General Microbiology Course Outline PDF

Welcome to General Microbiology Art by David Goodsell (Ebola) Microbes are ancient And they changed our planet How old do you think our planet is? Microbes are ancient How old is our planet? 2:46min Derek Muller - Veritasium Dec 13th Dec 31st 11:30pm Industrial revolution: 23:59:58 Feb 25th Land bas...

Welcome to General Microbiology Art by David Goodsell (Ebola) Microbes are ancient And they changed our planet How old do you think our planet is? Microbes are ancient How old is our planet? 2:46min Derek Muller - Veritasium Dec 13th Dec 31st 11:30pm Industrial revolution: 23:59:58 Feb 25th Land based fungi ~1 bya (that’s way before land plants!) Sept 17th (Sexual reproduction ~1.3 bya) July 15th Earth’s life calendar https://www.youtube.com/watch?v=I_ssiZCoRAs (start producing O2) March 21st Brock – Biology of Microorganisms, Chapter 1, Pearson. Microbes are numerous and ubiquitous Microbes are numerous World population: Over 8 billion or 8 x 109 as of Nov. 2022 (wikipedia) Microbial population: 5,000,000,000,000,000,000,000,000,000,000 or 5 x 1030 more then there are stars in the universe (~6-8 order of magnitude). If a microbe is 1µm in length, stacked next to each other in a row, microorganisms would cover the distance between the earth and the sun about 200 trillion times (1012). In 1ml of sea water, you can find up to 50 million viruses, and they kill about 20% of the living material in the oceans, every day! (ref Dr Patrick Keeling unverified) Ref: Prokaryotes: the unseen majority, PNAS, 1998, 95:6578-6583 Microbes are diverse Some have complex structures! Some bacteria are BIG! Complex organization Complex inner structures 100mm Myxobacteria Fisher, P.R. (2005). Microbial Development. In Encyclopedia of Molecular Cell Biology and Molecular Medicine. Vol. 8, pp. 289-342. Type 4 secretion system Alejandro Peña Ignacio Arechaga, Molecular Motors in Bacterial Secretion, J Mol Microbiol Biotechnol 2013;23:357–369 Their immense diversity allows them to be everywhere in huge numbers Use different sources of energy Use other organisms in symbiotic relationships “Developed” inventive chemistry and biochemistry in order to obtain or produce the metabolites they need “Developed” genetics that allow adaptation Microorganisms have evolved and adapted to every type of environment found on Earth Microbes can live in extreme environments Other planet extreme? Marina Cvetkovska NASA Hyperthermophiles can live in near boiling temperatures Blood falls: Psychrophiles pumping out Fe3+ in Antarctica Some acidophiles can live where the pH is close to 0 Alkaliphile can survive a pH range from 8.5-12 Halophilic microorganisms can survive concentration of salt up to 37% Conan the bacterium Deinococcus radiodurans Deeply branching bacteria Polyextremophile Can survive extreme heat, drought, vacuum, acidity, and radiation Owes its name to the fact that it can withstand doses of ionizing radiation that kill all other known bacteria unique mechanisms of DNA repair Microbiology v2 OpenStax Chap. 4.5 Fungi Zombified ant: Cordyceps fungal infection http://berkeleysciencereview.com/read/fall-2012/manipulative-microbes/ Choanoflagellates: beginnings of multicellularity? https://www.hhmi.org/research/choanoflagellates-and-origin-animals Dinoflagellate (eukaryote, protist) bioluminescent bloom in Hong Kong https://www.theatlantic.com/photo/2015/01/abioluminescent-bloom-in-hong-kong/384759/ Parasites Heart worm in dogs https://www.countryhillspets.com/heartworm-prevention/ https://erinmillsvet.ca/2015/06/18/the-truth-about-heartworm/ We are islands of microbes And that is a good thing! Source: Eighteen.B blogpost These living arrangements are in constant flux When the equilibrium fails between symbiotic communities, we call it dysbiosis. When one species takes over the community, it can sometimes result in pathogenesis. Gut Homeostasis, Microbial Dysbiosis, and Opioids, Wang and Roy, Toxicologic Pathology, 2017, Vol. 45 (1) 150-156. Humans and microorganisms relationships Where and how many? _______________________________________ Skin: 5-50 x 103/sq. inch Groin, axilla: 5 x 106/sq. inch Teeth: 5-50 x 106/sq. inch Colon: 300 x 106/g _______________________________________ Total bacteria (70Kg normal person): 40-70 trillion, 70 x 1012 Total human cells: ~30-40 trillion, 30-40 x 1012 That’s 2-6lbs of bacteria per 200lbs adult, not counting fungi and viruses. Not sure where I got this data, presumably Brock – Biology of Microorganisms, Pearson. or Wessner – Microbiology, Wiley. Animal symbiosis: Bacteria fermenting cellulose in ruminants are the animals major source of lipids (and farts!) Plant symbiosis: Native Mexican corn, mucus and nitrogen fixing bacteria https://atpeacewithpink.blogspot.com/2018/08/sierra-mixe-corn.html Humans and microorganisms relationships Taking into consideration the genetic potential of organisms only in our gut: provides 100X the genetic potential of all our cells Microbial genes 2 to 20 millions genes Grice, E. A. & Segre, J. A. The human microbiome: our second genome. Annu. Rev. Genomics Hum. Genet. 13, 151–170 (2012). Human genome ~23,000 genes (That’s or 99.9% of the genetic capacity of humans!) Environmental Microbiology Plant microbiology Microbial ecology Geomicrobiology Water and marine microbiology Aeromicrobiology Astro/Exo microbiology Known species: ~6 x 106 Predicted: 1 trillion (1012) species Of which about 104 have been cultured The entire Earth Microbiome Project (EMP) sequence bank covers less then 107 species, 29% of which have only been detected twice What we know about microbes Which means 99.999% of microbial species still need to be identified! And we haven’t even discussed viruses yet! Scaling laws predict global microbial diversity. Kenneth J. Loceya and Jay T. Lennon, PNAS May 24, 2016. 113 (21) 5970-5975; published ahead of print May 2, 2016. https://doi.org/10.1073/pnas.1521291113 Course Logistics Course Charter Request for a note-taker Note-Taker’s Responsibilities Maintain the confidentiality of the student for whom you are taking notes. Make sure that notes are legible and comprehensive. Attend each class. Provide a copy of your notes promptly following classes. Using a shared drive may facilitate notes-sharing. If for some reason you are unable to attend class or continue as a notetaker, contact the student so that other arrangements can be made. If you can be a note-taker, please email me ([email protected]) and I’ll forward your email to the person needing the notes. Textbook Microbiology – An Evolving Science, 5ed, by Slonczewski, Foster and Zinser, from WWNorton publishings. Buy paper or online version directly from Norton: Click on the following link https://digital.wwnorton.com/microbio5. Once the page opens, you will have the click on the “purchase options” button. The eBook with Smartwork option is $104CAD. The Smartwork only version (no hardcopy or ebook) is $39CAD. Rice University Openstax free textbook. uOttawa email!!!! If buying online If not sure whether you will stick with micro or don’t have the cash right now. Reenter your uOttawa email address Click on your address to enter the “student set” code Click on “Add yourself to a student set” Student set code is 715064 Help resources How to register to a Student Set: https://wwnorton.com/common/mplay/6.11/?p=/digital/registration/video/&f=adding-yourself-to-a-set&ft=mp4&cdn=1 How to register for Smartwork https://youtu.be/WPXcHtWBcSE See a whole list of tutorials here: https://wwnorton.knowledgeowl.com/help/student-help-notes-23c7139 Module 3 Module 2 Module 1 Course schedule: Subject Week Microbial world 4 Sept Introduction Bacterial cells 11 Sept Bacteria 2 Archaea and Eukarya 18 Sept Viruses 25 Sept Metabolism 2 Oct Smartwork Midterm 1 (10%) Metabolism Bacterial