Unit 1 4/18/17 11:50 AM

What are viruses in microbiology?

Microbiology: study of organisms too small to be seen without  magnification

Microorganisms include:

• Bacteria

o Prokaryotic  

o Cells made of polysaccharide (peptidoglycan)

o Reproduce asexually

o Most much smaller than eukaryotic cells

o Live alone, in pairs, clusters, chains

o Live anywhere w/enough moisture

• Viruses

o Smaller than the smallest prokaryote

o Acellular (not made of cells)

o Has little genetic material

• Fungi  

What are fungi in microbiology?

o Eukaryotic

o Get food from other organisms

o Have cell walls

o Microscopic Fungi: Molds & Yeasts  

▪ Molds  

???? Multicellular

???? Long filaments, intertwine

???? Reproduce w/sexual and asexual spores (make  

new individual w/out fusing to another cell)  

▪ Yeasts

???? Unicellular  

???? Oval – round

???? Reproduce by asexual budding (the daughter cell  

grows off the mother), some by sexual spores

• Protozoa  

o Single cell eukaryotes

What are microscopic fungi?

o Similar to animals in nutritional needs & cell structure  

o Capable of locomotion

▪ Cilia: Many short protrusions that propel the  We also discuss several other topics like What is the definition of macroevolution?

microorganism

▪ Flagella: few extensions, whip-like motion

o Live in water, & inside animal hosts

o Most reproduce asexually, some sexual

• Helminths (worms)

o A parasite that can be seen by the naked eye, studied by  microbiologists because eggs are microscopic

• Algae  

o Unicellular & Multicellular

o Photosynthetic eukaryotes = make their own food from  CO2 + water + energy from sunlight  

o Large Algae: kelp and seaweeds, found in oceans, Agar used  in lab media comes from these

o Small Algae: found in freshwater and oceans

• Archaea

o Prokaryotic

o Cell walls lack peptidoglycan  

o Reproduce asexually

o Live alone/in pairs/chains/clusters  

o Live in extreme conditions

▪ Highly saline and arsenic rich Mono Lake CA If you want to learn more check out What causes the trend of electronegativity?

▪ Acidic Hot Springs of Yellowstone National Park  

▪ In oxygen depleted mud at bottom of swamps

Microbiology led to:

• Immunology: Study of bodies defense against pathogens • Public Health Microbiology

• Epidemiology: study of the occurrence, distribution, spread of  disease in humans

o John Snow studied Cholera spread

• Food, Dairy, Aquatic Microbiology (Food Spoilage)

• Agricultural Microbiology: water pollution caused by microbes • Biotechnology

• Genetic Engineering & Recombinant DNA Technology: Microbes in  cheese making, alcohol, microbes interaction with immune system • Environmental Microbiology: Study of Microbes in soil, water, other  habitats

• Bioremediation: Use living bacteria, fungi, & algae to detox.  Polluted environmentsDon't forget about the age old question of If the government wanted to decrease unemployment, it could be?

• Industrial Microbiology: Microbes used to make products o Pasteurization: Heat something to kill contaminating bacteria Microbes are also involved in flow of Energy and Nutrients • Photosynthesis: convert CO2 to organic material

• Decomposition: Breakdown dead matter & wastes into simple  compounds

Microbes involved in infectious diseases

• Pathogens: microorganisms that harm (this is the minority of  microorganisms)

o 2,000 dif. Microbes cause harm

o 10 billion new infections worldwide per yr.

o 12 million deaths by infections per yr.

Science according to the organism studied

• Bacteriology: The study of Bacteria

• Phycology: The study of Algae

• Mycology: The study of Fungi

• Protozoology: The study of Protozoa

• Parasitology: The study of Parasites

• Virology: The study of Viruses

History Of Microbiology Don't forget about the age old question of What is the effect of a drug that is an agonist?

• Prominent Discoveries

o Microscopy

o Scientific Method

▪ Make observation

▪ Generate question

▪ Generate hypothesis We also discuss several other topics like What was the cancer rate in 1900?
We also discuss several other topics like What are the two criteria for science?

▪ Experiment (w/control groups)

▪ Accept, reject, or modify the Hypothesis

▪ If hypothesis repeatedly verified = Theory or Law (ex:  Theory of Evolution or Selective Pressure)

o Developed Medical Microbiology

o Microbiology Techniques

• Lifestyle of Microorganisms

o Exist freely in soil, water, surface

o Relatively harmless or even beneficial (decomposition)

o If associated with other organisms considered parasites and  live in a host

• Early Microscopy

o Robert Hooke - 1660

▪ First compound microscope (microscope w/2 lenses)  ▪ Saw cells of cork first and reminded him of a monk’s  quarters which were called a “cell” – how we got the  

name “cell”

▪ He examined living and non-living things

o Anton van Leeuwenhoek – 1632-1723

▪ Traded in linen & wanted to see the quality so made his  own magnifying glasses and achieved the level of  

microscopes

▪ Saw things that moved called them Animalcules

▪ When he died never passed on the secret to making the  lenses and lost ability to see microscopic things

o Edward Jenner – 1796

▪ Developed first vaccine

???? Knew about variolation (when take puss from  

someone w/smallpox & give it to someone else  

they either die or are protected)  

???? Decided when saw milkmaids hands w/something  

that looked like smallpox but they never  

contracted it, to take puss from their hands and  

give it to a boy

???? The boy survived & was given smallpox, he was  

protected, this was considered FIRST  

VACCINATION (“vaca,” from “cow”)

o Joseph Lister – 1867

▪ Founder of antiseptic surgery

▪ Opened field of research in antisepsis & disinfection o Ignaz Semmelweis  

▪ Introduced hand washing  

▪ People not ready for it, didn’t take on even when  

proved it worked

▪ Took on when Lister introduced it further

o Spontaneous Generation (Abiogenesis): Commonly held  theory that things just started growing randomly, until it was  disproved by the following people (proved Biogenesis: creation via biology)

o Louis Pasteur – 1822-1895

▪ Created flasks with bent necks that prevented microbes  from falling into sterile solution

???? If broke neck microbes got in and grew  

▪ Proved that grapes + yeast = alcohol & grapes +  bacteria = acid

▪ Proved Yeast can grow with & w/out oxygen & from  other cells  

o Robert Koch – 1843-1910

▪ Germ Theory: disease is caused by germs

▪ Pioneer of Etiology: How diseases is caused  

▪ Worked w/anthrax in animals & discovered endospores  (the resting stage of bacteria) – this was the 1st time  bacteria proven to cause disease

▪ Man who worked in lab developed Petri Dishes

▪ Walter Hess developed the media from algae – Agar ▪ Created framework to identify Pathogens: Koch’s  Postulates

???? 1. Find evidence of microbe in every case of the  disease

???? 2. Isolate microbe from infected subject & grow in  lab

???? 3. Inoculate healthy subject & cause same  

disease

???? 4. Re-isolate agent from subject

o Alexander Flemming – 1881-1955

▪ Discovered Penicillin 1929 (1st modern antibiotic) ???? Saw that fungi penicillian was killing staph he was  growing  

???? Penicillin enters widespread US – 1941

▪ Discovered Lysozyme from snot dripping & breaking  down bacteria

???? In snot, tears, egg whites

o Selman Waksman – 1944

▪ Discovered Streptomycin – first drug for tuberculosis

▪ Discovered first way to isolate soil bacteria & test  

antibiotics  

▪ Reference to soil bacteria on grave: “The Earth will  

open and bring forth salvation.”

5 I’s of Culturing Microbes

• Inoculation: Introduction of sample into container of media to  produce culture of observable growth

• Isolation: Separate one species from another

• Incubation: placed in a temperature controlled chamber and  microbe multiples for observable growth

• Inspection: Microscopic and Macroscopic Observation, what you can  see upon inspection:

o Pure Culture: growth of 1 single known species

o Mixed Culture: growth of 2 or more identified species

o Contaminated Culture: the mixture was once pure & was  mixed w/unwanted microbes growing in it.

