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Microbiology Thestudy of microorganisms: cellular and noncellularorganisms too small to be seen with naked eye.

Microbiologists study Bacteria, Viruses, Protozoans, Fungi,Algae, Diatoms, and more

Microbes can be:

not cellular (biophages & viruses)unicellular (Bacillus)multicellular (Anabaena)

Characteristics of Cells

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Ways microbes impact out lives

-agriculture-energy (biofuels)-food-disease (causes and treatments)

Agricultural Impacts

legumes: live in closeassociation with bacteria and form structures called nodules on their roots;convert atmospheric nitrogen into fixed nitrogen that the plants use forgrowth-reduces the need for costly and pollutingplant fertilizer.gutbacteria of ruminant animals such as cows-The bacteria can digest cellulose into glucose for a carbon andenergy source for the animal

Energy Impacts

Methaneis made by the anaerobic degradation of organic matter bymethanogenic microorganisms.Microbialfermentation turns sugarcane and cornstarch into ethanol.Microorganisms can be used to consume spilled oil, solvents, pesticides, andother environmentally toxic pollutants.

Food Impacts cheese, yogurt,buttermilk, sauerkraut, pickles, sausages, baked goods, and alcoholicbeverages



Disease Impacts

Drug resistant microbes are becoming morecommon.Biotechnologyis the use of microorganisms in industrial biosynthesis, typically bymicroorganisms that have been genetically modified to synthesize products (ex.Insulin) of high commercial value.

# of Microbial Cells on Earth 25x 10^29

Distribution of microorganisms on Earth

Marine Subsurface : 66%Terrestrial Subsurface : 26%Surface Soil : 4.8%Oceans : 2.2%All other habitats : 1.0%

3 Domains of Life-Bacteria (Eubacteria)-Archaea-Eukarya



How old is Earth? ~4.5 billion years

Characteristics of Early Earth• anoxic, lots of carbon dioxide• much hotter than the present Earth• abiotic synthesis of first biochemicalmolecules

Where were the first biological molecules made? they werelikely made in mounds of montmorillonite clayassociated with hydrothermal springs

What is LUCA? LastUniversalCommonAncestorfrom which cellular life evolved



Evidencefor RNA as 1stgenome? • RNAcan bind small molecules like ATP, nucleotides, amino acids•catalytic RNAs have enzymatic activity

Discovery of catalytic RNA

-first discovered in Tetrahymena (ciliated protozoan) by Thomas Cech-later by Syndey Altman studying RNAse P-catalytic RNA molecules are capable of cleaving phosphodiester bonds and others can removethe amino group from an amino acid.

When did cellular life evolve on Earth?

-3.8billion years ago – evidence of the first cells (microbes!) -Within the first half billion of Earth’s history cellular life evolved to give rise to the last universal common ancestor from which moderncellular life evolved.

Order of Beginning of Evolution on Earth

Biological Building Blocks (amino acids, sugars)RNA World (catalytic and self-replicating RNA)Protein Synthesis (RNA templated translation)DNA (Replication of transcription)Lipid Bilayers (cellular compartments)Divergence of Bacteria and Archaea



Primitive metabolism of early life•anaerobic (fermentative or anaerobic respiration)•autotrophic (CO2)• chemolithotrophic (H2, Fe2+)

Stromatolites

~3.5 billion years old-First evidence for microbial life can befound in rocks-Ancestors of filamentousphototrophic green nonsulfurbacterium Chloroflexus inancient stromatolites-Modern day stromatolitesin Shark Bay, Australia

Shift from anoxic to oxic environment:

~2.8 billion years ago• Oxygenic phototrophic cyanobacteriaappeared• Banded iron formations• Oxygen accumulated• Ozone (O3)layer formed•layers of ferric (Fe3+) iron oxides formed by ferrous (Fe2+) iron reacting with oxygen produced bythe cyanobacteria•Oxygen accumulation allowed aerobes toemerge•Short wavelength UV radiation reacts with oxygen to form ozone –about 400million years ago

TheHydrogen Hypothesis

Eukaryoticcell arose from the symbiotic association between a hydrogen-producing Bacteria (symbiont, aerobic respiring cell, gives risetothe mitochondria) and an Archaea (host that depended on H2 as an energy source). The nucleus formed after these two cellsformed a stable association and genes for lipid synthesis were transferred fromsymbiont to host, eventually enclosing theDNA (by need because of increasing genome size) and forming the organelles of amodern eukaryotic cell. Lateracquisition of a cyanobacteria (oxygenic phototroph, precursor to chloroplast) tobecome photosynthetic.



