Lecture #2

1. Lecture #2 Prokaryotes

3. Prokaryotes • microscopic single celled organisms • collective biomass – 10x of all eukaryotes!!!!! • vast genetic diversity among members • physical diversity • shapes: spheres (coccus), rods (bacilli) and spirals 1 µm 2 µm 5 µm Spiral Spherical (cocci) Rod-shaped (bacilli)

4. Prokaryotes • REMEMBER: adoption of a three domain system of superkingdoms • 1. Bacteria – prokaryotic (or Eubacteria) • 2. Archaea – prokaryotic • 3. Eukarya - eukaryotic • divisions into protist, fungi, plants and animals

5. Domain Eukarya Domain Archaea Domain Bacteria Proteobacteria Gram-positive bacteria Delta Alpha Epsilon Beta Chlamydias Korarchaeotes Euryarchaeotes Crenarcaeotes Nanoarchaeotes Gamma Cyanobacteria Eukaryotes Spirochetes Universal ancestor “The Tree of Life” • Applying molecular systematics to the investigation of prokaryotic phylogeny has produced dramatic results • lead to a phylogenetic re-classification of prokaryotes • development of Domain Bacteria • separation into 9 major taxa of prokaryotes - based on molecular systematics

6. Bacterial groups • Bacteria or Eubacteria • include the vast majority of prokaryotes that we are aware of • comprised of 5 major groups: • 1. proteobacteria: diverse group of gram negative bacteria • 5 major subgroups: alpha  epsilon • 2. gram-positive: very diverse • solitary and colonial • free-living and parasitic • e.g. Bacillus, Streptococcus

7. Bacterial Groups • 3. cyanobacteria: blue-green algae • photoautotrophs • O2-generating photosynthesis through chloroplasts • 4. chlamydias: parasitic bacteria • can only survive within animal cells • cell walls lack peptidoglycan entirely • 5. spirochetes: helical in structure • heterotrophs • most are free-living • some can be parasitic

8. Archaea • Archaea: share similarities with prokaryotes and eukaryotes • divided into four clades: Euryarchaeota, Crenarchaeota, Korarchaeota and Nanoarchaeota • 1996 - recent discovery of a new clade – Korarchaeota • koron = “young man” • found in hot springs in Yellowstone • 2002 – in hydrothermal vents off the coast of Iceland – found extremely small archaea • name Nanoarchaeota– smallest of the four • nanos = “dwarf” • smallest genome known – only 500,000 base pairs • three other species found since then – hydrothermal vents and hot springs

9. Archaea • first Archaea to be identified were found in extreme environments = extremophiles • 1. thermophiles (thermos = “hot”) • clade Crenarchaeota • thrive in very hot environments • 2. halophiles – high saline environments (halo = “salt) • clade Euryarchaeota • some tolerate the high salinity, others require it • red-brown scum possess a visual pigment called bacteriorhodopsin • 3. methanogens – named for the way they obtain energy • clade Euryarchaeota • use CO2 to oxidize H2 and produce energy - releases methane (CH4) as a waste • strictest of anaerobes – obligate anaerobes

10. Role of prokaryotes • chemical recycling • ecosystems depend on a continual recycling of chemical elements between the living and nonliving components of this planet • chemotrophic prokaryotes function as decomposers • prokaryotes also convert inorganic forms into organic forms for other living organisms • autotrophs use CO2 for energy but make organic compounds which are stored and passed up through the food chain

11. Role of prokaryotes • symbiotic relationships • prokaryotes can possess beneficial relationships with other prokaryotes in terms of metabolic cooperation • also hold beneficial relationships with other organisms like eukaryotes • known as symbiosis • GENERAL DEFINITION: symbiosis = ecologic relationship between organisms of different species • two major kinds: mutalism & parasitism

12. Role of prokaryotes • symbiotic relationships • mutalism – both organisms benefit • health benefit • parasitism – one organisms (parasite) benefits at the expense of the host • prokaryotes cause 50% of diseases in humans • cause illness through the production of endotoxins or exotoxins