Growth 9 Oct Sketch script Growth 1 Growth 2 Spatial organization 16 Oct Regulation 1 Case study Spatial Organization Quorum sensing 23 Oct Molecular regulation 30 Oct Sketch storyboard Regulation 2 Gene transfer 6 Nov Smartwork Midterm 2 (20%) Genes Microbial genetics 13 Nov Microbiome1 Case study whooping cough Microbiome 20 Nov Sketch delivery Microbiome2 TBD Host-pathogen interactions 27 Nov Sketch peer evaluations Host-pathogen 1 Final exam prep 4 Dec Smartwork No class, Final exam (cumulative) Monday Tuesday Wednesday Sketch subject with references Thursday Bacteria 1 Archaea Virus Reading week Friday Case Studies due date Case study Abx resistance Case study Sporulation Revision (quorum sensing case study) Monday schedule Case Studies due date Case Studies due date Revision Host-pathogen 2 Course evaluations Midterm 1: 10% of final mark Midterm 2: 20% of final mark Final Exam: During final exam period: 45% of final mark Case studies : 10% Smartwork or Sketch project : 15% Sketch evaluations: Bonus Point (1%) Summative (in person exams) Formative The average of all the midterms and final put together must be 50% or over in order to pass the class, regardless of the grades obtained from coursework and homework. Course evaluations Does the cumulative mark of all my exams average over 50%? No Yes You have not met basic course expectations and need to redo the course. You have met basic course expectations! Your Smartwork or science sketch mark will be added to your total course mark. Your case study mark will be added to your total course mark. Case studies These will be mostly replacing Zoom classes (home assignments) Assignments will be posted in Brightspace You will need to submit all your answer on a pdf in the BS assignment. All assignment questions must be answered to be marked. Not all questions will be marked, and you will not know which questions will be marked. Worth 10% of your total course mark. Each Case study will be 20 or 30 points. The cumulative number of points you obtain over the semester will dictate your overall mark for this part of the course. For example, if there is a total number of 130 points to accumulate over the semester and you have accumulated 115 points: 115*100/130 = 88.5% No late submission will be accepted. Smartwork Quizzes in Smartwork platform ($$ - 21 days free trial). Almost weekly, 10-20 questions per quiz. You have 2 tries for each questions. Worth 15% of your total course mark. The cumulative number of points you obtain over the semester will dictate your overall mark for this part of the course. For example, if there is a total number of 130 points to accumulate over the semester and you have accumulated 115 points: 115*100/130 = 88.5% No late submission will be accepted. Is a Science Sketch project for you? https://www.sciencesketches.org/ Science Sketch (in competition with Carleton) There is a limited number of students will be able to choose to do a 2min video sketch on a microbiology topic of their choice. If you choose to do a video Sketch, it will replace the Smartwork (quizzes) component and be worth 10% of your total course mark. You can work as a pair or on your own, but not 3 or more. ONLY 50 Projects available! Limited inscriptions! We estimate it will take 15 to 20h to create the sketch. Detailed info and forms on BS You will need to create a 2min video +/- a couple of sec (strict time limit!) All the instructions are posted on BS. A dedicated TA will help you scaffold your project in several steps: Find the subject and appropriate literature Write a script Write a storyboard Submit your video No late submission will be accepted. Your final video will be posted on YouTube (unpublished list) will be evaluated by myself, the TA and 5 students (uOttawa or Carleton). Is this for you? Sign up before Friday the 14th September! Sign on google sheet: https://docs.google.com/spreadsheets/d/11ksJhiRilzFZZoabr-Eq281OaeAYXboxwBg4ROhwZU/edit?usp=sharing Sign in the uOttawa student part of the document Add your full name so I can recognize you There are only 50 spots, when the list is full, the list is full. Play nice, don’t kick others off the list. Once your name is on the list, assume you are officially “registered”. You will have to submit your subject with references on Tuesday 19th, so don’t wait for the confirmation. Absences During midterms: You do not need to submit a medical note or even let me know why you haven’t been able to do the midterm. The weight of the midterm will automatically be added to the final exam. During final exams: Contact the Faculty asap. Prolonged absence affecting coursework: Come talk to me or email me if you are absent for a week or more and you worry it will affect your course performance, we’ll see what we can do. Course expectations Teamwork You will not be assigned specific teamwork. However, case studies can be done in small groups, but each member must submit their own copy, stating who they have worked with, and your answers on your copies must be in your own words, ie, you can’t simply copy off each other. If you are doing the Science Sketch in a pair, I expect you to share ideas and skills and truly work as a team, where both team members are implicated in all aspects of the works. Individual work I expect you to do the Smartwork quizzes individually. I expect you to keep a growth mindset. Ask questions when something isn’t clear, be accountable for your learning. I am utterly dedicated to your learning and the relationship between students and me is a two way relationship. We are partners in your education, lets do this together. Don’t forget to email me if you can take notes for someone else. If you can be a note-taker, please email me ([email protected]) and I’ll forward your email to the person needing the notes. Bacteria BIO3124 General microbiology Textbook Chapter 3 Openstax Microbiology Chap 3 Art by David Goodsell (E.coli) Chapter 3 Overview § A synopsis of the bacterial cell § The cell membrane and transport § The cell envelope and cytoskeleton § How bacterial cells divide § The nucleoid: structure and expression § Specialized structures: vesicles, nanotubes, pili, and more § Bacterial flagella and chemotaxis 2 Morphology: Size of microorganisms Three domains of life: Bacteria Archaea Eukarya (protists (algae and protozoa), fungi, molds) Helminths (multicellular but microscopic) Viruses and other acellular microbes (virions and prions) Chap 1.3 Morphology: shapes How do bacteria know what shape they have to be? What molds their shape? Morphology: Cell arrangements Figure 3.14 General structure of a bacterium A typical bacterial cell contains: a cell membrane, chromosomal DNA that is concentrated in a nucleoid and extrachromosomal DNA called plasmids ribosomes, and a cell wall. Some prokaryotic cells may also possess flagella, pili, fimbriae, and capsules. Features of the cytoplasm Structure Composition Function Nucleoid DNA, RNA, protein Genetic information storage and gene expression Extra chromosomal DNA DNA Variable, encode non-chromosomal genes for a variety of functions Enzymes Protein Replication of the genome, transcription Metabolism, Cell signaling Regulatory factors Protein, RNA Control of replication, transcription, and translation Ribosomes RNA, protein Translation (protein synthesis) Cell inclusions Various polymers Storage or reservoir Gas vesicles Protein Buoyancy Magnetosomes Protein, lipid, iron Orienting cell during movement Cytoskeleton Protein Guiding cell wall synthesis, cell division, and possibly partitioning of chromosomes during replication Modified from ©2018 John Wiley & Sons, Inc. 3.2 Membrane Molecules and Transport Membrane Lipids Membrane Proteins Molecules Cross the Cell Membrane 3.2 The Cell Membrane (plasma membrane is the same) Lipid bilayer Separate