EndosymbiosisTheoryMitochondria and chloroplasts of todaycame from the stable incorporation of a chemoorganotroph and a cyanobacterium into the same cell.

Supportive Evidence for Endosymbiotic Theory

•Mitochondria and Chloroplasts–havetheir own genomes–containtheir own ribosomes---samesize as prokaryotes (70S)---antibioticsare effective---rRNAsequences are similar to prokaryotic rRNA•Eukaryotic nuclear genome has bacteriallyderived sequences.

Ampicillin

Ampicillin,like other β-lactam antibiotics, not only blocks the divisionofbacteria, but also the division of chloroplasts of the Glaucophytes(called cyanelles) and chloroplasts of themoss Physcomitrella patens, a bryophyte. Incontrast, it has no effect on the plastids of the higherdeveloped vascular plant Lycopersicon esculentum L. (tomato).

Phylogeny

•evolutionary relationships of organisms• measured by evolutionary chronometers–genes like those for the ribosomal RNAs(16S & 18S) or enzymes like RecA– sequence comparison used to determinethe evolutionary distancebetween organisms



SSU (smallsubunit)RNA sequencing

• sequencingthe small subunit of the ribosomal RNA gene–16Sin prokaryotes–18Sin eukaryotes• usedto identify organism• usedto determine phylogeny•If16S rRNAsequence differs by more than 3%, then different species.•If 16S rRNA sequence differs by more than 5%, thendifferent genera.

Why use the SSU RNA instead of anothergene?

– universallydistributed–functionally constant–sufficiently conserved (slow changing)–adequate length

NextGeneration Sequencing

•a culture-free method that enablesanalysis of the entire microbial community within a sample•For example–Sequencethe Campus Microbiomeproject–Mikereauxbiomepilot study

Phylogenetic trees •Illustration to represent evolutionarydistance



Taxonomic Heirarchy Domain , Phylum, Class, Order, Family, Genus, Species, (Strains)

Identifying organisms base on phenotypicproperties

1. Isolation and MicroscopyIsolation -> pure culture ->gram rxn/morphology2. General PhysiologyGram neg. rod -> facultative -> ferments lactose to acid/gas3. Detailed PhysiologyFacultative lactos fermentor ->perform series of biochemical tests -> positive (idol, methyl red, mutate); negative (citrate, H2S)4. ConclusionE.coli

Phenotypic Analysis morpholgy, motility, metabolism, physiology, cell lipid chemistry, cell wall chemistry, etc.

Molecular taxonomy (chemotaxonomy)1.DNA:DNA hybridization2.Multilocus Sequence Typing (MLST)3.Fatty Acid Methyl Ester (FAME)analysis



DNA:DNA Hybridization

•Tests ability of denatured DNA in singlestrand form from 2 organisms to bond (anneal) to one another•Same species – 70%-100% of the DNA willanneal•Same genus – at least 25% of the DNA will anneal

MultilocusSequence Typing •Sequencecomparison of several housekeeping genes•Usefulfor determining different strains of the same species

Fatty Acid Methyl Ester (FAME) Analysisbacterial culture -> extract fatty acids -> derivative to form methyl esters -> gas chromatography -> campare pattern of peaks with database -> identify organisms

Binomialsystem ofnomenclature

descriptive genus name and species epithet.-ex. BacillussubtilisThe International Code ofNomenclature of Bacteriaregulates naming of prokaryotes.-Major taxonomic compilations of Bacteria and Archaea:•Bergey's Manual of Systematic Bacteriology•The Prokaryotes



Formalrecognition of a new prokaryotic species requires:

1.Deposition of a sample of the organism in2 international culture collections (ATCC – American Type Culture Collection; www.atcc.org)2.Official publication of the new speciesname and description in InternationalJournal of Systematic and Evolutionary Microbiology (IJSEM).