13. Key Prokaryotic Adaptations • 1. Cell surface structures • evolution of the cell wall • 2. Motility • evolution of flagella • 3. Internal organization of DNA • evolution of the chromosome and plasmid DNA • 4. Reproduction • evolution of binary fission, conjugation, transformation and endospores

14. The Bacterial Cell Wall • key feature – prokaryotes are surrounded by a cell wall • maintains cell shape, provides physical protection and allows the cell to control its osmolarity • in a hypertonic environment – most prokaryotes will lose water and shrink = plasmolysis • cell wall is NOT like the cell wall of plants and fungi – which are made of cellulose or chitin • encloses the entire prokaryote

15. The Bacterial Cell Wall • roles of the bacterial cell wall • structural: forms an anchor for the attachment of many intracellular subsatnces • counteracts the osmotic pressure created by the cytoplasm • changes in OP can result in the loss of water and plasmolysis • involved in binary fission (reproduction) • protection against changes in ion and pH levels, foreign enzymes, phagocytosis by foreign pathogens

16. Cell Wall & Peptidoglycans • most prokaryotic cell walls contain peptidoglycans (murein) • presence is used to classify the two types of bacteria: gram negative and gram positive • thicker in gram positive bacteria than gram negative • peptidoglycan: • sugar polymer modified with amino acids • cross-linked in gram-positive bacteria • forms a crystal lattice organization

17. Peptidoglycan • peptidoglycan layer is a crystal lattice or mesh-like structure • formed from linear chains of two alternating sugars called N-acetyl amino sugars • N-acetyl glucosamine (GlcNAc or NAG) • N-acetyl muramic acid (MurNAc or NAM) - 3 to 5 amino acids attached • interactions occur between these amino acids = cross-linking • cross-linking results in a 3-dimensional structure that is strong and rigid • high degree of cross-linking in gram-positive bacteria

18. Antibiotic actions • Antibacterial drugs such as penicillin interfere with the production of peptidoglycan by binding to the enzymes that perform the cross-linking • for a bacterial cell to reproduce – new cell walls must be made • this requires the assembly of more than a million new peptidoglycan subunits • these subunits must be cross-linked – by enzymes called transpeptidases • penicillin & vancomycin –inhibits cell wall synthesis by preventing NAM and NAG cross-linking

19. Lipopolysaccharide Outer membrane Peptidoglycan layer Cell wall Cell wall Peptidoglycan layer Plasma membrane Plasma membrane Protein Protein Gram- positive bacteria Gram- negative bacteria 20 µm Gram-negative Gram-positive Gram positive bacteria • retain the crystal violet stain used in a Gram stain – so they stain purple • simpler wall construction with larger amounts of peptidoglycans • high peptidoglycan content of the cell wall take up the crystal violet dye – not washed away in subsequent steps • most pathogens in human are gram +ve • divided into cocci and bacilli forms

20. Gram negative bacteria • do not retain the crystal violet dye - dye is washed away • cell wall of gram negative bacteria is comprised of a PG layer PLUS an outer membrane • located outside the PG layer • comprised of lipopolysaccharides, lipoproteins and porins • the lipopolysaccharides are toxic to humans – endotoxin layer Peptidoglycan layer Plasma Membrane

21. Gram negative bacteria • SOME WELL KNOWN GRAM NEGATIVE BACTERIA • coccobacilli: H. influenzae, B. pertussis, L. pneumophilia • cocci: N.meningitidis, N. gonorrhae • bacilli: E.coli, V. cholerae, H. pylori, S. dysenterae, Salmonella

22. Gram staining 1. Place a slide with a bacterial smear on a staining rack. 2. STAIN the slide with crystal violet for 1-2 min. 3. Pour off the stain and rinse with water thoroughly.4. Flood slide with Gram's iodine for 1-2 min. 5. Pour off the iodine and rinse with water thoroughly.. 6. Decolourize by washing the slide briefly with acetone (2-3 seconds) – alternatively use 95% ethanol 7. Wash slide thoroughly with water to remove the acetone 8. Flood slide with safranin counterstain for 2 min. 9. Wash with water. 10. Blot excess water and dry by hand over bunsen flame. • both Gram-positive and Gram-negative bacteria take up the same amounts of crystal violet (CV) and iodine (I). • in Gram-positive bacteria - the ethanol used in washing the bacteria dehydrates the bacteria and traps the CV-I in the cell wall– PURPLE STAIN • in gram negative bacteria – the thinner cell wall does not prevent extraction of the CV-I complex • plus the outer membrane limits the amount of CV-I complex that can reach the PG layer – CLEAR STAIN http://www.youtube.com/watch?v=OQ6C-gj_UHM