• Identification:  

o Use micro/macroscopic appearance to identify

o Perform biochemical tests to identify

o Use genetic characteristics to identify  

o Use immunological tests to identify

Media to Isolate Microorganisms

• Physical Form of the Media

o Nutrient Agar (made from red algae)  

▪ Solid Agar: Solid at room temp and body temp

▪ Holds moisture & nutrients (beef extract, peptone,  

agar)

▪ Not digestible for most microbes

▪ Semisolid Agars: help determine if the organism is  

motile or not, if it is then it will move in this media  

???? Used in clinical tests

o Nutrient Broth: Liquid medium w/beef extract & peptone

• Different Chemical Compositions of Media

o Complex or Non-synthetic: Media has nutrients released by  partial chemical breakdown by beef, soy, proteins, has at  least 1 ingredient that is not exact (chemically defined)

o Defined: Media in which the exact chemical composition is  known  

o Synthetic: Contains pure organic and inorganic compounds in  an exact amount (chemical formula)

o General Purpose Media: Grows many microbes, considered  non-synthetic

o Enriched Media: complex organic substances (blood, serum,  hemoglobin, and special growth factors that picky microbe  require)  

▪ Used to grow less abundant microbes

• Different Functions of Media

o Selective Media: media that contains nutrients that promote  the growth of one microorganism and inhibit the growth of  others

o Differential Media: media made so there is a change in  appearance of media or the organisms that helps the  

microbiologists tell the difference between different bacteria  ▪ Agar has dye that changes color based on pH

o Blood Agar considered a differential and complex  medium

Isolation Techniques

• Streak Plate: Serial dilution on a solid surface, use loop to spread  on one quadrant then go on to quadrant 2 and go back into  quadrant 1 slightly to dilute the bacteria, repeat 2 more times • Pour Plate Technique:

o Serial dilution of liquids

o Allows growth of different types of microbes because of  different environment

o Used for fewer colonies

Colonies: Culture visible on the surface

CFU: Colony Forming Unit which pure culture comes from (comes from a  single progenitor)

How to Dispose of Cultures

• Steam Sterilization

• Incineration

Key Characteristics of a Microscope

• Magnification: ability to enlarge objects

• Resolving Power: ability to show detail (Resolution)

o If particle size is smaller it has a better resolution

Compound Microscope  

• Hooke used

• Binocular – 2 lenses

o Objective Lens: the lens right above the object, can have a  series of lenses (4x, 10x, 40x, and 100x)  

▪ Low Power: 10x10=100

▪ High Power: 10x40=400

▪ Oil Immersion: 10x100=1000 used to reduce light  

refraction

o Ocular Lens: lens closest to the eye (can be monocular or  binocular) – is 10 or 12.5 power (this is the 10 being  

multiplied by the objective lens powers above)  

• Light needs to be focused on specimen for best resolution Types of Microscopy

• Bright Field Microscopy

o Used in medical labs

o Image made w/light through the object

o Image absorbs light, darker than the background

o Used for live & fixed material

▪ Stain to improve contrast but kills microorganisms

• Dark Field Microscopy

o Illuminates objects from side

o Object white against dark background

o No stain used

o Living objects can be seen  

• Fluorescence Microscopy

o UV light used w/filters to protect eyes

o Use dyes that fluoresce when exposed to UV light

▪ Have specificity to certain cell structures

▪ Can be coupled to antibodies to specifically target an  

object  

▪ Used to target specific bacteria or look for specific viral  antigens  

• Transmission Electron Microscopy (TEM)

o Uses a wavelength of an electron beam of ~0.5nm and  creates a resolution of ~0.3nm = greatest magnifying  

resolution

o Use very thin sections (20-100nm) for the beam to go  

through specimen

o Use heavy metal salts to stain image & make contrast

o Nuclei diffract electrons

o Max magnification is 120,000x = can see what is inside things • Scanning Electron Microscope (SEM)

o Makes 3D image

o Sample is plated w/thin layer of a metal w/large nuclei (ex.  gold)

o Electrons scanned across surface of sample & detector detects  reflected electrons  

o Image shown on TV screen

o Magnification not as good as TEM, ~10nm

Contrast: difference in intensity between two objects or between the object  & the background

• Enhance Contrast by Staining  

o Thick cell walls retain blue/purple stain (The Principle stain) o Thin cell walls lose principle/1st stain and allow to see the  Secondary Stain = cells are pink  

o Staining also deals with charges

▪ Positive Staining: Stains the organism

▪ Negative Staining: Stains the background, acidic dyes  

used are repulsed by the negative charges on the cells  

and don’t stain them (the capsules of bacteria are  

negatively charged) –AKA Capsule Stain

o Simple Staining: made of single basic dye, soak the smear in  dye for 30-60 seconds and rinse

o Differential Staining: Use more than one dye to see different  cells & structures

Taxonomy

Taxonomy: organizing, classifying, and naming living things, was created by  Carl Von Linne (Linneaus)

Classification: The orderly arrangement of organisms into Groups Nomenclature: Assigning names

Identification: determining and recording traits of organisms for placement  into taxonomic schemes

Old Whittaker System

• Set up with Binomial (Scientific) Nomenclature, everything had two  names – 1st name = Genus & 2nd name = Species  

o Ex: Homo Sapiens

• Thought originally only 2 Kingdoms: Plantae and Animalia o Noticed certain organisms didn’t fit and created kingdoms  Protista, Bacteria (Monera), and Myceteae for fungi

7 Main Taxa

• Species: the most specific, organisms that can successfully  interbreed

• Genera: Similar species

• Families: Similar genera

• Orders: Similar families

• Classes: Similar orders

• Phyla: Similar classes

• Kingdoms: similar phylas

• The classification is determined by genetic/cellular level, use rRNA to compare things, Woes and Fox were the first to do  this

There are 3 Domains which cover all organisms and then they are  categorized by the 7 main taxa starting with kingdoms and getting more  specific from there

• Archaea: Odd bacteria that live in extreme conditions

• Bacteria: True bacteria (*Prokaryotes fall under Bacteria and  Archaea)

• Eukarya: have nucleus and organelles

How to Assign Names

• Binomial (Specific) Nomenclature

o 2 Names  

▪ Genus – capitalized

▪ species – lowercase

o Both italicized or underlined  

o Sometimes shorten first name with first letter and period.  o Inspiration for names varied & imaginative  

o Names usually mean something

▪ Ex: Staphylococcus aureus (S. aureus)

???? Staphyl = bunch of grapes (tells plane of division)

???? Coccus = “berry,” spherical shaped cell

???? Aureus = “golden,” yellow color  

Evolution of Microorganisms

• Phylogeny: the natural relatedness between groups  

• Evolution

o All new species come from an existing

o Close relatives have similar features, evolved from a common  ancestor  

o Usually progress towards a greater complexity  

Characteristics of Cells

• Eukaryotic Cells: (Animals, Plants, Fungi, & Protists)

o Membrane-bound organelles (perform specific functions and  compartmentalize cytoplasm)

o Has double-membrane bound nucleus with DNA in  

chromosomes

• Prokaryotic Cells: (Bacteria & Archaea)

o No nucleus or other membrane bound organelles

o How to classify Prokaryotes:  

▪ Microscopic Morphology: individual cocci or bacilli  

appearance  

▪ Macroscopic Morphology: Colony appearance

▪ Bacterial Physiology

▪ Serological Analysis: use liquid portion of blood after  

clotting factors removed to determine the  

microorganisms

▪ Genetic and Molecular Analysis

▪ If something causes a similar disease they are  

often named something similar

Bacterial Taxonomy based on Bergey’s Manual of Systematic Bacteriology • Classification based on genetic information = phylogenic • Not all organisms have the same amount of G+C content (chemical  content), affects DNA H-bonding

• (Remember that bacteria are Prokaryotic) There are 2 main  Domains

o Archaea  

o Bacteria

• 5 Major Subgroups w/25 different phyla in each one

*Within the 3 Domains (Eukarya, Archaea, and Bacteria) there are  Organelles in the Bacteria Category because of Endosymbiosis (The idea that  eukaryotic life evolved from prokaryotic life, will have some of the same  characteristics)

Species and Subspecies

• Species: bacterial cells w/overall similar pattern of traits • Strain/Variety: A culture from one parent that differs from the  others of that species (different structure or metabolism)

o Ex: Biovars and morphovars

• Type: subspecies w/differences in antigenic  

makeup/immunoresponse (serotype or serovar), susceptibility to  viruses (phage type) & pathogenicity (pathotype)

Diagnostic Scheme for Medical Use

• Phenotypic qualities to identify

• Limited to bacterial disease agents

• Categorizes bacteria based on shape, arrangement, physiological  traits, cell wall structure

Dimensions of Bacteria (Prokaryotic)

Morphology: shape of organism

• Cocci: roughly spherical  

o Staphylo: clusters of cocci, form when planes of cell divide  randomly

o Strepto: Long chains of cocci, form when cocci cell divides  down the center plane & the resulting cells stay connected  ▪ Diplococcus: cocci that divide down the center plane &  

stay connected in pairs

o Tetrad: the cocci divide in two planes and the four resulting  cells stay connected

o Sarcina: Cocci that divide in 3 planes, stay connected to make  a 3D tetrad  

• Bacillus: non-spore forming rods

o Gram Positive (Thick peptidoglycan = retains purple dye) o Obligate Intracellular Parasite are Bacilli

▪ Can’t grow outside the host

▪ Very small

▪ Pleomorphic (shape varies) or Coccobacilli  

▪ Ex: Rickettsia and Chlamydia

• Endospore Producing Bacilli

o Soil organisms

o Gram Positive (Thick peptidoglycan = retains purple dye) o Ex: Bacillus & Clostridium

• Helical Bacteria

o Spirochetes: Flexible twisting bacteria, rod-shaped, have  flagella at both ends that tightly spiral around cell

▪ Move by creeping motion

▪ Has 70 turns (wound less tight)  

o Spirilla: rigid twisting rod shaped structure

▪ Flagella helps move with swimming motion (like

corkscrew)

▪ Have 20 turns (wound tightly)

Size of microorganism: measured in micrometers

• Largest bacteria 0.75mm, could see w/naked eye

• Smallest bacteria 140 nm

Prokaryotic Structures: cell membrane, cytoplasm, ribosomes, one or a  few chromosomes

• External Structures: Appendages and Glycocalyx

o Appendages (2 groups)

▪ Motility  

???? Flagella: long structures, extend beyond the  surface of the cell and glycocalyx, used to propel  through the environment,  