in/out Fluidity Membrane proteins Integral or peripheral Transport Osmosis Energy (cell respiration) Sensing Secretion Membrane Lipids Charged head Hydrophobic tail FIGURE 3.5 Phospholipids. A. Phosphatidylglycerol consists of glycerol with ester links to two fatty acids, and a phosphoryl group linked to a terminal glyceride. B. Phosphatidylethanolamine contains a glycerol linked to two fatty acids, and a phosphoryl group with a terminal ethanolamine. The ethanolamine carries a positive charge. Membrane Lipid Diversity Microorganisms live in vary different environments (hot, cold, salty, etc.), membrane lipid diversity helps to respond to these conditions. Cardiolipin (diphosphatidylglycerol) Palmitic and oleic acid Saturated or unsaturated, add fluidity to the membrane in cold temp. Cyclopropane fatty acid Localizes to the cell poles Binds certain environmental stress proteins, such as a protein that transports osmoprotectants when the cell is under osmotic stress Stiffens the cell membrane wikipedia The conversion of unsaturated fatty acids to cyclopropane is an important process for Mycobacterium tuberculosis pathogenesis Hopanoids Cholesterol Stabilizes membranes like cholesterol wikipedia 11 Membrane Lipids: Hopanoids Figure 3.8 Hopanoids add strength to membranes. Hopanoids limit the motion of phospholipid tails, thus stiffening the membrane. Hopanoids Cell membrane function Polar and charged molecules must be transported. Transport proteins accumulate solutes against the concentration gradient Holds transport proteins in place Generation of proton motive force 3.3 The Envelope and Cytoskeleton The Cell Wall Is a Single Molecule Cell Envelope of Bacteria Cell Envelope—Gram-Positive Cell Envelope—Gram-Negative Mycobacterial Cell Envelope Bacterial Cytoskeleton The Cell Wall Is a Single Molecule § The cell wall (or envelope) confers shape and rigidity to the cell and helps it withstand turgor pressure. § The bacterial cell wall (or envelope), consists of a single interlinked macromolecule. Like a flexible mesh bag or scaffold This mesh is made of peptidoglycan. 100+ distinct peptidoglycans have been described 15 Cell wall function In prokaryotic cells, the cell wall provides some protection against changes in osmotic pressure, allowing it to maintain its shape longer. The cell membrane is typically attached to the cell wall. Rigid sugar-protein coat Figure 3.16 Peptidoglycan Structure Rigid layer that provides strength typically composed of: alternating modified glucose N-acetylglucosamine and N-acetylmuramic acid in β-1,4 linkages amino acids L-alanine, D-alanine, D-glutamic acid, and either L-lysine or diaminopimelic acid (DAP) Can be destroyed by lysozyme enzymes that cleave glycosidic bond between sugars found in human secretions, major defense against bacterial infection http://library.open.oregonstate.edu/micr obiology/chapter/bacteria-cell-walls/ How peptidoglycan are arranged to form the wall Gram - Gram + Sugar backbone Bridge between 2 peptides Copyright ©2018 John Wiley & Sons, Inc. A. Peptidoglycan crosslinking in E. coli B. Peptidoglycan crosslinking in S. aureus Note: Amino acids have two forms that are mirror opposites, D and L, of which only the L form is incorporated by ribosomes into protein. The D-form amino acids, however, are used by microbes for many nonprotein structural molecules. Peptidoglycan Structure – cont’d § Peptidoglycan is unique to bacteria. Thus, the enzymes responsible for its biosynthesis make excellent targets for antibiotic. – Penicillin inhibits the transpeptidase that cross-links the peptides. – Vancomycin prevents cross-bridge formation by binding to the terminal D-Ala-D-Ala dipeptide. Unfortunately, the widespread use of such antibiotics selects for evolution of resistant strains. 19 Peptidoglycan Structure – cont’d § Growth of peptidoglycan occurs via a synthesis complex that extends the chains of amino-sugars. § So-called penicillin-binding proteins catalyze the formation of peptide cross-bridges. § Overall direction of cell wall extension is organized by a protein complex that includes MreB (actin homologue). 20 Peptidoglycan Growth in Different Species § Bacterial species differ in where they synthesize new peptidoglycan in their growing cell walls: In dispersed zones At the septum At the poles 21 Cell Envelope: Gram-positive and Gram-negative Gram positive Gram negative Stains Blue with Gram stain Stains Pink with Gram stain S-layer is not part of the cell wall per say, but an outer layer present in some Bacteria and Archaea. 22 Gram stain Most well established method for distinguishing bacterial morphology and cell wall composition Gram + retain the crystal violet dye but Gram – do not. A counterstain is added (fuchsine or safranin) and whilst Gram + stay violetblue, Gram - retain the red of the safranin. http://stanleyillustration.com/latestwork/2015/2/8/ngoo8tdfmqo4tyh0v ksu37vqroxnvs Exoenzymes: Digests nutrients that are too large to pass Has multiple layers of peptidoglycan threaded by teichoic acids Teichoic Acid: Glycopolymer Neg. charged (helps generate proton motive force) Rigidity, cell shape Protects against high temp. or high salt conditions Protects against antibiotics (Abx) like β-lactams lipoteichoic acids: teichoic acids covalently bound to membrane lipids LPS consists of core polysaccharide: O-polysaccharide and lipid A Multiple serotype exist and defines the O- in bacterial serotype: Ex. E.coli O157:H7 H is for flagellar protein. Sometimes K is used for capsular polysaccharide antigens. Other Differential Stains § Acid-fast stain: carbolfuchsin used to stain Mycobacterium species § Spore stain: malachite green used to detect spores of Bacillus and Clostridium § Negative stain: colors the background, which makes capsules more visible 26 Acid-fast bacteria Acid-fast stained M. tuberculosis sputum Hydrophobic layer (mycolic acids are lipids) Looks light blue with Gram stain but bright pink with special stain Pathogen Recognition and Innate Immunity, Akira et al., Cell 124, 783–801, February 24, 2006 Why so much detail for these structures? *The cell wall is ESSENTIAL for most bacteria* Antimicrobials target cell structures and cell processes Antibiotics are for bacteria only Antimicrobials include drugs against viruses, fungi and parasites. Bactericidal KILLS bacteria Bacteriostatic INHIBITS growth Nosocomial infection: acquired in hospital care Antibacterial drug targets Subject of your first Cases study! Important to know what is targeted by antimicrobials Openstax Microbiology Chap 14.3 How antibiotic resistance happens Resistance is a genetically acquired tool to resist the toxic effects of an antibiotic. The organisms’ genome is changed, and the genetic material can be shared with other microbes (mostly same species, but cross-species sometimes). From CDC website https://www.cdc.gov/drugresistance/about.html https://www.youtube.com/watch?v=plVk4NVIUh8 Why is it such a problem? People die of infectious diseases (not even drug resistant ones) ALL THE TIME! If all these infectious agents became drugs resistant, then we are back to the medieval ages of medicine! Planet wise, 25% of deaths are attributable to microbial infections 11 to 12 millions of deaths per year WHO 2016 Ischaemic heart disease and stroke are the world’s biggest killers, accounting for a combined 15.2 million deaths in 2016 (26.7% of all deaths). Lower respiratory infections remained the deadliest communicable disease, causing 3.0 million deaths worldwide in 2016. The death rate from diarrhoeal diseases decreased by almost 1 million between 2000 and 2016, but still caused 1.4 million deaths in 2016. In 2012 0.1/10 death < 15 yo 4/10 death < 15 yo 7/10 death > 70 yo 2/10 death > 70 yo 6.5 millions of children deaths < 5 ans. 99% of those in low-income