23. Bacterial capsule • found in many prokaryotes – both +ve and –ve • found outside the cell wall • also called the glycocalyx • if it is less organized = slime layer • resists dehydration • roles in adherence to surfaces • participates in colonization • may make the bacteria resistant to the immune system

24. Bacterial adhesion • via the glycocalyx or capsule • also through the development of specialized appendages • fimbrae – more numerous and shorter than pili • pili – some can be specialized for the reproduction of the bacteria

25. Bacterial motility • half of all bacteria exhibit taxis – the ability to move towards a specific signal • movement towards a chemical signal = chemotaxis • movement toward light = phototaxis • major mobility mechanisms: flagellar and gliding • gliding: movement of cells over surfaces without the aid of flagella • not completely understood

26. Bacterial Flagellae • most motile bacteria propel themselves by flagella that are structurally and functionally different from eukaryotic flagella • major types of flagellar bacteria: • monotrichous (one flagella) • lophotrichous (tuft at one end) • peritrichous (found evenly over the surface)

27. Bacterial Flagella • consist of three parts:the motor, the hook and the filament • the filament consists of a hollow, rigid cylinder composed of a protein called flagellin • attaches to a curved structure called the hook • hook is attached to the basal body or basal apparatus • basal body: embedded in the cell wall down to the plasma membrane hook filament basal body stator http://www.youtube.com/watch?v=Ey7Emmddf7Y rod rotor

28. Bacterial Flagella • a portion of the basal body is called the motor • made up of stationary ‘stators’ and a rotating ‘rotor’ connected to the hook via a rod • ATP-driven proton pumps pump protons out of the bacteria • when the protons diffuse back in through the stator – turns the rotator and the attached rod • the hook and attached filament also rotate • anticlockwise rotation of flagella thrusts the cell forward with the flagellum trailing behind hook hook filament filament basal body basal body stator stator http://www.youtube.com/watch?v=Ey7Emmddf7Y rod rod rotor rotor

29. Prokaryotic genome organization • lack the compartmentalization of eukaryotic cells • do have specialized membranes that perform specific functions • genome is a single circular chromosome contained in a nucleoid region • located in a nucleoid– a region of the cytoplasm • can also have several smaller circular pieces of DNA = plasmids

30. DNA replication – the prokaryotic players • prokaryotic replication requires 3 things: • 1. initiation sequence– DNA sequence that initiates DNA synthesis – called oriC • region of DNA that the replication machinery recognizes • 2. initiators – proteins that recognize the oriC region • DnaA –binds to oriC and unwinds a small area of the DNA helix (20 bps) • DnaB–unwinds the DNA further - acts as a helicase • two DnaB molecules move in opposite directions  replication bubble • 3. termination sites – DNA synthesis stops when the regions of DNA being replicated meet each other • alternatively – can stop at specific sequences of DNA = termination sequences

31. Prokaryotic replication • bacterial chromosome is a helix • unwinding will produce two parent strands • sense and anti-sense • parent strands are used a templates for the creation of new “daughter” strands • DNA daughter strands can only be made in one direction – 5’ to 3’ • the anti-sense strand can be replicated continuously = creates the leading daughter strand • the sense strand is replicated discontinuously = in fragments (Okazaki fragments) and creates the lagging daughter strand • both parental strands are replicated at the same time by a large complex of proteins • so the sense strand needs to be “looped” so that is “runs through” this complex in the same orientation as the anti-sense strand

32. Prokaryotic DNA replication • at the oriC – a replication complex forms: • 1. helicase – DnaB– unwinds the DNA helix into separated parental strands • 2. single, strand binding proteins (SSBs) – bind to the unwinding DNA to prevent rehybridization back into a helix • 3. primase – DnaG (or RNA polymerase II) - makes a small RNA primer for the binding of DNA polymerase III • 4. DNA holoenzyme complex – complex of several proteins including DNA polymerase III • 5. DNA ligase – links together Okazaki fragments into one continuous daughter strand topoisomerase DNA Pol III replicated DNA DnaB primer DnaG Replication Complex SSBs Replication Direction – daughter DNA made 5’ to 3’