• 3 parts

o Filament: long, thin, hollow, helical  

structure made of protein called  

Flagellin

o Hook: curved structure that connects  

the filament to the basal body, made  

of different proteins

o Basal Body: Stack of 2 and 4 rings of  

protein firmly anchored into cell wall,  

varies between gram (-) and gram  

(+) cells, it doesn’t extend into the  

cytoplasm  

• Rotates 360 degrees to move prokaryotic  cells like a boat propeller

• Number and arrangement of flagella varies o Monotrichous: one flagella at one end  

of the cell

o Lophotrichous: group of flagella at  

one end of the cell

o Amphitrichous: one flagella at both  

ends of the cell

o Peritrichous: Flagella all around the  

perimeter of the cell  

• Flagellar Responses: Guide bacteria in  

certain direction in response to outside  

stimulus

o Chemotaxis: guided by chemical  

stimuli (either positive or negative)

o Phototaxis: guided by light stimuli

o Signal sets flagella in rotary motion in  

either clockwise or counterclockwise  

direction, moving towards nutrients

▪ Counterclockwise: smooth,  

linear direction = Run, if  

bacteria in high concentration  

will run more

▪ Clockwise: Tumbles, if bacteria  

in low concentration will tumble  

more

▪ Attachments or Channels

???? Fimbriae: fine proteinaceous, hair-like bristles on  

cell surface

• Function: Adhesion to other cells and  

surfaces

???? Pili: Rigid tubular structure

• Function: join bacterial cell for partial DNA  

transfer = Conjugation (an exchange of  

genetic material in mating types)

• 1-3 present per cell

• Found only in Gram Negative (-) cells (thin  

peptidoglycan layer and outer membrane,  

doesn’t hold purple stain = pink cell)

o Glycocalyx: surface coating of cell wall

▪ Capsule: tight grouping

???? Chemicals in these are similar to the host so the  

white blood cells don’t recognize them and cant  

perform phagocytosis on them

▪ Slime Layer: loose grouping

▪ Functions of both are similar (both made of glycoprotein  and polysaccharide)

???? Keep cell from drying out (desiccation)

???? Help bacteria stick together to form Biofilms

(aggregates of cells stuck together in a film)

???? Prevent WBC from phagocytosis

• The Cell Envelope: External covering outside of the cytoplasm, 2  layers, helps maintain cell integrity

o Cell Wall: provides structure, protects from osmotic forces,  helps attach to other cells, resists antimicrobial drugs

o Cell Membrane: Selectively permeable, nutrients flow in and  wastes flow out

o 2 Groups of Cell Envelopes

▪ Gram Positive (+) Bacteria: Thick cell wall made of  peptidoglycan around the cell membrane.  

???? Contains Teichoic Acid: in part of peptidoglycan  layer, does not extend all the way through

???? Contains Lipoteichoic Acid: extend through the  whole peptidoglycan layer & attached to cell  

membrane

???? 1 Periplasmic Space

???? Peptidoglycan layer 20-80nm thick  

???? Ex: Staphylococcus aureus

▪ Gram Negative (-) Bacteria: Outer cell membrane, thin  peptidoglycan layer and then the cell membrane inside ???? Has Porins on the outside

???? 2 Periplasmic spaces  

???? Peptidoglycan layer 8-10nm thick  

???? Ex: Escherichia coli (E. coli)

o Peptidoglycan Structure:  

▪ Polymer of NAM/NAG chains  

???? NAM: N-acetylmuramic acid

???? NAG: N-acetylglucosamine  

▪ Cross links between adjacent NAMs give strength, these  differ between Gram(+) and Gram(-)

???? Gram(+) Cross Linkage

• There is an Interbridge of repeating Glycine  

units between the Lysine of one NAM & the  

Alanine of the NAM next to it

???? Gram(-) Cross Linkage

• There is a direct bond between the DAP of  

one NAM & the Alanine of the NAM next to it

▪ Penicillin affects the direct bonds between adjacent  chains, but the outer membrane of Gram(-) make it  hard for penicillin to penetrate  

▪ Lysozyme also breaks down NAM-NAG chains

o Non-typical Cell Walls 

▪ Bacteria that don’t have typical cell wall structure  

(Mycobacterium & Nocardia)  

???? Gram(+) cell wall with Mycolic Acid give cell wall  

waxy nature because it’s a lipid = this wall called  

an Acid-Fast Cell Wall

• Gives it pathogenicity & high resistance to  

chemicals & dyes

• Must use Acid-Fast Stain to diagnose  

infections caused by these microorg.  

▪ Bacteria w/No Cell Wall (Mycoplasma)

???? Cell membrane stabilized w/sterols (ex:  

cholesterol)  

???? Pleomorphic (less structured, more flexible)  

• Internal Structures of Bacteria (Reminder: Prokaryotic) o Cytoplasm: gelatinous solution of sugars, amino acids & salts,  70-80% water (solvent used in all cell functions)

o Chromosomes: Single, circular, double-stranded DNA, has all  the genetic material, in prokaryotes. not packaged around  histones but packaged into Nucleoid

o Plasmids: small circular, double-stranded DNA, not essential  for bacteria to growth or metabolism

▪ There are variations in Segregation of these, some  linear some circle

▪ Sometimes harbor in antimicrobial resistance genes o Ribosomes: Made of 2 subunits of protein & rRNA

▪ 60% made of rRNA (the biggest portion and most  

important component, makes peptide bonds, can be  

enzymatic making enzymes included the one that  

translates)

▪ Function: synthesis of proteins

▪ Different than Eukaryotic Ribosomes because smaller & sediment faster when centrifuged  

▪ Two Types:  

???? 30S + 50S = 70S Bacterial Ribosomes, found  

inside organelles

???? 40S + 60S = 80S Eukaryotic Ribosomes, found  outside organelles

• Prokaryotes have BOTH types of  

ribosomes

o Inclusions & Granules 

▪ Intracellular storage bodies

▪ Size, number & their content varies

▪ When environmental sources are depleted the bacterial  cell can use these

▪ Ex: glycogen, poly-β-hydroxybutyrate, gas vesicles for  floating, sulfur & phosphate granules (metachromatic  granules), particles of iron oxide

o Cytoskeleton: Internal network of proteins associated w/cell  wall

o Endospores: formed by microbes, toughest & longest living  form of life

▪ High resistance due to high levels of Calcium &  Dipicolinic Acid

▪ Longevity = ~250 million yrs.

▪ Resistant to ordinary cleaning & boiling

▪ Destroyed by pressurized steam @ 120°C for 20-30  min.

▪ When dehydrated = metabolically inactive – is how they  live for so long  

▪ Sporulation: Formation of an Endospore, depends on  the species vegetative cell can form endospore  

centrally, subterminally, or terminally

???? Step 1) In a Vegetative Cell (single cell develops  into an endospore when one or more nutrients are  limited, this endospore reactivates to transform  

into a vegetative cell) DNA is replicated

???? Step 2) DNA aligns along cell’s long axis

???? Step 3) The cytoplasmic membrane invaginates to  form the Forespore

???? Step 4) the cytoplasmic membrane grows and  

engulfs the forespore in a 2nd membrane,  

vegetative cell’s DNA disintegrates

???? Step 5) A Cortex of Calcium and dipicolinic acid is  

deposited between the two membranes

• the cell holding the Endospore at this point  

is called the Sporangium

???? Step 6) Spore coat forms around the endospore

???? Step 7) Endospore matures, spore coat  

completed, resistance increases

???? Step 8) Endospore released, divides again under  

right conditions

Eukaryotic Structures (Major difference from Prokaryotic is contains a  membrane-bound nucleus & has membrane-bound organelles, can also be  unicellular or multicellular)

• Organisms Studied

o Protozoa: Always Unicellular

o Fungi & Algae: Unicellular or Multicellular

o Helminths: animals w/unicellular egg or larval forms, animals  themselves are multicellular

• External Structures

o Glycocalyx: outermost layer of cell, comes in direct contact  w/environment

▪ Made of polysaccharides, is a network of fibers  

▪ Can be Slime Layer or Capsule

▪ Eukaryotes not as structurally organized as prokaryotes ▪ Function: adhere cells to one another, strengthen cell  

surface, protect against dehydration, cell to cell  

recognition & communication

o Under Glycocalyx, some Eukaryotes have Cell Wall some don’t ▪ Fungi & Algae – thick rigid cell wall

???? Cell Wall: Provide support & shape

???? Fungi: thick layer of polysaccharide fibers made of  

chitin or cellulose + thin layer of mixed glycans

???? Algae: Variation of chemical composition, some  

common ones: cellulose, pectin, mannans, silicon  

dioxide, calcium carbonate

???? Some cells that have cell walls can also have  

things like Sterols in the cell membrane

▪ Protozoa, some algae, all animal cells – no cell wall,  only Cell Membrane  

o Cytoplasmic/Cell Membrane:  

▪ Bilayer of phospholipids & proteins

▪ Function: selectively permeable barrier for transporting  material into & out of the cell, proteins are like  

recognition molecules, receptors, carriers, enzymes, or  channels.