countries. CDC’s biggest threats list (2013) https://www.cdc.gov/drugresistance/biggest_threats.html Urgent threats: Serious threats: C.difficile Multidrug resistant Acinetobacter Carbapenem-Resistant Enterobactericeae (CRE) Drug-resistant Campylobacter Neisseria gonorrhoeae Extended Spectrum Enterobacteriaceae (ESBL) Fluconazole-resistant Candida Vancomycin-resistant Enteroccocus (VRE) Multidrug resistant Pseudomonas Aeruginosa Drug-resistant Non-Typhoidal Samonella Drug-resistant Salmonella Serotype Typhi Drug-resistant Shigella Don’t memorize this! Methicillin-resistant Staphylococcus Aureus (MRSA) Drug-resistant Streptococcus pneumonia Drug-resistant Tuberculosis Concerning threats: Vancomycin-resistant S. aureus Erythromycin-resistant Group A Streptococcus Clindamycin-resistant Group B Streptococcus Clostridium difficile 223900 Hospitalization/year 2019 AR threats report From 2013 -2019 13,000 Carbapenem-resistant Enterobacteriaceae (CRE) bacteria Stable 2013-2019 Neisseria gonorrhoeae Fact sheet: https://www.cdc.gov/drugresistance/pdf /drug-resistant-gonorrhea-508-final.pdf Drug-resistant infections nearly doubled to 550,000 from 2013 to 2019 Antibiotic resistance monitoring in Canada Canadian Antimicrobial Resistance Surveillance System Report 2022 Antibiotic resistance monitoring in Canada 42.8% 5.6% 18% Non-GP/FP and non-physiscian: dentist, nurse practitioners, optometrists, pharmacists, veterinarians Canadian Antimicrobial Resistance Surveillance System Report 2022 Canadian Antimicrobial Resistance Alliance (CARA) The Canadian Antimicrobial Resistance Alliance (CARA) started the CAN-R project in 2007 to track antimicrobial resistance across Canadian hospitals. Link: http://can-r.com/study.php?study=canw2018&year=2018 Back to our cell surface structures… …next class. I hope we made it this far! Chapter 3 Overview § A synopsis of the bacterial cell § The cell membrane and transport § The cell envelope § Other cell surface structures § The cytoskeleton § How bacterial cells divide § The nucleoid: structure and expression § Specialized structures: vesicles, nanotubes, pili, and more § Bacterial flagella and chemotaxis 43 Other cell surface structures Glycocalyces not considered part of cell wall because these do not confer significant structural strength polysaccharide layers (may be thick or thin, rigid or flexible) Capsules Tightly attached, tight matrix; visible if treated with India ink Slime layer loosely attached, easily deformed (e.g., Leuconostoc) assist in attachment to surfaces role in development and maintenance of biofilms virulence factors: protect against phagocytosis prevent dehydration/desiccation Acinetobacter calcoaceticus The capsular material surrounding these bacteria (Acinetobacter calcoaceticus) is revealed in a suspension of India ink and viewed through a light microscope (magnified about 2,500×). From W.H. Taylor and E. Juni, “Pathways for Biosynthesis of a Bacterial Capsular Polysaccharide,” Journal of Bacteriology (May 1961) https://www.britannica.com/science/bacteria/Capsules-and-slime-layers Cell surface structures § S-layer An additional protective layer commonly found in free-living Grampositive and Gram-negative bacteria and archaea Crystalline layer of thick subunits consisting of protein or glycoprotein May contribute to cell shape and help protect the cell from osmotic stress 45 Cell surface structures Fimbriae and pili filamentous protein structures ~2–10 nm wide Fimbriae enable organisms to stick to surfaces or form pellicles (thin sheets of cells on a liquid surface). Source: Khan Academy Pili are typically longer, and fewer (1 or a few) found per cell than fimbriae. Conjugative/sex pili facilitate genetic exchange between cells (conjugation). Type IV pili adhere to host tissues and support twitching motility (e.g., Pseudomonas and Moraxella). Figure 3.30 The bacterial cytoskeleton shapes bacteria Implicated in cell division Shapes the morphology of the cell FtsZ: tubulin-like protein MreB: actin-like protein Crescentin: filament-like protein Skin and bones: the bacterial cytoskeleton, cell wall, and cell morphogenesis. Matthew T. Cabeen and Christine Jacobs-Wagner, The Journal of Cell Biology, Vol. 179, No. 3, November 5, 2007 381–387 Bacterial cytoskeleton Necessary for the movement of ParM: actin-like protein molecules to the right location within the cell Example of Par system: Segregates a dividing chromosome to both polls of the cell in preparation for cell division. Also segregates extrachromosomal DNA such as plasmids. Plasmid partitioning system (wiki) 3.4 Bacterial Cell Division Cell Division by Septation DNA Is Organized in the Nucleoid DNA Replication Regulates Cell Division 3.4 Bacterial Cell Division § Bacterial cell division, or fission, requires highly coordinated growth and formation of all the cell’s parts. Unlike eukaryotes, prokaryotes synthesize RNA and proteins continually while the cell’s DNA undergoes replication. § Bacterial DNA replication is coordinated with the cell wall expansion and ultimately the separation of the two daughter cells. § Bacteria do not undergo mitosis or meiosis. 50 Cell Division by Septation § As DNA synthesis terminates, the cell divides by a process called septation, the formation of the septum. § The septum grows inward from the sides of the cell, at last constricting and sealing off the two daughter cells. § FtsZ subunit assembly circles around the septum in a “treadmilling” pattern, stepwise around the cell, that directs septal growth. Figure 9.3 Openstax Microbiology 51 Cell Division by Septation – cont’d § Septation requires rapid biosynthesis of all envelope components, including membranes and cell wall. § The overall process of septation is managed by a protein complex called the divisome. One component of the divisome is FtsZ, which polymerizes to form the Z-ring. § To avoid the “guillotine” of the cell, septation is coordinated with DNA replication. 52 DNA Is Organized in the Nucleoid Microbiology OpenStax, Chap 3.3 Prokaryotic cells have a nucleoid region that extends throughout the cytoplasm and is not enclosed by a membrane. 53 DNA Is Organized in the Nucleoid – Cont’d In most bacterial species, the DNA is attached to the envelope at the origin of replication, on the cell’s equator. In prokaryotes, translation is tightly coupled to transcription. The ribosomes bind to mRNA and begin translation even before the transcription of the mRNA strand is complete. 54 DNA Replication Regulates Cell Division § In prokaryotes, a circular chromosome begins to replicate at its origin, or ori site. § Two replication forks are generated, which proceed outward in both directions. At each fork, DNA is synthesized by DNA polymerase, with the help of accessory proteins. – This protein complex is called the replisome. § As the termination site is replicated, the two forks separate from the DNA. § Completion of replication triggers Z-ring formation. 55 DNA Replication FIGURE 3.28 (Part 1) Replisome movement within a dividing cell. The DNA origin-of-replication sites (green) move apart in the expanding cell as the two replisomes (yellow) stay near the middle, where they replicate around the entire chromosome, completing the terminator sequence last (red). As the terminator sequence nears completion, FtsZ proteins assemble the Z-ring organizing septum formation. Source: Top 2 insets: Ivy Lau et al. 2003. Mol. Microbiol. 49:731. Bottom inset: Jackson Buss et al. 2015. PLoS Genet. 11(4). 56 DNA Replication FIGURE 3.28 (Part 2) Replisome movement within a dividing cell. The DNA origin-of-replication sites (green) move apart in the expanding cell as the two replisomes (yellow) stay near the middle, where they replicate around the entire chromosome, completing the terminator sequence last (red). As the terminator sequence nears completion, FtsZ proteins assemble the Z-ring organizing septum formation. Source: Top 2 insets: Ivy Lau et al. 2003. Mol. Microbiol. 