First person to describe microorganisms 1665-Robert Hooke

First person to describe bacteria 1676-Antoni van Leeuwenhoek

4 Types of Light Microscopy

1. Brightfield2. Phase contrast3. Darkfield4. Fluorescent



Brightfield-Simplest-Specimenilluminated, magnified 1000x-Specimenstained to increase contrast

Phase constrast and Darkfield

-Visualize live samples-No staining required-Image contrast derived from cellstructuresPhase : dark gray backgroundDark field : black background

Fluorescent visualization of autofluorescent molecules or fluorescent stains

Compound light microscopes

– brightfield, phase-contrast, darkfield, fluorescent– optimize image resolution by using lenses with highlight-gatheringcharacteristics– limit of resolution ~ 0.2 mm



Electron microscopes

–Limit of resolution 0.2 - 4nm–Two types of electron microscopy1. Transmission electron microscope (TEM) for observing internal cellstructure2. Scanning electron microscope (SEM) for three-dimensional imaging andviewing surfaces

Significanceof Size of Microbes

-Microorganismsare as small as 0.2μm.-Sizeaffects physiology, growth rate, and ecology-Highsurface area–to–volume ratio aids in nutrient and waste exchange with theenvironment

Basic Components of All Microbial Cells

DNAcytoplasmcytoplasmicmembraneribosomes

Cytoplasmic membrane

–“fluid” highly selective permeabilitybarrier– made of lipids and proteins– Phospholipids form a lipid bilayer with polar exteriors and a nonpolarinterior.



Archaealipid bilayer •Phytanyl instead of fatty acids

Other lipids in cytoplasmic membrane helpto strengthen: – sterols in Eukarya– hopanoidsin Bacteria

Archaea Lipid monolayer More resistant to peeling apart than bilayers

Functionsof the cytoplasmic membrane1. Permeability barrier2. Energy conservation3. Protein anchor



Permeability barrier-Prevents leakage and functions as a gateway for transport of nutrients into, and wastes out of, the cell

3 Classes of Transport Systems1. Simple - proton motive force2. Group - phosphoenolpyruvate3. ABC - ATP

Types of Transport Eventsuniporterantiportersymporter

Examples of simple transporters

-sulfate symporter-potassium uniporter-phosphate symporter-sodium-proton antiporter-lac permease (symporter)



Energy ConservationSiteof generation and use of the proton motive force-Generationand use of a proton motive force bythe electrontransport chain & ATP synthase

Protein anchor Siteof many proteins that participate in transport, bioenergetics, and chemotaxis

Cytoskeleton

– “skeleton” of the cell– Eukaryotic cytoskeleton– microfilaments• polymers of actin• define, maintain, change cell shape• cell motility – cytoplasmic streaming– microtubules• polymers of tubulin• maintain cell shape• cell motility – form flagella and cilia• movement of organelles and chromosomes

Prokaryotic cytoskeleton

– FtsZ• protein structure similar to tubulin• determines where cell division will occur– FtsA and MreB• proteinstructures similar to actin• Fts A proteins help hold Fts Z proteins in place• Mre B proteins determine where new cell wallwill be inserted in an expanding/dividingcell.• Coccus shaped bacteria lack MreB gene.