???? Endocytosis: Active Transport, membrane  

distends to form pseudopods around a substance  

and bring it into the cell

• Phagocytosis: Bringing in a solid substance

• Pinocytosis: Bringing in a liquid substance  

• Stuff brought in contained in a Food Vesicle  

• Amoeboid Action: some use this for  

locomotion by extending pseudopod &  

streaming into it

???? Exocytosis: how things exported from the cell,  

vesicles w/stuff inside fuse to cytoplasmic  

membrane & dump it out

▪ Sterols help with stability

▪ Eukaryotic Cells contain membrane-bound organelles,  these are 60-80% of cell volume

• In Cytoplasm/Internal Structures

o In TEM microscopic image, the varying darkness in the  cytoplasm is varying density

▪ Euchromatin = light, less dense chromatin fibers

▪ Heterochromatin = dark, more dense chromatin fibers ▪ The more tightly packed DNA (more dense) is being  silenced

???? DNA is packaged around Histones ???? beads on a  string (Nucleosomes) ???? chromatin fiber ???? form  

loops ???? compress to form Chromatid

o Rough Endoplasmic Reticulum: 

▪ Ribosomes adhere to it

▪ assemble proteins into their secondary, tertiary, and  quaternary structure

▪ transport proteins throughout the cell

▪ modifies protein w/glycoprotein (sugar wrapped around  the protein)

o Smooth Endoplasmic Reticulum: 

▪ Make Lipids

▪ Release Calcium ions

▪ Detoxify organic chemicals

▪ Produce steroid hormones

o Golgi Complex: 

▪ Structure: flattened hollow sacs in a phospholipid  bilayer

???? Cisternae: Flattened membrane disk, Golgi

contain 3-20 most contain 6

???? Transitional Vesicles: a sac with molecules that  comes from the ER

???? Condensing Vesicles (Secretory Vesicles):

packages secretions into these sacs that fuse to  

the cytoplasmic membrane & dump contents  

outside cell w/exocytosis

▪ Function: modifies proteins to be sent other places,  receives & packages large molecules to export from the  cell  

o Lysosomes: vesicles with enzymes that come from the Golgi ▪ Involved w/intracellular digestion & protects against  microbes

o Vacuoles: membrane bound sacs with particles to be  digested, excreted, stored

o Phagosome: A food vesicle (vacuole) that is taken into the  cell by endocytosis, when it fuses with a lysosome from the  Golgi called a phagolysosome

o Mitochondrion: 

▪ Shape: Spherical to elongated, has 2 membranes ▪ Structure:  

???? Cristae: Inner bilayer that has many folds to  

increase the surface area, where most of ATP  

produced in eukaryotic cells produced

???? Matrix: contains (prokaryotic) 70S ribosomes &  circular DNA (has genes for some RNA & few  

mitochondrial polypeptides made by mitochondrial  ribosomes)

▪ Function: Makes ATP, part of the Krebs Cycle (Citric  acid cycle), works w/electron transport

o Chloroplast: light harvesting organelle, found in  photosynthetic eukaryotes

▪ Structure: two phospholipid bilayer membranes & DNA,  make a few polypeptides w/own 70S ribosomes.

???? Thylakoids: membranous sacs that provide a lot  of surface area for chloroplasts to  

photosynthesize. Chlorophyll found here.  

???? Grana: Stacks of Thylakoids

???? Stroma: Space enclosed by the inner membrane,  contains mix of metabolic products, enzymes &  

ions

o Cytoskeleton 

▪ Function: Anchor organelles, help cytoplasmic  streaming, move organelles within the cytosol, move  cytoplasmic membrane for endocytosis & amoeboid  action

▪ Structures:  

???? Microtubules made of tubulin, are spindle fibers  and are also part of the Eukaryotic Flagella, also  

transport things

???? Microfilaments made of actin, used for amoeboid  

movement, cytokinesis, cell movement  

???? Intermediate filaments made of various proteins

Kingdom Fungi:  

• Majority unicellular/colonial, few have cellular specialization • Been around for 650 million yrs.

• Species number difficult because mating forms look different than  non-mating forms, could look different & be the same thing • 100,000 species divided into 2 groups  

o Macroscopic Fungi (Mushrooms, puffballs, gill fungi)

o Microscopic Fungi (molds, yeast)

▪ Two Morphologies = Dimorphic (Transitions between  

two forms to become pathogenic)

???? Yeast: round ovoid shape, reproduce asexually,  

the form that is pathogenic

???? Hyphae: long filamentous fungi or molds, the  

form that is benign

• When in individuals called “hyphae,” when  

make a mat of them called, “Mycelium”

(filamentous fungal growth)

• Fungal Organization

o Grow in loose colonies

o Yeast: soft, uniform in texture & appearance

o Filamentous Fungi: mass of hyphae = Mycelium (cottony,  hairy, velvety texture)

▪ Septate: when hyphae is divided with cross walls, each  separate part has its own nucleus

???? Aseptate Hyphae: no cross walls dividing

???? Septate Hyphae: cross walls dividing

▪ Vegetative Hyphae: digests & absorbs nutrients

▪ Reproductive Hyphae: makes spores for reproduction

Unit 2 4/18/17 11:50 AM

Microbial Nutrition

Nutrition: the process of acquiring chemical substances or nutrients from the  environment and using them in cellular activities

• Essential Nutrients

o Macronutrients: elements that make up the majority of the  cell, CHONPS  

▪ Carbon

▪ Hydrogen

▪ Oxygen

▪ Phosphorus

▪ Sulfur

o Micronutrients: elements that are required in small amounts  and involved in cell function. Ex: Mg used in chlorophyll.  

• Nutrients can be:

o Organic: carbon, carbohydrates, proteins

o Inorganic: CO2, water, gases like oxygen

• Microbial cytoplasm made up of  

o 70% water

o Proteins  

o 96% of cell made up of 6 elements (CHONPS) – this is in a  Dry Weight Perspective

• Chemical Composition of E. Coli

o Dry Weight

▪ Proteins: 50%

▪ RNA: 20%

▪ DNA: 3%

▪ Carbohydrates: 10%

▪ Lipids: 10%

o Wet Weight

▪ Proteins: 15%

▪ RNA: 6%

▪ DNA: 1%

▪ Carbohydrates: 3%

▪ Lipids: 3%

• Sources of Essential Nutrients

o Carbon Sources

▪ Heterotroph: must obtain Carbon from organic sources  (proteins, carbohydrates, lipids, nucleic acids)  

???? Chemoheterotrophs: Microorganisms that use  organic compounds for both energy and carbon  

(Chemotroph get energy from chemical sources)  

▪ Autotroph: organism that gets carbon through inorganic  sources, make organic compounds from CO2 –

(photosynthetic organisms = photoautotroph)  

???? Chemoautotroph: microorganism that uses CO2  as a source and catabolizes organic molecule for  

energy  

o Nitrogen Sources

▪ Reservoirs

▪ It is a component of air and chitin

▪ Some organisms can use it as Nitrate and other can  make nitrogen through the nitrogen cycle  

o Oxygen Sources

▪ It is a component of the air, carbohydrates, lipids,  nucleic acids, proteins, salts

▪ It can be directly used

???? Anaerobes don’t get oxygen in the same form  (gas) but still use it.  

o Hydrogen Sources

▪ Can get it from water, gases and salts  

▪ Roles of Hydrogen are to maintain pH and is an  acceptor of oxygen during cellular respiration

o Phosphorus/Phosphate Sources

▪ Component of nucleic acids (essential to genetics) ▪ Used in energy transfers (ATP)  

o Sulfur Sources

▪ Found in amino acids and vitamins  

▪ It is widely distributed in the environment

▪ Used in protein stability

o *Other Nutrients that are Important in Microbial Metabolism ▪ Potassium

▪ Sodium

▪ Calcium

▪ Magnesium

▪ Iron

▪ Zinc, copper, nickel, manganese, etc.  

• Classification of Nutritional Types

o Carbon Source

▪ Autotroph: organisms that use inorganic sources of  carbon as their only source of carbon and make organic  compounds from CO2

▪ Heterotroph: catabolize organic molecules they get from  other organisms.

o Energy Source  

▪ Chemotroph: organisms that get energy from inorganic  and organic chemicals through redox reactions  

???? Chemoautotroph: microorganism that use CO2 as  

their carbon source but chemical compounds as  

their energy source  

???? Chemoheterotroph: Microorganisms that use  

organic compounds for the carbon source and  

chemical compounds as their energy source  

▪ Phototroph: organisms that use light as a source of  energy

???? Photoautotroph: use CO2 as source of carbon and  

light as source of energy

???? Photoheterotroph: use organic compounds as  

source of carbon and light as source of energy  

• Transport Mechanisms

o Passive Processes – no energy used  

▪ Simple Diffusion: net movement of chemical down its  concentration gradient, requires no spending of energy  ▪ Facilitated Diffusion/Transport: proteins act as channels  or carriers to transport down the concentration  

gradient, into or out of the cell, no energy spent,  

???? Non-specific channel proteins don’t have a  

preference for what they transport

???? Permeases: channels that carry only specific  

chemicals  

▪ Osmosis: diffusion of water across semipermeable  

membrane, down concentration gradient  

o Active Processes – uses energy, can move against or with the  concentration gradient