49:731. Bottom inset: Jackson Buss et al. 2015. PLoS Genet. 11(4). 57 DNA Replication Regulates Cell Division 58 3.5 Cell Asymmetry, Membrane Vesicles, and Extensions Bacterial Cell Differentiation Growth Asymmetry and Polar Aging Membrane Vesicles Membrane Extensions and Nanotubes 3.5 Cell Polarity, Membrane Vesicles, and Nanotubes § Even superficially symmetrical bacilli such as E. coli show underlying chemical and physical asymmetry. Such as possession of a chemoreceptor array at the “forward” pole § Other species, such as Caulobacter crescentus, develop different structures at either pole, and their cell division generates two different cell types. § And many kinds of bacteria extend their cytoplasm in surprising ways. 60 Bacterial Cell Differentiation Some bacteria generate two kinds of daughter cells: one stationary (sessile) and one mobile (swarmer). Example: the flagellum-to-stalk transition of the bacterium Caulobacter crescentus 61 Growth Asymmetry and Polar Aging § The actual process of cell division itself determines that the poles of each daughter cell differ chemically from each other. Polar aging is increased by stress. § A major form of asymmetrical growth is endospore formation by Firmicutes such as Bacillus and Clostridium species. Endospores can remain dormant but viable for thousands of years. 62 Extracellular Membrane Vesicles and Nanotubes Surprisingly, microbial cells export bits of cytoplasm in membrane vesicles. These carry proteins and nucleic acids (RNA,DNA). Both Gram – and Gram + bacteria can shed membrane vesicles. 63 Extracellular Membrane Vesicles and Nanotubes – Cont’d § Some bacteria and archaea can form membrane extensions that merge directly with the membranes of neighboring organisms. These nanotubes allow bacteria to directly share proteins and mRNA useful under hostile conditions, such as when exposed to antibiotics. 64 3.6 Specialized Structures Thylakoids, Carboxysomes, and Storage Granules Pili and Stalks Rotary Flagella Endospores 3.6 Specialized Structures: Thylakoids Thylakoids: extensively folded intracellular membranes found in photosynthetic bacteria 66 3.6 Specialized Structures: carboxysome Carboxysome: polyhedral bodies packed with the enzyme Rubisco for CO2 fixation 67 3.6 Specialized Structures: Gas vesicles Gas vesicles: protein-bound gas-filled structures that increase buoyancy 68 3.6 Specialized Structures: cell inclusions Inclusions function as energy reserves, carbon reservoirs, and/or have special functions. Enclosed by thin membrane Reduces osmotic stress Carbon storage polymers glycogen: glucose polymer poly-β-hydroxybutyric acid (PHB): lipid polymer, stored as lipid droplets. PHB is produced by species of Bacillus and Pseudomonas. Industrially, PHB has also been used as a source of biodegradable polymers for bioplastics. Other types of stored energy or material Cell inclusions or storage granules 69 3.6 Specialized Structures: Pili § Pili (also called fimbriae) are straight filaments of pilin protein. Used in attachment § Gram-negative enteric bacteria use sex pili for conjugation. Openstax, Microbiology, Chap. 3.3. 70 3.6 Specialized Structures: stalks and holdfast § Stalks are membraneembedded extensions of the cytoplasm. Stalk § Tips secrete adhesion factors called holdfasts. C. crescentus Holdfast Wagner, Jennifer K, PNAS, 2006. https://doi.org/10.1073/pnas.0602047103 Cell motility Several modes of locomotion: Flagella, Archaella, and Swimming Motility Gliding Motility Chemotaxis and Other Taxes Rotary flagella and Swimming Motility Flagellar and flagellation In bacteria long, thin appendages (15–20 nm wide) different arrangements: polar, lophotrichous, amphitrichous, peritrichous Figure 3.32 Microbiology OpenStax Flagella structure Rotation! helical in shape consists of several components Filament composed of flagellin. reversible rotating machine increase or decrease rotational speed relative to strength of proton motive force Note: flagella rotate either clockwise (CW) or counterclockwise (CCW) relative to the cell. Is this a Gram-negative or Gram-positive bacterial cell? Chemotaxis § Chemotaxis is the movement of a bacterium in response to chemical gradients. § Attractants cause CCW rotation. Flagella bundle together Push cell forward “Run” § Repellents (or absence of attractants) cause CW rotation. Flagellar bundle falls apart. “Tumble” – Bacterium briefly stops, then changes direction. Figure 3.33 Microbiology Openstax Movement of flagellated bacteria - Chemotaxis Attractant concentration increases and prolongs run = more likely to run toward chemoattractant Chemotaxis Animation 77 Chemotaxis and other taxes Taxis: directed movement in response to chemical or physical gradients chemotaxis: response to chemicals phototaxis: response to light aerotaxis: response to oxygen osmotaxis: response to ionic strength hydrotaxis: response to water monitor/sample environment with chemoreceptors that sense attractants and repellents Endospores Bacterial cells are generally observed as vegetative cells, but some genera of bacteria have the ability to form endospores, structures that essentially protect the bacterial genome in a dormant state when environmental conditions are unfavorable (not to be confused with the reproductive spores formed by fungi). Figure 3.19 Endospores Structure and features many layers: exosporium (outermost), spore coats, cortex, core wall contains dipicolinic acid and is enriched in Ca2+, both form the calcium-dipicolinic acid (DPA) complex DPA complex help the cells to cope with dehydration and stabilize DNA Core contains small acid-soluble spore proteins (SASP), which bind and protect DNA and function as carbon and energy source for outgrowth. Core also contains the cytoplasmic membrane, cytoplasm, ribosomes and other cellular essentials. Endospores Figure 3.20 Formed during endosporulation or sporulation (again, not to be confused with the reproductive spores formed by fungi) Highly differentiated cells resistant to heat, harsh chemicals, and radiation Survival structures to endure unfavorable growth conditions “Dormant” stage of bacterial life cycle Ideal for dispersal via wind, water, or animal gut Present only in some gram-positive bacteria, (e.g., Bacillus and Clostridium), none in archaea, suggesting this process evolved in bacteria after split. Endospore Formation Animation 82 Bacterial taxonomy Bacteria are named using a binomial system: Escherichia coli Genus Species Taxonomic level Example Phylum Proteobacteria Class Gammaproteobacteria Order Enterobacteria Family Enterobacteriaceaea Genus Escherichia Species coli Genus: group of closely related species Species: group of organisms sharing common features while differing considerably from other organisms Strain vs serotype Strain: Distinct subtype of species that differs genetically, and often phenotypically, from other subtypes. Serotype (or serovar): Strain identified by serotyping (identifying surface antigens particular to a subspecies) Escherichia coli O147:H7 Genus Species Strain or serotype Escherichia coli BL21 Genus Mus musculus BALB/c Genus Species Influenza A virus Influenza virus A H1N1 Species Strain Bacteria Strain Genus Animal Species Virus Strain or serotype Serotyping Study.com Bacteria Patient serum Agglutination means patient is infected (has developed Ab against bacteria) Two serotypes 1a and 1b with antigens 2a and 2b on surface, which are recognized by two distinct antibodies, 3a and 3b, respectively Wikipedia Bacterial taxonomy Bacteria are typically classified according to shared characteristics: Morphology (colony appearance on a plate) Gram colouring Size and shape (rod or