Eukaryotic cells can contain several membrane-enclosedorganelles: nucleus, endoplasmic reticula, Golgi complex, lysosome, mitochondrion or hydrogenosome, chloroplast

Nucleus

•contains DNA•has pores– join the outer and inner nuclear membranesto allow for import and export of proteins and nucleic acids•contains nucleolus– where rRNA is made

Endoplasmicreticula (ER)

•RoughER–continuouswith nuclear outer membrane–containsribosomes; site of protein synthesis–makesglycoproteins and new membrane material•SmoothER–continuouswith the rough ER–lipidsynthesis and carbohydrate metabolism

Golgicomplex

– chemicallymodifies carbohydrates on ER glycoproteins– sortsmolecules from the ER– secretedfrom the cell– usedwithin the cell



Lysosome

–Producedby budding of the Golgi complex–pH 5–containsdigestive enzymes (lipases, nucleases, proteases, etc.)–recyclemacromolecules, kill foreign bacteria, apoptosis (cell suicide)

Lysozyme

• enzyme that destroys peptidoglycan bycleaving β-1,4-glycosidic bonds between the N-acetyl-glucose sugars• found in animal secretions• major line of defense against infectionby Bacteria

Peroxisome

•Organellesite of oxidation– oxidase enzymes break down lipids and other molecules forenergy•Containscatalase, superoxide dismutase to degrade H2O2 and O2-respectively•Beta-oxidationof lipids– broken down a 2-carbon acetyl unit at a time– occurs in theperoxisome– Acetyl-CoA is thentransported into the cytosol

Mitochondrion

•outermembrane–rigid–somewhatpermeable (ions, small organic molecules)•innermembrane(cristae - folds)–lessrigid and folded–lesspermeable (transport through specific proteins)–sitesof electron transport and ATP production •matrix–aqueousinside of the mitochondrion–containsenzymes of the citric acid cycle



Hydrogenosome•replacesmitochondria in some anaerobic eukaryotes•siteof ATP production but no electron transport•nocitric acid cycle

Chloroplast

•foundin phototrophic eukaryotes•surroundedby double membrane•stroma–aqueousinside of the chloroplast–siteof Calvin-Benson cycle•Ribulosebisphosphatecarboxylase/oxygenase(RuBisCO)fixesCO2 intoorganic compounds

Grana of thylakoidmembranes–stacksof flattened membrane discs–containlight harvesting pigments such as chlorophyll–siteof photosynthetic electron transport and ATP production

Cellwall

Structure•Proteins,Glycoproteins, Polysaccharides•Somelack cell wallsFunctions•Protection•Maintaincell shape•Preventsosmotic lysis•Interactwith environment



Eukarya cell walls

-Glycoprotein(widespread)-GlcA(β1→3)Gal(βl→3)Gal(βl→4)Xyl(β1→O)Ser-Found in the extracellular matrixof many eukaryotes-celluloseis a polymer of glucose- Chitinis a polymer of N-acetylglucosamine

Bacteria cell walls •Mycoplasma& Chlamydiaspecies lack cell wall•Planctomycesspecies have protein cell walls•Peptidoglycanis found in all other groups

Gram-negative Gram-negative Bacteria have only a few layers of peptidoglycan(only 10% of cell wall)

Gram-positive Gram-positive Bacteria have several layers of peptidoglycan (asmuch as 90% of cell wall).



Teichoicacids negativelycharged polymers containing glycerophosphate or ribitolresidues, bind Ca2+ andMg2+

Lipoteichoicacids teichoicacids covalently linked to membrane lipids

OuterMembrane of Gram-Negative Bacteria

outer membrane, periplasm, cytoplasmic membraneLipopolysaccharide(LPS) on outer cell wall surface-makes cell wall look scraggly and uneven-makes gram neg. bacteria harder to kill

Archaea cell walls •made of pseudomurein (Methanobacterium),protein, glycoprotein, or polysaccharide (Halococcus)



S-layercell wall -atwo-dimensional array protein or glycoprotein; selective sieve-Foundin most Archaea, usually in addition topolysaccharide

Prokaryotic cell surface structures: fimbriae pili capsules slimelayers

Fimbriae short protein filaments; attachment

Pili longer protein filaments;conjugation, motilityadhesionof pathogens to host tissue



Capsule polysaccharide or polypeptidelayer; attachment

Slimelayer similarto capsule but more loose layer of polysaccharide that is easily sloughed off,used for gliding

Gliding

• Slow, smooth movement of cell across asurface by non flagellated prokaryotes– slime secretion, ex. Cytophaga– twitching motiltiy (extension/retraction of type IV pili), ex. Myxococcus– ratchet-protein mechanism, ex. Flavobacterium

Flagellaand cilia

• move back and forth by dynein motor proteins• madeof microtubules (9 pair surrounding 2 central)• Filamentrotates like a boat motor propeller.• Motordriven by proton motive force (1000 H+ per rotation) is somewhat analogous to arevolving door.• Electrostatic forcesfrom the Mot proteins large positive charge on the Rings of the basal bodycause the basal body to rotate.