33. The big picture animation • for the “big picture”: http://www.youtube.com/watch?v=-mtLXpgjHL0 Leading Daughter- Antisense parent Sense parent Beta clamp Anti- Sense parent alpha helicase primase alpha SSBs RNA primer #2 RNA primer #1 Okazaki Fragment #1 Beta clamp

34. Leading Daughter- Antisense parent Polymerase direction Sense parent Anti- Sense parent alpha RNA primer #3 helicase alpha primase SSBs RNA primer #2 Okazaki Fragment #2 RNA primer #1 Okazaki Fragment #1

35. Prokaryotic Reproduction • once the DNA is replicated – the bacteria must divide • bacterial reproduction is through binary fission = asexual reproduction • each replicated chromosome attaches to the plasma membrane • the cell elongates and causes the two chromosomes to separate. • the plasma membrane invaginates, or pinches inward toward the middle of the cell • when it reaches the middle - the cell splits into two daughter cells • limited by resource availability and competition from other microorganisms (produce antibiotics)

36. Prokaryotic Reproduction • Budding helps some prokaryotes to replicate. • The bud is an outgrowth of the parent cell. • The bud has an exact duplicate copy of the parent cell’s genome. • The bud falls off and a mature parent cell arises.

37. Mixture Mutant strain arg+trp– Mutant strain arg–trp+ Mixture Mutant strain arg+trp– Mutant strain arg–trp+ No colonies (control) No colonies (control) Colonies grew Genetic recombination in prokaryotes • prokaryotes can transfer information to each other • Experiment: two mutant strains of E.coli with different nutritional requirements grown on minimal media (sugars, salts, no amino acids) • one strain trp- will NOT grow in the absence of tryptophan • second strain arg- will NOT grow in the absence of arginine • mix the two strains and grow in minimal media (lacks arginine and tryptophan) • growth of the colony is observed • transfer of genetic information between the two strains to create an arg+trp+ strain New strain arg+ trp+

38. Genetic Recombination in Prokaryotes • Three processes bring prokaryotic DNA from different individuals together: • 1. Transformation • 2. Transduction • 3. Conjugation

39. Transformation • Transformation = the uptake of naked, foreign DNA from the surrounding environment • Experiment: transformation of harmless Streptococcus pneumoniae bacteria into pneumonia-causing cells • mix a live, nonpathogenic strain with a dead strain (lysis in dead strain results and the release of genetic material into the surrounding environment) • non-pathogenic strain takes up a piece of DNA carrying the allele for pathogenicity • foreign allele becomes incorporated into the non-pathogenic hosts chromosome • can be artificially induced in the lab • either through chemical weakening of the plasma membrane • OR electrical weakening

40. Phage DNA A+ B+ A+ B+ Donor cell A+ Crossing over A+ A– B– Recipient cell A+ B– Recombinant cell Transduction • bacteriophages carry bacterial genes from one host cell to another • bacteriophage – virus that infects a bacterium • infection of another bacterium results in the introduction of the new piece of DNA – if it contains a new gene – alters the genetic makeup of the recipient cell • if this is a random event = generalized transduction • in specialized transduction – phage picks up only a few bacterial genes

41. Conjugation • Conjugation: bacterial “sex” • conjugation is the direct transfer of genetic material between bacterial cells that are temporarily joined • requires the formation of a mating bridge – sex pilus • the transfer is one-way: One cell (“male”) donates DNA, and its “mate” (“female”) receives the genes • “Maleness,” the ability to form a sex pilus and donate DNA, results from a gene called = F (for fertility) factor • F factor can be part of the chromosome or found on a plasmid (F plasmid)