▪ Active Transport: use transmembrane permease  

proteins to move molecules across the membrane, has  

to use energy to do so, use gated channels or ports

???? Uniport: one thing moving in one direction

???? Antiport: move two things at the same time but in  

opposite directions

???? Symport: move two things at the same time in  

the same direction  

▪ Group Translocation: The substance transported across  the membrane is also chemically changed during the  

transport  

???? Ex: when take glucose into the cell and it is  

phosphorylated at the same time

• Solution Types

o Isotonic: when solutions on both sides of the semipermeable  membrane have the same concentration of solutes

o Hypertonic: concentration of solutions unequal, the solution  with higher concentration of solutes is called this

o Hypotonic: when concentration of solutions unequal, the  solution with the lower concentration of solutes is called this  Microbial Growth  

*Prokaryotes do not perform phagocytosis, only Eukaryotes do, Prok. send  enzymes out to break down things and then import them via active transport  and facilitated diffusion  

• Population Growth

o Generation Time: the time required for a cell to double

▪ Cells divide by binary fission (form of exponential  

growth, to calculate the number of cells that one cell  

can produce = 2^n, n being the number of generations,

to calculate the total number of cells = multiply number  of original cells by 2^n )

???? Exponential Growth: regular spaced division, very  rapid increase in growth

▪ Bacteria Cells synthesize a new cell wall while  weakening the outside cell wall to form two new cells  from the original.  

o Growth Curves: graph that plots # of organisms in a growing  population over time  

▪ Lag Phase: time period where the cells are adapting to  the new environment, do not reproduce right away,  synthesize enzymes to use nutrients in the medium

▪ Log Phase: phase when cell population increases  logarithmically

▪ Stationary Phase: nutrients become depleted and the  number of dying cells equal the number of cells being  produced, population remains constant  

▪ Death Phase: the number of cell deaths rises about the  number of cells produced

o Diauxic Shift: Discovered by Jacques Monod 1941. different  sugars produce different growth curves, the growth curves  are typically based off of how the bacteria would be grown in  a laboratory setting, with a single carbon source. The true  growth curves would be different than these standard ones  because the organism would have many different carbon  sources in real life.  

o Methods for Analyzing Growth:  

▪ Hemocytometer: sample under a glass slide with an  etched grid on the bottom, the grid is 25 squares and  designed to suspend the microbes in 0.02mm of liquid,  volume of suspension of microbes is 1mm x 1mm x  0.02mm = 0.02mm^3

???? Not good for motile things  

▪ Coulter Counter: as cells pass through triggers an electronic sensor that tallies their numbers

???? Useful to count larger cells (yeast, unicellular  

algae, protozoa), and for bacterial counts because  

of debris in the media.

▪ Flow Cytometer: uses a light sensitive detector, detects  changes in light transmission through the tube as cells  pass, used to tell between differentially stained cells  

that have been stained with fluorescent dyes or tagged  w/fluorescent antibodies

???? Detect and sort different populations of cells from  

blood  

???? Counts bacteria and host cells  

???? Used to look at fungal cells to determine if they  

are haploid or diploid

▪ Pour Plate Technique: step-wise dilution of a culture,  dilution factor the same every time, scientist plates set  amount of dilution onto agar plate and counts the  

number of colonies resulting on a plate.  

▪ Turbidity: cloudiness of the solution, more bacteria the  cloudier the solution

???? Spectophotometer measure the amount of light  

transmitted through a culture under standard  

conditions. Higher concentration of bacteria in a  

broth more light will be absorbed and scattered,  

less passing through and hitting back sensor.  

???? Technique only useful density above 1 million per  

mL.

• Environmental Factors Influencing Growth

o Environment – enzymes adapt to whatever condition it grows  at

▪ Temperature

???? Psychrophile: microorganism requiring cold  

temperatures below 20°C

???? Mesophile: microorganism that requires  

temperatures ranging from 20°C to 40°C

• Optimum Growth (can grow at other  

temperatures but grows best at optimum  

temp) = 37°C

???? Thermophile: grows at temperatures above 45°C • In compost piles and hot springs

▪ Oxygen Requirements

???? Aerobic: organism that uses oxygen as a final  electron acceptor in ETC

• Obligate Aerobe: they are obliged to use  

oxygen, they can’t survive below depth that  oxygen penetrates

???? Microaerophilic: Microorganism that requires low  levels of oxygen

???? Facultative Anaerobe: Can live with/without  oxygen, prefers oxygen  

???? Aerotolerant Anaerobe: microorganisms that  prefer anaerobic conditions but can tolerate  

exposure to low levels of oxygen.

• Expect relatively even growth until it  

reaches its point of oxygen tolerance.  

???? Anaerobe (Strict): An organism that cannot tolerate oxygen.

▪ pH

???? Acidophile: organisms that grow best in acidic  conditions

???? Neutrophile: grow best in a narrow range around  a neutral pH (between 6.5 and 7.5, which is also  the pH range of tissues and organs in the human  body)  

???? Alkalophile: organisms that live in alkaline soils  and water up to 11.5pH

▪ Osmolarity  

???? Halophile: need high salt concentration

???? Osmotolerant: organism can tolerate salt, doesn’t need it to survive  

• Ex: Staphylococcus epidermidis

• Ecological Associations  

o Symbiotic: organism lives in close nutritional relationship, one  or both of the organisms involved require the relationship.  ▪ Mutualism: both organisms involved require the  

relationship and both benefit from it.  

???? Ex: bacteria in cow’s rumen & interaction between  

pea plants and Rhizobium & E.coli

▪ Commensalism: one organism benefits from the  

relationship, the other is not harmed or benefitted.  

???? Ex: Satellitism between Staphylococcus aureus

and Hemophilus influenza

▪ Parasitism: the relationship is not required by both  

organisms, the one that requires it is doing damage to  

the other  

???? The host is always harmed – levels of damage  

may differ

???? Ex: any pathogen that can get you sick  

o Non-Symbiotic: organisms are free-living, the relationships  are not required for survival

▪ Synergism: members benefit from each other but are  

still able to survive on their own  

???? Works in either biosynthesis of a certain end  

product or the breakdown of waste

• Provide extra things to get to the end  

product  

▪ Antagonism: relationships don’t help survival, compete  for nutrition and space  

???? Some members are inhibited or destroyed by  

others  

???? Organisms that produce antibiotics, one microbe  

makes antibiotic and the other is hurt by it

• Led to penicillin  

Metabolism of Microbes

• Metabolism: chemical and physical workings of a cell

o Anabolism: biosynthesis, forms larger molecules, requires  energy input  

o Catabolism: degradative, breaks bonds to form smaller  molecules, releases energy  

o *The intermediates can be used for other reactions at  multiple stages in the reaction  

• Enzymes (biological catalysts, lower energy of activation to increase  the rate of chemical reaction) – in biological reactions  o Energy of Activation: the resistance to a reaction, amount of  energy needed to trigger a chemical rxn

o Enzymes are Not permanently altered in the rxn  

o They promotes rxn by having a physical site for substrates to  bind to  

o Most are made of protein and may have cofactors

▪ Cofactors could be metallic cofactor like iron in  

Hemoglobin or non-metallic cofactors

o They are organic catalysts  

▪ Catalyst: chemicals that increase the likelihood of a rxn  but are not permanently changed in the process.

o Have unique characteristics (shape, function, specificity) that  doesn’t allow them to react with just any type of substrate o Have an active site – where the substrate binds and the  reaction happens

o The enzyme itself is not part of the reaction substrate or  products  

o It is not used up in the reactions or recycled  

o The rxns are often controlled mechanisms  

o Lock and Key Model: shape of the substrate is complementary  to the shape of the active site, describes the enzyme

substrate specificity.  

o Induced-fit Model: lock and key analogy not entirely correct  because the enzyme’s active site changes shape when the  substrate binds, describes enhanced enzyme-substrate  specificity

▪ Shape of the substrate is kind of specific to the active  site but not exact  

o Structure

▪ Apoenzyme: the protein portion of enzyme, enzyme  without its cofactor, is inactive unless bound to one or  more cofactors.  

▪ Holoenzyme (Conjugated): binding of an apoenzyme  and its cofactor that makes an active enzyme.

???? Cofactors: inorganic ions or organic molecules  that are essential for the enzyme action.

• Coenzymes are an example of organic  

cofactors, organic cofactors can also be  

vitamin based

o three important ones are the electron  

carriers (NAD+, NADP+, FAD)

• Metallic Cofactors

▪ Exoenzymes: enzyme that functions outside the cell  that produced it

???? Can secrete things and damage things  

???? Eukaryotes and Prokaryotes can secrete things so  there are proteins that end up outside the cell for  specific reasons  

▪ Endoenzymes: enzyme that functions inside the cell  that produced it

???? Most enzymes are these, intracellular

▪ Constitutive Enzymes: enzymes that are always  present, they are always produced regardless of  

physiological demand because their function in cell  processes is indispensible.

