the ETC- 1000 ATP molecules to turn motor around one loop- filament is made of 1 protein, but multiple subunits- rotation determined by environmental signal

Chemotaxis Driven by Flagellar Movement- how bacteria determines where it wants to go- does not move straight to food source (move and tunble)- once attractant detected, flagella form bundle to move but quickly loose direction