42. F plasmid Bacterial chromosome F+ cell F+ cell Mating bridge F– cell F+ cell Bacterial chromosome Conjunction and transfer of an F plasmid from and F+ donor to an F– recipient The F Plasmid and Conjugation • bacteria containing the F plasmid are designated F+ cells (male) • function as DNA donors during conjugation • F+ cells transfer DNA to an F recipient cell (female) through conjugation: • 1. formation of the mating bridge • 2. a single strand of the F plasmid breaks at a specific point and begins to move into the female/F- bacteria • 3. the missing piece of DNA is regenerated in the male/F+ by replication • 4. the female replicates the incoming DNA – two new double stranded circular plasmids result • 5. two cells result that are F+ - therefore bacterial sex converts the F- into an F+ bacteria

43. Chromosomal F factors and Conjugation • -if the F gene is part of the chromosome = cell is called the Hfr cell (high frequency of recombination) • the Hfr cell forms a mating bridge with the F- cell • single strand of the chromosome breaks and moves into the F- cell • -movement of the F factor “carries” additional genes into the F- cell • e.g. A+ and B+ alleles • DNA replication begins in the Hfr and F- cell – to create double stranded DNA Hfr cell F+ cell F factor Hfr cell F– cell Temporary partial diploid Recombinant F– bacterium Conjugation and transfer of part of the bacterial chromosome from an Hfr donor to an F– recipient, resulting in recombination

44. Chromosomal F factors and Conjugation • 4. the mating bridge usually breaks before complete transfer of the chromosome • -so just the F factor and a few downstream genes move into the F- cell • 5. homologous recombination can result – B+ allele (from the Hfrcell) is switched for the B- allele in the F- cell • 6. extra piece of DNA outside the chromosome is degraded over time • 7. new bacteria remains F- and is called a recombinant bacteria Hfr cell F+ cell F factor Hfr cell F– cell Temporary partial diploid Recombinant F– bacterium Conjugation and transfer of part of the bacterial chromosome from an Hfr donor to an F– recipient, resulting in recombination

45. Bacterial adaptation • prokaryotes are very successful because they are able to adapt to many environments • because of rapid reproduction rates – natural selection in overdrive • numerous metabolic adaptations have evolved in prokaryotes

46. Adaptations in Food Metabolism • one adaptation is in “food metabolism” • broken down into two major categories: • 1. Autotrophs: “self”, “nourishing” • producers in the food chain • able to make their own food • use the energy from either light (photo) or from electron donors in chemical reactions (chemo) to make this food • so they do NOT need organic carbon sources as a source of energy • 2. Heterotrophs: “different”, “nourishing • consumers in the food chain • have to “eat” – must obtain organic food • cannot “fix carbon” – i.e.must use organic sources of carbon as an energy source

47. Categories of Adaptations • 1. photoautotrophs: photosynthetic organisms that capture light energy and use it to drive synthesis of organic compounds from inorganic carbon sources (e.g.CO2) • e.g. blue-green algae & plants • 2. chemoautotrophs – also need CO2 as a carbon source • use electron donors as their energy source – such as hydrogen sulfide, ammonia or iron • e.g. green sulfur bacteria • 3. photoheterotrophs:use light for energy but must obtain their carbon from outside organic sources • 4. chemoheterotrophs: must consume organic molecules for both energy and carbon • e.g. parasitic bacteria

49. Metabolism in Prokaryotes • prokaryotes also vary with respect to O2 utilization • 1. obligate anaerobes – cannot use O2 and are killed by the presence of O2 • some live exclusively by fermenting their carbon sources • some extract energy by using something other than O2 as the ultimate electron acceptor - called anaerobic respiration • e.g. nitrate ions or sulfate ions • 2. obligate aerobes – require O2 for cellular respiration & growth • 3. facultative anaerobes – use O2 but only if its present • can also carry out fermentation and anaerobic respiration

50. Metabolism in Bacteria • prokaryotes can also utilize nitrogen for metabolic pathways = nitrogen metabolism • nitrogen is essential for the production of amino acids and nucleic acids in all organisms • eukaryotes are limited in the nitrogenous compounds they can derive this nitrogen from • prokaryotes have more options available: • some can convert atmospheric N2 to ammonia through a process called nitrogen fixation • e.g. cyanobacteria - blue-green algae • this fixed nitrogen is capable of being used biochemically