▪ Induced Enzymes: molecules induce or enhance the  production of these enzymes

▪ Synthesis or Condensation Reactions: anabolic rxns,  form covalent bonds between smaller substrate  

molecules, require ATP, release one molecule of water  for each bond formed

▪ Hydrolysis Reactions: Catabolic reactions, breakdown substrates into smaller molecules, need water to break  bonds  

o Environmental Sensitivities – activity influenced by  environment  

▪ Labile: describes some enzymes when they are  chemically unstable  

▪ Increased temperature = Denatured Protein: protein  heated up = bonds are broken. If cooled it won’t go  back to normal because it didn’t start out complete,  they fold as they are made  

???? Functional Protein: needs structure to get  

catalytic site in place  

▪ pH: denatures protein when ions released from acids  and bases interfere w/h-bonding and disrupt secondary  and tertiary structure

▪ Enzyme/Substrate Concentration: As concentration of  substrate increases the enzyme activity increases until  all active sites are bound = saturation point, more  substrate will not increase rate of activity

o Regulation of Enzymes  

▪ Competitive Inhibition: inhibitory molecules, similar in  shape to the substrate molecules, compete for & block  the active sites. This can be permanent or reversible, if  it is reversible it can be overcome by increasing the  concentration of substrate = increasing the likelihood  that substrate will bind before inhibitor.  

▪ Noncompetitive Inhibition: Allosteric, regulatory  inhibition  

???? Allosteric Inhibition: The regulatory site is  

separate from the active site, when the inhibitor  binds it distorts the active site and doesn’t allow  the substrate to bind  

???? Allosteric Activation: the activator reverses this  ▪ Feedback Control: when end product of a series of  reactions is an allosteric inhibitor of an enzyme in an

earlier part of the pathway, prevents synthesis of more  of this end product.  

???? There is enough product already made, signals  that they don’t need anymore

▪ Control of Gene Synthesis – this is a slower response ???? Enzyme Repression: controls the synthesis of key  enzymes and inhibits at the genetic level

???? Enzyme Induction: enzymes are made only when  the right substrates are present  

o Pursuit and Utilization of Energy  

▪ Energy: the capacity of a system to do work

???? Forms of energy: Thermal, radiant, electrical,  

mechanical, atomic, chemical  

▪ Exergonic: get energy out  

???? X + Y ????(Enzyme)???? Z + Energy  

▪ Endergonic: not going to happen unless you put energy  in

???? Energy + A + B ????(Enzyme)???? C  

▪ ATP is the energy currency in the Cell  

???? 3 part molecule

• Adenine – nitrogenous base

• Ribose – 5-carbon sugar

• 3 phosphate groups attached  

???? using and replenishing of ATP is constantly  

happening in the cell

???? Remove terminal phosphate = energy released  ???? Formation of ATP through three different  

mechanisms:  

• Substrate Level Phosphorylation: transfer  

phosphate from phosphorylated compound  

(substrate) directly to ADP

• Oxidative Phosphorylation: series of redox  

reactions occur during respiratory pathway  

• Photophosphorylation: ATP is made by using  

the energy of sunlight

o Biological Oxidation and Reduction

▪ Redox Reactions: reduction/oxidation reactions, always  in pairs (electron donor and acceptor involved), the  

process saves electrons and their energy, the energy  

that is released can be used to phosphorylate ADP or  

another compound

???? Oxidation: rxn where electrons are donated (often  in the form of Hydrogen)  

???? Reduction: rxn where electrons are gained

???? (OIL RIG = oxidation is loss & reduction is gain)

???? Reduction Potential: measure of tendency of a  

chemical species to acquire electrons therefore  

being reduced (measured in Volts)  

▪ Electron and Proton Carriers: repeatedly accept and  release electrons and hydrogen to help transfer redox  energy  

???? These are Coenzymes (NAD+, NADP, FAD,  

coenzyme A, and compounds of respiratory chain)  

• Aerobic Respiration: series of enzyme-catalyzed reactions, electrons  are passed from fuel molecules to the last electron acceptor,  oxygen.

o Glycolysis: glucose is split and produces two molecules of  pyruvic acid, while at the same time producing small amounts  of ATP, NADH is also generated  

▪ Step 1: Glucose is phosphorylated by ATP into glucose 6-phosphate (G6P)

▪ Step 2: G6P is rearranged and another phosphate is  added to make Fructose-1,6-bisphosphate (F1,6P)

▪ Step 3: F-1,6-P is divided into DHAP (which is  

converted to G3P) and G3P

▪ Step 4: An inorganic phosphate is added to the two G3P  molecules to make two molecules of 1,3BPG and a  

NAD+ is reduced to NADH (two times)

▪ Step 5: (two molecules so this step happens two times)  ADP steals a phosphate from the 1,3BPG and that  

becomes 3PG

▪ Step 6: 3PG is rearranged to become 2PG

▪ Step 7: Water molecule released when 2PG is converted  to Phospho-enolpyruvic acid (happens 2 times for the  two molecules)  

▪ Step 8: ADP steal the phosphate off the P-enolpyruvic  acid and make pyruvate, and ATP (rxn happens 2x for 2  molecules)  

▪ NET TOTAL: 2 ATP and 2 NADH

▪ ALTERNATE PATHWAYS (Instead of Glycolysis)

???? Pentose-Phosphate Pathway

• The enzymatic formation of phosphorylated  

pentose sugars from glucose-6-phosphate

• alternate way to breakdown glucose

• yields NADPH instead of NADH

• has different intermediates such as Ribose

5-phosphate which feeds into the synthesis  

of nucleotides  

• Yields less ATP than glycolysis

???? Entner-Dourdoroff Pathway

• Produces NADPH instead of NADH

• Yields half ATP than glycolysis (1 ATP)

• Pseudomonads use this pathway to  

catabolize glucose

o Synthesis of Acetyl-CoA: Decarboxylation, enzymes remove a  carbon from the pyruvic acid as CO2. Enzyme then joins the  remaining two-carbon molecule (acetate) to coenzyme A to  make acetyl-CoA and also produced a molecule of NADH (From NAD+).

▪ Because glycolysis made 2 molecules of pyruvic acid  this is done 2 times 

o TCA/Citric Acid Cycle/Krebs Cycle: processes pyruvic acid and  generates 3 CO2 molecules , NADH and FADH2 are generated ▪ Occurs in the cytosol of prokaryotes and matrix of  mitochondria. in eukaryotes. Cells also use for  

catabolism of lipids and proteins

▪ Step 1: oxaloacetate combines with acetyl-CoA

▪ Step 2: citrate is converted to isocitrate so the right  isomer is in place

▪ Step 3: isocitrate is converted to α-ketoglutarate  (NAD+ is converted to NADH and a CO2 molecule is  released)

???? *Isocitrate can also take the Glyoxylate

???? Bypass which produces 2 carbon compounds,  without this there would not be enough carbon  

left to survive at the end of the cycle  

▪ Step 4: α-ketoglutarate interacts with CoA to form  succinyl-CoA (NAD+ and CoA are used to form Succinyl CoA and NADH and CO2 are released)

▪ Step 5: succinyl-CoA releases CoA to form succinate  (two molecules of acetyl-CoA pass through the Krebs  cycle from every one glucose molecules creating two  pyruvates, from these two molecules two molecules  of ATP are produced by substrate level  

phosphorylation, GTP is an intermediary at this step) ▪ Step 6: Succinate is converted to fumarate (FAD is  reduced to FADH2)

▪ Step 7: fumarate gains water to generate malic acid ▪ Step 8: malic acid is converted to oxaloactetate (NAD  +is converted to NADH)

▪ SUMMARY: overall the two molecules of acetyl-CoA that  are gained from the original glucose molecule, end up  forming: 6 NADH, 2 FADH2, 2 ATP

???? Glycolysis and TCA created ATP, Carbon  

intermediates to make other molecules, reduced  

electron carriers (NADH, FADH)

o Electron Transport Chain: Final processing of electrons and  hydrogen and the major generator of ATP  

▪ occurs in the cytoplasmic membranes of prokaryotes  and the inner mitochondrial membranes of eukaryotes ▪ Accepts electrons from NADH and FADH2, this  generates energy through redox reactions and this

energy is captured by ATP synthase to produce ATP =  oxidative phosphorylation

???? Oxidative Phosphorylation: protons flow down  their electrochemical gradient through protein  channels called ATP synthases and these channels  phosphorylate molecules of ADP to ATP.

• Specifically, Hydrogen ions diffuse back  

through the ATP synthase complex causing  

it to rotate, causing a 3-dimensional change  

resulting in the production of ATP  

o Form of Chemiosmosis:

▪ as the electron transport  

carriers shuttle electrons, they  

actively pump hydrogen ions  

(protons) across the membrane  

setting up a gradient of  

hydrogen ions – Proton Motive  

Force

▪ Membrane Set Up – membrane includes  

???? flavoproteins: integral proteins, FMN is an  example of these and is the first carrier molecule  of the transport chain that NADH passes electrons  to.  

???? Ubiquinones are lipid-soluble nonprotein carriers  found in the membrane, Coenzyme Q is an  

example here.

???? Metal containing proteins are a mix of integral  proteins. Cytochromes are integral proteins  

associated with hemes  

▪ Bacterial Process – involves 7 enzymes that rapidly  undergo redox reactions  

???? 1. Complex I – NADH dehydrogenase/FMN ???? 2. Complex II  

???? 3. Coenzyme Q

???? 4. Complex III

???? 5. Cytochrome C

???? 6. Complex IV – cytochrome a/a3 and ATP  

Synthase  

???? The final step: Oxygen accepts 2 electrons from  

ETS and forms water with 2 hydrogen ions from  

the solution. Oxygen is the final electron acceptor  

???? OUTPUT:

• From the 2 NADH from Glycolysis through  

the ETS = 6 ATPs

• From the 6 NADH from the TCA through the  

ETS = 18 ATPs

• From the 2 FADH2 from the TCA through  

the ETS = 4 ATPs

• TOTAL from ETS: 28 ATP

o Complete aerobic oxidation of one molecule of glucose  by a prokaryote is 38 molecules of ATP. Eukaryotes have  to use 2 ATP to get NADH from the cytoplasm to the  

mitochondria so their net ATP is 36.