coccus, single, chain or cluster, …) Presence of structures (such as magnetosomes or flagella) Metabolic traits (energy source, enzymatic capacities,…) DNA sequence Bacterial taxonomy 84 phyla identified so far Only 32 of these have been described on the basis of strains in cultivation 90% of strains in cultivation belong to one of only four phyla: Firmicutes Proteobacteria Bacteroidetes Actinobacteria Sample of known bacterial phyla For more info, see chapter 18 in your textbook. Copyright ©2018 John Wiley & Sons, Inc. Next class: Archaea Archaea cell biology © ISTOCK.COM/NANOSTOCKK Overview Discovery of Archaea Distinctive properties of Archaea Archaeal cell structure Diversity of Archaea Wooclap The phylogenetic tree (tree of life) has evolved through time Originally based on morphology, anatomy, biochemistry, … 2 main domains of life: prokaryotes and eukaryotes Ernst Haeckel’s rendering of the tree of life, from his 1866 book General Morphology of Organisms Archaea and the tree of life https://youtu.be/hw-ij3822DY Molecular biology reveals a new vision of microbiology and microbial diversity Used genetics to compare different organisms Common cellular ancestry means certain molecules are shared between all living organisms Such as molecules that form parts of the transcriptional machinery, including ribosomal RNA. I recommend The Tangled Tree by David Quammen Carl Woese Archaea phylogeny Seven major phyla identified (so far) Only 5 of these phyla have species we can grow in a lab Most know are: Euryarchaeota (methanogens, halophiles, thermophiles) TACK (Crenarchaeota, thermophiles) Asgard (ancestral eukaryotes?) DPANN (obligate symbionts?-gene loss) To learn more: Archaea and the tree of life, by Dipti Nayak, iBiology v 19.1 Archaeal Diversity at a glance Archaea share many metabolic traits, such as redox metabolism, with bacteria. Other core traits of DNA-RNA machinery are shared with eukaryotes. Whilst gene regulation looks more like that of bacteria. Key traits such as ether-linked membrane lipids are found only in archaea, and in a few bacteria that received them by horizontal gene flow from archaea. These distinctive features are called “archaeal signatures.” 8 Archaeal Traits The cell wall (envelope) is the most distinctive feature of Archaea 9 Distinctive properties of Archaea Often associated with extreme environments, but they do grow in temperate marine, soil and freshwater (a few in or on us) Structure Size is usually 0.5 to 5 μm in diameter. Similar shapes to Bacteria/Eukarya Archaea, as for bacteria, are prokaryote, ie have no nucleus The Archaea plasma membrane structure is unique to this domain. Halococcus salifodinae, microbiologyonline.org Thermococcus gammatolerans, wiki commons Sulfolobus, microbiologyonline.org Methanosarcina rumen (green with red cell walls), microbiologyonline.org Distinctive properties of Archaea Genetically different from bacteria and eukarya, with a distinct genome structure: Both Archaea and Bacteria usually possess singular, circular chromosomes and lack a membrane-bound nucleus. Archaeal DNA is complexed with histones (like eukaryotes). Many of the DNA replication enzymes of Archaea look like those of Eukarya. http://www.biologyreference.com/Ar-Bi/Archaea.html Archaea cytoplasm: general properties Inclusion bodies such as gas vacuoles or carbon storage have been observed in some Archaea. Protein content similar as in Bacteria (ribosomes, enzymes, etc…). Cytoskeleton formed of protein that have homologues in both bacterial and eukarial cells. No Archaea is known to form spores. No nucleus means the DNA is within the region called the nucleoid. Inclusion bodies (lipids) Rice Uni Openstax Chap 3.3 Archaeal cytoplasm: cytoskeleton Cytoskeletal homologues are found in both Bacteria and Archaea. Homologues/orthologues: FtsZ/TubZ/tubulin MreB/crenactin/actin Microtubules CreS/ - /IF ESCRT no homologues, completely different system Intermediate filaments (IF) Actin microfilament The evolution of the cytoskeleton, Bill Wickstead, Keith Gull, The Journal of Cell Biology Aug 2011, 194 (4) 513-525; DOI: 10.1083/jcb.201102065 Archaeal cell membrane Archaeal cytoplasmic membranes have different lipid constituents and chemistry but are structurally similar Archaea can form membranes with lipid bilayers but with monolayers too! Source: Khan Academy Archaeal cell membrane Isoprene chains instead of fatty acids in bacteria and eukaryotes https://courses.lumenlearning.com/sunyosbiology2e/chapter/structure-ofprokaryotes-bacteria-and-archaea/ Ether linkages instead of ester linkage in bacteria and eukaryotes Archaeal cell wall: pseudomurein With pseudomurein S-Layer Source: IPY programs is MERGE: Microbiological and Ecological Responses to Global Environmental No peptidoglycan! Euryarchaeota Pseudomurein: found in cell walls of certain methanogenic Archaea polysaccharide similar to peptidoglycan Immune to lysozymes and penicillin composed of NAT and NAG: Nacetylglucosamine (in peptidoglycan) and N-acetyltalosaminuronic acid (different, replaces N-acetylmuramic acid) β-1,3 glycosidic bonds instead of β-1,4 amino acids all L-stereoisomer Archaeal cell wall: pseudomurein With pseudomurein S-Layer Source: IPY programs is MERGE: Microbiological and Ecological Responses to Global Environmental No peptidoglycan! S-layer: most common cell wall type consist of protein or glycoprotein paracrystalline structure of various symmetry: hexagonal, tetragonal, or trimeric in many organisms, S-layers present in addition to other cell wall components, usually polysaccharides always Generally outermost layer Resist osmotic pressure in extreme environment, more flexible than peptidoglycan cell wall Archaeal cell wall No peptidoglycan layer! Other proteins or glycoproteins possible Methanochondroitin: looks a little like chondroitin found in connective tissues in animals Protein sheath: Often found covering multiple cells forming a chain instead of a single cell. http://library.open.oregonstate.edu/microbiology/chapter/archaea/ Cell surface structures Hami Archaeal “grappling hooks” assist in surface attachment, forming biofilms. Sandy Y. M. Ng et al. J. Bacteriol. 2008;190:6039-6047 Structure of Archaella Simpler then bacterial or eukaryotic flagella Uses ATP rather then the proton motive force as energy source Rotating flagella (eukaryotes have whip-like flagella) Archaea are the only known organisms capable of methanogenesis Whether they are is swamps or in animal guts, archaea are responsible for the methane production in our atmosphere. Chemolithotrophy: ATP H2 + CO2 Chemoorganotrophy: CH4 ATP Methanol CH4 ATP Acetate CH4 Methanogens contribute to gas house emissions UAF - 2010 - Hunting for methane with Katey Walter Anthony. 1:54min Retinal-Based Photoheterotrophy Most haloarchaea are photoheterotrophs. Haloarchaea respire with oxygen or anaerobically with nitrate. Supplement their utilization of organic substrate energy by using light-driven ion pumps. Do not fix carbon (like photosynthesis) Do not produce O2 Use light-driven ion pumps capture light energy. Bacteriorhodopsin (BR) pumps out H+. Halorhodopsin (HL) pumps in Cl–. Both increase proton motive force. Other rhodopsins signal to the archellum. Phototaxis Blue versus red light alter archellar rotation. 