Bacterial flagellaIncludes filament, hook, motor/basal body-Proton motive force drives the flagellar motor to rotate the flagellum filament.

Archaea flagella•Multipleproteins make up filament•Smaller diameter flagellum less torque,slower speeds•Poweredby ATP not protons

Location of flagella

•Polar– flagella are attached at one or bothends of the cell– movement more rapid, spinning aroundfrom place to place• Peritrichous– flagella are inserted at many locationsaround the cell surface– movement typically slow and in astraight line

Types of Flagellation

monotrichous-single polar flagellalophotrichous-several polar flagellaamphitrichous -2 polar flagella ; one on each end of the cellperitrichous -many flagella on all sides of the cell body



Examples of different types of flagellated bacteria

Monotrichous – Vibrio, PseudomonasLophotrichous – Rhodospirillum photometricum Amphitrichous - SpirillumPeritrichous – Proteus, Salmonella, Escherichia

Movement with polar flagella

reversible flagella-counterclockwise and clockwise rotationundirectional flagells-clockwise rotation-cell can stop, reorient and then use cw rotation again to move about

Movement with peritrichous flagella

Run-bundled flagella (counterclockwise rotation)Tumble-flagella pushed apart (clockwise rotation)

Howdoes a motile cell determine where to go?

Theycompare the current environment with the environment they sensed momentsbefore.–Needed/Favorable – move towards–Toxic/Unfavorable– move away–Indifferent– no net movement



Taxes

directedmovements toward or away from a chemical or physical signal1. Aerotaxis - oxygen2. Chemotacis - chemicals3. Phototaxis - lightNoattractant – random movementAttractantpresent – directed movement

Gas vesicles

-small gas filled structures made of protein thy confer buoyancy on cells-aquatic microorganism slide Cyanobacteria-orient for optimum light harvesting or in response to other environmental stimuli

Magnetosomes-intercellular particles of magnetite (Fe3O4)-aquatic microbes like Magnetospirillum-orient cells in a particular direction

Carboxysome-organelle found in some prokaryotes-contains ~250 molecules of RuBisCO used for CO2 fixation by the Calvin Benson Cycle



Cell inclusion bodies

Storage depots-carbon---glycogen---Poly-B-hydroxyalkanoate-phosphate---poky phosphate granules-sulfur---sulfur granules stored in periplasm by gram neg. bacteria like purple sulfur bacteria---Theelemental sulfur can be used by the purple sulfur bacteria as an electron donorfor photosythesis.

Ferdinand Cohn

1876-In studying heat resistance in bacteria he discoveredendospores.-Described the life cycle of Bacillus subtilis fromvegetative cell↓endospore↓vegetative cell

Endospore formation

– called sporulation–survivalmechanism by some Gram-positive Bacteria like Bacillusand Clostridium– when cell growth stops due to harshconditions– within and at the expense of the vegetative cell-Multipleendospores can form within a vegetative cell in some species such as Metabacterium polyspora (a guinea pig intestinal symbiont).

Endosporeintracellular location1. terminal - vegetative2. subterminal -sporulating3. central - mature endospore



Endospore

–dormant, survival structure–contains essential macromolecules–contains calcium dipicolinic acidand small acid-soluble proteins not present in vegetative cells–dehydrated

Layersof an Endospore (outermost listed 1st)

-spore coat-cortex-exosporium-core well-DNA-core

Spore– released from vegetative cell– can survive indefinite periods of time– resistantto heat, chemicals, radiation, dessication

Germination–Sporetransforms into vegetative cell genetically identical to the vegetative cellthat made the spore–Occurswhen conditions become favorable



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