▪ 10 molecules of NADH from glycolysis, synthesis  of acetyl-coA, and TCA = 30 ATP

▪ 2 molecules of FADH2 from TCA = 4 ATP

▪ 2 ATP netted from glycolysis

▪ 2 ATP from TCA

• Anaerobic Respiration: use inorganic chemicals (oxygen containing  ions) rather than oxygen as the final electron acceptor in the ETC  o Examples of what is used as the final electron acceptor =  Nitrate (NO3-), Nitrite (NO2-), Sulfate, Methanogens may use  CO2 and end up with methane, some use methyl  

o Most obligate anaerobes use H+ made during glycolysis and  Krebs cycle to reduce the compound  

• Fermentation: partial oxidation of sugars (glucose or other  carbohydrates when there is no oxygen present) to release energy o Final electron acceptors in these pathways are organic  molecules

o These yield a small amount of ATP

o Examples:

▪ Ethanol Pathway: Decarboxylation of pyruvic acid  

produces Acetaldehyde, with oxidation of NADH to  

NAD+ ethanol is produced.

???? Yeasts ferment glucose to produce ethanol (then  

complete aerobic respiration)

▪ Lactic Acid Pathway: NADH reduces pyruvic acid to  create lactic acid

???? Various bacteria acting on pyruvic acid forms acid,  gas and other products

▪ Both pathways recycle the electron carrier NAD+  to be used again in Glycolysis to produce more  

energy, benefit to fermentation is ATP production  without a final electron acceptor or cellular  

respiration.

• Amphibolic Pathways: pathways can be catabolic and anabolic o Catabolic pathways can have molecular intermediates that are  diverted into anabolic pathways  

▪ Pyruvic Acid converted into amino acids through  

amination

▪ Amino acids converted into energy sources through  deamination

???? Polypeptides can be broken down into their  

component amino acids by Proteases, the amino  

acids can then be deaminated to create an  

intermediate that feeds into the Krebs Cycle

???? Reactions that make and convert amino acids

• Amination: add a free amine to a molecule  

to produce an amino acid  

o Ex: NH2 + Oxaloacetic Acid ????

Aspartic Acid/Aspartate

• Transamination: switch an amine group  

from one molecule to another

o Ex: Glutamic Acid + Oxaloacetic Acid  

???? α-ketoglutaric acid + Aspartic  

Acid/Aspartate

▪ Glyceraldehyde-3-phosphate converted into precursors  for amino acids, carbohydrates, and fats  

???? Side note: energy that comes from 6 carbon fatty  

acid is about 50 ATP while energy that comes  

from 6 carbon sugar is about 38 ATP

Microbial Genetics  

• Genome: all of the genetic material in a cell

o Made up of

▪ Chromosomes: A molecule of DNA that is associated  

with proteins  

???? Prokaryotes: circular and in the nucleoid

???? Eukaryotes: threadlike (chromatin structure,  

when packed it is wrapped around histones)  

???? Genes: divisions of the chromosome, the unit of  

heredity responsible for a given trait, are a  

specific sequence of nucleotides that code for RNA  

or polypeptide molecules  

• Three Categories

o Structural Genes: genes that code for  

proteins

o Regulatory Genes: genes that control  

gene expression

o RNA Genes: Genes that code for RNA  

• Genotype: the actual set of genes in an  

organisms genotype

• Phenotype: the physical features &  

functional traits of an organism

▪ Plasmids: small circular molecule of DNA, replicates on  its own with its own genes, has genes for nonessential  

functions like resistance to antibiotics  

▪ Mitochondria & Chloroplasts: eukaryotes have  

extranuclear DNA in these & plasmids  

o Prokaryotic Genome Structure: haploid, chromosomal DNA,  circular, all extrachromosomal DNA in the form of plasmids.

o Eukaryotic Genome Structure: nuclear chromosomal DNA,  one or more linear chromosomes, plus all extranuclear DNA in  mito, chloroplasts, and plasmids

o The genome is a thousand times longer than the cell and  must package the DNA efficiently  

• DNA: composed of two strands twisted into a double helix, the basic  unit is a nucleotide

o Nucleotide has three parts:

▪ 5 carbon sugar - deoxyribose

▪ Phosphate Group

▪ Nitrogenous Base – adenine, guanine, thymine, cytosine o The backbone: the sugar-phosphate linkages

▪ Each sugar attaches to a phosphate at its 5’ carbon and  at its 3’ carbon

o The Nitrogenous Bases hydrogen bond with their  

complements in the center

▪ Adenine bonds to Thymine with 2 hydrogen bonds, a  section of DNA that is AT rich is weaker and easier to  

separate

▪ Guanine bonds to Cytosine with 3 hydrogen bonds, a  section that is GC rick is stronger and harder to  

separate  

o As the helix winds it creates a Minor and Major Groove ▪ Minor Groove: information poor

???? Here the proteins can only detect what the base is

▪ Major Groove: information rich  

???? Here proteins interact and based on their ability  

to form bases can tell the difference between A or  

T and G or C, they can also methylate the strand  

and use base flips to find a specific sequence  

without ever having to separate the strands.  

▪ In these grooves there are h-bond donators and h-bond  acceptors they are labeled as D and A respectively.

o Watson and Crick – 1953: These are the two scientists given  credit for discovering the structure of DNA, however, they

would never have gotten it right if it weren’t for Roslin  Franklin’s crystallography data.  

o Significance of this structure:

▪ Code must be maintained during reproduction, this  constancy of base pairing makes sure the code will be  kept (if don’t have constancy then we have mutation)  ▪ Order of base pairs provides variety which is  

responsible for the unique qualities of organisms  

• DNA Replication – takes 30 different enzymes to make exact  duplicate

o Begins at Origin of Replication – this is usually an AT rich  region because of the weaker hydrogen bonding  

▪ Prokaryotes: one origin

▪ Eukaryotes: multiple sites present  

o Helicase unwinds & unzips the DNA by breaking hydrogen  bonds between complementary bases – creates Replication  Forks and Replication Bubble  

▪ Theta Structure: when circular DNA is being replicated  the fork is opening/replicating in both directions until  meeting and forming two new DNA Molecules

o Stabilizing proteins (ssb protein) keep strands from coming  back together

o At origin of replication the enzyme RNA Primase makes a  short RNA sequence that is complementary to the DNA strand  being copied – Primer – provides hydroxyl group for DNA  Polymerase III to add nucleotides in the 5’ to 3’ direction (DNA Polymerase III also proofreads – nuclease activity - as  it adds bases and can back up to delete incorrect ones and  replaces with correct)  

▪ Leading Strand: DNA is replicated towards the  

replication fork, 5’ to 3’ continuously starting from one  primer

▪ Lagging Strand: DNA is replicated away from the  

replication fork, synthesized in short 5’ to 3’ segments  with many primers (overall the direction is 3’ to 5’)

???? Okazaki Fragments: fragments of replicated DNA,  

must be replicated this way on the lagging strand  

to maintain 5’ to 3’ direction  

o DNA Polymerase I: removes RNA primers and replaces with  DNA segments  

o DNA Ligase: link the DNA fragments of the lagging strand to  complete the synthesis  

o The two strands of the original DNA molecule have now  separated into two daughter strands each with one original  parent strand and one new strand – Semiconservative  

• From DNA to Protein – Transcription (DNA into RNA) and  Translation (RNA into Polypeptides/Proteins)  

o Rules for the Standard Code:  

▪ Redundant but not ambiguous: Many codons can code  for one amino acid, but each codon only codes for one  amino acid.  

▪ Code is Universal

o RNA – single stranded molecule made of nucleotides (instead  of deoxyribose the 5 carbon sugar is Ribose, and uracil  replaces thymine)

▪ mRNA: molecules which carry genetic information from  chromosomes to ribosomes as codons (mRNA contains  nucleotides and a set of three nucleotides makes up one  Codon)

???? In Prokaryotes:

• mRNA carries start codon, codons for other  

amino acids in a polypeptide, and one of 3  

stop codons

???? In Eukaryotes:  

• The mRNA is first called pre-mRNA because  

it has to be processed first  

• Contains instructions for only one  

polypeptide

▪ tRNA: molecules that deliver the right amino acids to the ribosomes based on the sequence of nucleotides in  the mRNA.

???? Anticodon: bottom loop of the tRNA that is  

complementary to the codon in the mRNA

???? Acceptor Stem: holds the specific amino acid  

designated by the anticodon

???? tRNA can recognize more than one codon because  of Wobble – a change in the angle of the molecule  can cause the third nucleotide to bond with  

another besides its normal complement

▪ rRNA: part of ribosomes where protein synthesis occurs o Transcription: Information stored in DNA transcribed into  mRNA  

▪ Initiation: 

???? RNA Polymerase binds to promoter region  

???? RNA Polymerase made of 3 subunits: Alpha, Beta,  Beta prime  

• In Prokaryotes: RNA binds to the promoter  

with the help of RNA subunit called Sigma  

Factors

o Sigma factors bind to the promoter so  

RNA polymerase can recognize it  

easier, how tightly the sigma factor  

binds varies, this can lead to variation  

in amount and kind of polypeptide  

formed.  