23 Light-Driven Ion Pumps and Sensors 24 Retinal-Based Photoheterotrophy – Animation 25 Some Archaea can use light to power ATP production without doing photosynthesis Using bacteriorhodopsin: structurally similar to rhodopsin, which is a lightgathering pigment found in vertebrate eyes cytoplasmic membrane proteins that can absorb light energy and pump protons across the membrane 1. Bacteriorhodopsin absorbs light (replaces ETC) 2. Retinal (actually purple) is excited by rhodopsin and 3. Changes conformation from trans to cis 4. Retinal transformation coupled to one proton being pumped outside of the cell 5. ATP synthase can use proton gradient to produce ATP Brock Microbiology, Pearson, Chap 17 Bacteriorhodopsin in haloarchaea and proteorhodopsin in bacteria. Go figure! Archaeal Gene Structure and Regulation Genomes of archaea resemble those of bacteria in gene size and density. However, certain features of genome structure more closely resemble those of eukaryotes: Certain tRNA genes are interrupted by introns. DNA and RNA polymerases and transcription factors are similar to those of eukaryotes Archaea contain histone homologs. Certain features are unique to archaea: Certain thermophiles possess a reverse gyrase that introduces positive supercoils in chromosomal DNA, which helps protect it against high temperatures. 27 Genetics of archaea Stacked nucleosomes Histones Chromatin structure: Histones form tetramers around which DNA wraps around (instead of the eukaryotic octamers). The nucleosome structure allows stacking of units, or superhelix (something prevented by histone structure in eukaryotes) Histone tails may allow for some epigenetic modifications (Dec 2018, one DNA Nucleosome Archaeal nucleosomes HHMI.org paper) Eukaryotic nucleosomes Methanococcus jannaschii complete genome Genetics of archaea Genomic structure: Not a whole lot of whole genome sequencing, ~150 genomes (mostly Euryarchaeota) Presence of introns In Methanocaldococcus jannaschii: 1.66Mbp, circular genome ~1700 genes Homology to bacterial genes (mostly genes encoding central metabolic pathways and cell division) Homology to eukaryotic genes (genes encoding molecular processes like transcription and translation) 40% of genome with no homology to any living organism (methanogenesis and unknown functions) http://bacmap.wishartlab.com/maps/NC_000909/index.html Genetics of archaea Equivalent to general transcription factors (like TFIIB) in eukaryote Gene structure : Promoters resemble that of eukaryotic cells: Presence of a TATA box Presence of a BRE Formation of a pre-initiation complex (RNA pol II needs to be recruited by general transcription factors (TBP, TFBs)) Transcription Regulation in the Third Domain, Elizabeth A. Karr, Advances in Applied Microbiology, Volume 89, 2014, Pages 101-133 BRE: B-recognition element TBP: TATA-binding protein TFB: Archaeal transcription factor B In shorts, bacteria-like regulators must interact with a scaled-down version of a eukaryotic transcription machinery and gene structure Archaeal Gene Regulation RNA pol II structure similar to that of eukaryotes Multiple regulatory elements (binding sites) direct gene expression Activators and repressors resemble more that of bacterial DNA binding proteins then eukaryotic transcription factors However, the domain arrangements and sensing domains (in response to environment) are often unique to the archaeal domain of life Distinctive modified bases in their tRNA molecules Archaeosine, a guanosine analog 31 Cell division in Archaea Cell cycles and cell division in the archaea. Rachel Y Samson and Stephen D Bell, Current Opinion in Microbiology Volume 14, Issue 3, June 2011, Pages 350-356. Very much research in progress. Only possible to study archaea we can culture, lack of genetic tools, growth in esoteric conditions. Members of distinct phyla have very different chromosome copy numbers, replication control systems and even employ distinct machineries for cell division. Sometimes these processes seem more bacteria-like or more eukaryote-like depending on archaeal species. Why don’t Archaea cause disease? Next class Viruses! BIO3124 - Viruses BIO3124 General Microbiology Textbook (Norton) Chapter 6 OpenStax Chap.6 Image by David Goodell Zika virus Chapter Overview § What is a virus? § The ecology of viruses § The structure of viruses § What agents are even smaller than a virus? § How viruses are classified § The life cycles of bacteriophages § The life cycles of animal and plant viruses § How viruses are cultured 2 What is a virus? § A virus is a noncellular particle that infects a host cell and directs it to produce progeny particles (more viruses). § The virus particle, or virion, generally consists of a viral genome (DNA or RNA) contained within a protein capsid. 3 Viruses Infect Specific Hosts § Viruses are typically specific to their hosts and a range of cells within these hosts. Bacteriophages (or phages) are viruses that infect bacteria. Their replication is observed as a plaque of lysed cells on a lawn of bacteria growing in a Petri dish. An example of a human virus is the measles virus. An example of a plant virus is the tobacco mosaic virus. Plaques Measles TMV 4 We have more degenerate viral sequences then protein coding sequences in our genome “Ghost of infections past” – Dr Paul Turner Integrated Viral Genomes § Some viruses do more than replicate within a cell; they integrate their genomes into that of the host. In effect, such viruses become a part of the host organism. § A bacteriophage that integrates its genome into its bacterial host’s genome is called a prophage. § Within a human cell, an integrated viral genome is called a provirus. § A permanently integrated provirus transmitted from one human to another via the germ line is called an endogenous virus. Source: Wikipedia 6 Dynamic Nature of Viruses § We now know that a virus may interconvert among three very different forms: Virion, or virus particle – An inert particle that does not carry out any metabolism or energy conversion. Intracellular replication complex – Within a host cell, the viral gene products direct the cell’s enzymes to assemble progeny virions at “virus factories” called replication complexes. Viral genome integrated within host DNA – Some types of viral genomes may integrate within a host chromosome as a prophage or provirus. This may be a permanent condition. 7 Dynamic Nature of Viruses § The inert nature of the virion particle, which lacks metabolism and the ability to reproduce independent of its host, argues that viruses are nonliving. § The virion assembly process argues that viruses are living organisms. § The genomes of large viruses show evidence of reductive evolution (evolutionary loss of genes) from a cellular origin § Genomes of small RNA viruses indicate they may have been built up from mere parts of a cell. These claims are debated regularly by virologists. Would you like to know more? Dr Paul Turner Yale University https://www.ibiology.org/speakers/ paul-turner/ 8 Size of viruses Most viruses are smaller than prokaryotic cells; range from 0.02 to 0.3 µm. Size of viruses Giant viruses Up to 2500 genes! Bigger the some bacteria. Viral structure capsid: the protein shell that surrounds the genome of a virus particle Naked viruses (e.g., most bacterial viruses) have no other layers. Enveloped viruses (e.g., many animal viruses) have an outer layer consisting of a phospholipid bilayer (from host cell membrane) and viral proteins. Nucleocapsid: nucleic acid + protein in enveloped viruses Brock Microbiology, Pearson. Virion morphology: Spherical Complex Spherical Icosahedral Helical Filamentous Image Source: Pinterest 6.3 Viral Genomes and Classification § Viral genomes can be: DNA or RNA Single- or double-stranded (ss or ds) Linear, circular, or segmented § The form of the genome has key consequences for the mode of infection, and for the course of a viral disease. § Viral genomes are used as the basis of virus classification. 13 6.4 Viral replication § Viruses display a remarkable diversity of ways to replicate within a host cell. § Bacteriophage replication § Animal virus replication § Naked virus § Enveloped virus 14 Overview of the Virus Life Cycle Major difference between prokaryotic and eukaryotic viruses is nucleic acid entry in prokaryotes and virion entry in eukaryotes. The replication cycle of a bacterial virus. Source: Brock Microbiology, Pearson. Phases of viral replication in a permissive (supportive) host attachment (recognition) of the virion penetration (entry, injection) of the virion nucleic acid synthesis of virus nucleic acid and protein by host cell metabolism as redirected by virus assembly of capsids and packaging of viral genomes into new virions release of mature virions from host cell Bacteriophages Infection § Bacteriophages exhibit two different types of replication cycles: 1. Lytic cycle 2. Lysogenic cycle § The “decision” of which replication cycle to utilize is dictated by environmental cues that either activate or repress transcription of genes for virus replication. In general, events that threaten host cell survival trigger a lytic burst. 16 Entry mechanism of bacteriophage Attachment: major factor in host specificity Microbiology, Wessner, Dupont, Charles and Neufeld, 2nd ed, Wiley, Chap.5, p.139. The bacteriophage injects its genetic material with a spring-like needle into the host cell! Virions attach to cells via tail fibers that interact with polysaccharides on E. coli LPS layer. Tail fibers retract, and tail pins contact cell wall. T4 lysozyme forms small pore in peptidoglycan. Tail sheath contracts, and viral DNA passes into cytoplasm. Capsid stays outside. Source: Brock Microbiology, Pearson. Video of bacteriophage infection Video by Biolution Viral life cycle Virulent: Viruses always lyse and kill host after infection. Temperate: Viruses replicate their genomes in tandem with host genome and without killing host, establishing long-term, stable relationship. can be lytic/virulent can enter lysogeny: most viral genes are not transcribed, viral genome is replicated with host chromosome and passed to daughter cells lysogen: host cell that harbors temperate virus can result in lysogenic conversion with new properties (e.g., virulence in pathogens) The lytic cycle of a bacteriophage Rice Uni chap 6.2 The lysogenic cycle of a bacteriophage Rice Uni chap 6.2 Bacterial Defenses § Bacteria have evolved several forms of defense against bacteriophage infection: Genetic resistance – Restriction endonucleases – Altered receptor proteins Cleave viral DNA lacking methylation CRISPR integration of phage DNA sequences – Clustered regularly interspaced short palindromic repeats – A bacterial immune system 23 Bacterial Defenses – Genetic resistance Modification of cell-host recognition Bacteriophage resistance mechanisms. Labrie SJ et al, Nature Reviews Microbiology volume 8, pages 317–327 (2010) Bacteria changes cell surface molecule recognized by phage Phage adapts to recognize new surface molecule Bacteria can produce a molecule that masks the phage target 24 Bacterial Defenses – Restriction enzymes Restriction enzymes cat and mouse Going from a to i: a. Normal infection b. Bacteria digest viral DNA/RNA using restriction enzymes c. Virus counteract with undigestible modified nucleic acid d. Bacteria finds a way to digest modified nucleic acids e. Virus finds another way to protect its nucleic acids f. Bacteria finds another way to get around it g. Etc… h. Etc… i. Etc… Bacteriophage resistance mechanisms. Labrie SJ et al, Nature Reviews Microbiology volume 8, pages 317–327 (2010) 25 Bacterial Defenses – CRISPR CRISPR: Clustered Regularly Interspaced Short Palindromic Repeats Protect from bacteriophage infection Regions contain short repeats of DNA sequences alternating with short variable spacers corresponding to “memory” of viral or other foreign DNA. Some viruses have evolved to avoid CRISPR. mutation of PAM regions production of Cas inhibitors When immunized cell encounters same virus, Cas proteins destroy incoming DNA. PAM: protospacer adjacent motif, crRNA: CRISPR RNA Brock Microbiology, Pearson 6.5 Animal and Plant Viruses § Animal and plant viruses solve problems similar to those faced by bacteriophages: Host attachment, genome entry and gene expression, virion assembly, and virion release § However, eukaryotic cells have a more complex structure than prokaryotic cells. Therefore, animal and plant viruses have greater complexity and diversity of viral replication cycles than we see in bacteriophages. § Animal viruses bind specific receptor proteins on their host cell. Receptors determine the viral tropism, or ability to infect a particular tissue type within a host. For example, Ebola virus exhibits broad tropism by infecting many kinds of host tissues, whereas papillomavirus shows tropism for only epithelial tissues. 27 Entry mechanism of animal viruses A. Endocytosis of non-enveloped virus B. Membrane fusion of an enveloped virus C. Endocytosis of an enveloped virus Two key differences with bacterial viruses: Entire virion enters the animal cell. Eukaryotic cells contain a nucleus, the site of replication for many animal viruses. Microbiology, Wessner, Dupont, Charles and Neufeld, 2nd ed, Wiley, Chap.5, p.138. Animal Virus Replication Cycles § The primary factor that dictates the details of a replication cycle of an animal virus is the form of its genome. § DNA viruses Utilize some or all of the host replication machinery § RNA viruses Use an RNA-dependent RNA polymerase to transcribe their mRNA § Retroviruses Use a reverse transcriptase to copy their genomic sequence into DNA for insertion in the host chromosome (Most human viral diseases are caused by RNA viruses) 29 Lytic cycle in animal cells Rice Uni Openstax Chap 6.2 Lysogenic cycle in animal cells HIV infection: The adhesion of the virus gp120 envelop protein to the cell surface marker CD4 (only present on T helper cells) and its coreceptor is mandatory for infection. The viral envelop fuses with the lipid bilayer of the host cell, the viral capsid is delivered in the cytoplasm. The capsid is digested by proteases and the ssRNA genome delivered into to the nucleus with the reverse transcriptase enzyme necessary to convert ssRNA in cDNA which can then be converted to dsDNA by the host cell. An integrase, also delivered with the virus, helps the transduction of the virus into the host genome. New viral mRNA and proteins are produced by the host cells, virions assemble within the cytoplasm and envelop proteins are integrated into the host plasma membrane. Encapsulated new viral particles exocytose, the vesicle with which they exocytose forms the new envelop, the virions are mature and can infect new cells. Rice Uni Openstax Chap 6.2 HIV life cycle (vimeo in collab with David Goodsell) Animal and Plant Host Defenses § Since viruses are ubiquitous, a wide range of defense mechanisms have evolved in animals and plants. § Genetic resistance – Hosts continually experience mutations. § Immune system – “Innate immunity”— interferons – “Adaptive immunity”— antibodies § RNA interference (RNAi) – Alters gene expression and degrades viral RNA 33 Chapter Summary – 1 § Viruses fill important niches in all ecosystems. § A virus is a noncellular particle that can reproduce only within a living cell. § An infectious viral particle, or virion, is composed of a nucleic acid genome surrounded by a protein capsid and, in some cases, an envelope. § Viruses can be divided into two main types: Symmetrical Asymmetrical § The Baltimore system classifies viruses mainly on their means of mRNA synthesis. 34 Chapter Summary – 2 § Phages may undergo a lytic or lysogenic cycle. § The more complex structure of eukaryotic cells leads to greater complexity and diversity of life cycles in animal and plant viruses than in phages. § The life cycles of retroviruses and pararetroviruses are especially complex. § Plant viruses enter host cells mechanically and are transmitted to other cells via plasmodesmata. § Both bacterial and animal viruses can be cultured. Plaque assays are employed for this purpose. 35