• In Eukaryotes:  

o Many Transcription Factors (TFIIH,  

TFIID) are used to bind RNA  

Polymerase to the promoter

o Have 3 types of RNA Polymerase (kind  

to transcribe mRNA, rRNA, tRNA and  

other small rRNA)  

▪ Elongation: 

???? RNA Polymerase adds nucleotides complementary  to the template strand of DNA in 5’ to 3’ direction

???? Uracil placed as adenine’s complement

???? Only one DNA strand is transcribed

▪ Termination: 

???? RNA polymerase recognizes signals and releases  the transcript (100 – 1,200 bases long)  

???? In Prokaryotes:

• Self-Termination: RNA polymerase reaches  

terminator sequence (rich in GC followed by  

rich in AU) , when reaches GC portion

transcription slows and gives time for the  

RNA transcript to bind together and form a  

hairpin, this causes tension that the AU  

portion of the terminator cannot withstand  

and the transcript breaks off.  

• Rho-dependent Termination: Rho protein  

binds to end of RNA transcript and works its  

way towards the growing 3’ end, the Rho  

protein wedges in between polymerase and  

DNA strand and forces them apart,  

releasing the transcript, the Rho protein and  

the RNA Polymerase.

???? In Eukaryotes:

• After termination and before Translation the  

pre-mRNA must be processed into  

mRNA(helps export the RNA from the  

nucleus, stabilizes the RNA in cytoplasm,  

and aids in translation) – Eukaryotic  

transcript more stable than prokaryotic  

o Capping: adds Guanine nucleotides to  

the 5’ end of mRNA

o Polyadenylation: after termination,

proteins add 100-250 adenine  

nucleotides to the 3’ end  

o Splicing: spliceosome acts as  

ribozyme and removes introns,  

splicing together exons  

o Translation: mRNA is used by ribosomes to translate the  genetic code into polypeptides

▪ Initiation: Initiation complex forms (2 ribosomal  subunits, mRNA, several protein factors, the initiator  tRNA)

???? In Prokaryotes:  

• Might happen while cell is still transcribing  RNA from DNA  

• Steps:

o Smaller ribosomal subunit attaches to  

mRNA at ribosome-binding site near  

start codon (AUG)  

o Anticodon of initiator tRNA aligns  

w/start codon on mRNA, bound in  

place w/energy from GTP

o Larger subunit attaches to form  

complete initiation complex

???? In Eukaryotes:

• Same process but initiation happens when  small ribosomal subunit binds to the 5’ cap  

not a specific nucleotide sequence

▪ Elongation:  

???? tRNAs deliver amino acids to the A site

(accommodates a tRNA delivering an amino acid)  as directed by the codons of the mRNA  

???? rRNA (enzymatic RNA molecule – ribozyme) in the  large subunit makes a peptide bond between  amino acid at A site & growing polypeptide in P  site (holds tRNA and growing polypeptide)

???? the ribosome moves ahead one codon & the tRNA  that was in the P site exits through the E site,  leaving the polypeptide attached to the tRNA that  was in the A site but is now in the P site, the A  site is now open for the next amino acid to be  delivered  

???? Elongation Factors escort the tRNA along with a  GTP molecule

???? When the empty tRNA is released back into the  cytosol an enzyme recharges it with another  

molecule

???? As elongation goes on and the ribosome passes  over the start codon, it is exposed for other  

ribosomes to attach and begin translating  

identical polypeptides (a group of these ribosomes  = polyribosomal complex)

▪ Termination:  

???? Termination factors recognize the stop codon

(UAA, UAG, UGA codons that don’t have a tRNA)  

and modify the large subunit to activate another  

ribozyme that cuts the polypeptide from the final  tRNA – ribosome dissociates

???? The polypeptides released can function alone as  proteins or w/others in quaternary proteins  

???? *Eukaryotic Note: ribosomes attached to  

endoplasmic reticulum can synthesize  

polypeptides into the cavity of the RER

▪ Protein Folding  

▪ Processing

o Regulation of Protein Synthesis

▪ Rules of Genetic Regulation

???? If the cell doesn’t need it, turn it off

???? If the cell needs it, get an enzyme there as quick  as possible

???? The rate of synthesis can be controlled w/DNA  sequence

???? Enzymes needed only under certain conditions

???? In prokaryotes regulation pathways can be  

controlled as operons  

▪ Operons: set of genes that are regulated as a single  unit

???? made up of promoter, set of genes that code for  enzymes and structures, & an operator

???? Inducible Operons: (Enzyme Induction) operon  not normally transcribed must be turned on by  inducers

• Ex: Lac Operon – normally off

o Made up of 3 segments  

▪ Regulator: gene that codes for  

repressor

▪ Control Locus: contains  

promoter & operator

▪ Structural Locus: 3 genes that  

each code for an enzyme that  

catabolizes lactose  

???? β-galactosidase:

hydrolyzes lactose

???? Permease: brings lactose  

across the cell membrane

???? β-galactosidase  

transacetylase: uncertain  

fxn

o Activated by:

▪ 1. positive regulation by protein  

called CAP

???? When glucose is low 

increases [cAMP] which  

binds to CAP (catabolite  

activator protein), CAP  

binds to CAP-binding  

sites, RNA polymerase  

more attracted to the lac  

operon promoter =  

increased transcription of  

lactose catabolizing genes

▪ 2. deactivation of repressor  

molecule  

???? When lactose is present 

and glucose is absent,

allolactase acts as the  

inducer, binds to the  

repressor, changes its  

shape and causes it to fall  

off the operator so RNA  

polymerase can bind to  

the promoter & lactose  

catabolism genes can be  

transcribed  

???? Absence of lactose =  

repressor binds the  

operator locus and blocks  

RNA Polymerase  

transcription of lactose  

catabolism genes

o Lac Operon inactivation/activation a  

form of Catabolite Repression

(inducible operons involved in  

controlling catabolic pathways whose  

polypeptides are not needed unless a  

particular nutrient is available)

▪ Following is repetitive:

▪ Low lactose & High glucose =  

low cAMP = few CAP-cAMP  

complexes = inactivation of lac  

operon

▪ High lactose & low glucose =  

high cAMP = many CAP-cAMP  

complexes = activation of lac  

operon

???? Repressible Operons: operons that are continually  transcribed unless repressor deactivates it

Unit 3 4/18/17 11:50 AM

Repressible Operon: Operon that is always active unless repressed • Tryptophan Operon

o No Tryptophan = inactive repressor and transcription of  

operon continues

o Tryptophan present = tryptophan binds to the repressor and  activates it, causing it to bind to the operon and block  

transcription

o Inside the RNA transcript there is a leader region with two  

Tryptophan codons that will need to be translated before  

structural genes are translated, keeping this in mind…

▪ If Tryptophan is LOW there isn’t many tRNA charged  

with Trp so as transcription moves translation/the  

ribosome is close behind but when it gets to the leader  

region with 2 Trp codons it stalls because there is not a  

lot of charged tRNA. The RNA polymerase/transcription  

gets ahead and before the ribosome can catch up to  

translate it the mRNA #2 segment and the #3 segment  

bind together. This loop is far enough away from where  

transcription is happening that it doesn’t cause tension  

and transcription can continue.  

???? Trp Low = 2-3 bind = Transcription continue

▪ If Tryptophan is HIGH transcription is proceeding and  

translation/ribosome is close behind, there is enough  

tryptophan charged tRNA that the ribosome gets  

through the Trp part of the leader region fine and  

instead of 2-3 binding segment #3 and #4 bind  

together. This creates a termination loop close to where  

transcription is occurring that causes tension on the  

transcript and halts transcription.

???? Trp High = 3-4 bind = Transcription stops  

Mutations: Change in phenotype due to change in genotype (nucleotide  base sequence)  

• Wild-type: natural, non-mutated characteristic

• Mutant: organism with a mutation, its morphology, nutritional  characteristics, genetic control mechanisms, resistance to chemicals  and more vary from the norm.  

• Causes of Mutations

o Spontaneous Mutations: Random change in the DNA  sequence that happens without a known cause  

o Induced Mutations: change in the DNA sequence that is  caused by exposure to known mutagens, either physical or  chemical agents (Chemicals, nitrogen base analogs, radiation)  • Categories of Mutations  

o Substitution Mutations

▪ Missense Mutation: change in a base pair that results in  a codon that codes for a different amino acid in  

translation  

▪ Nonsense Mutation: change in a base pair that results  in a stop codon and early termination of the polypeptide  during translation

▪ Silent Mutation: change in a base pair that changes the  codon but still codes for the same amino acid due to the  redundancy of the genetic code  

o Back Mutation: Reversion, when a mutated gene reverses  back to its original base composition

o Frameshift Mutation: insertion or deletion of a single base  that causes the nucleotides after the mutation to shift  (reading frame of mRNA shifts) resulting in change in codons  and different polypeptides  

▪ It is a given that stop codons will be produced  

downstream in this form of mutation, this happens  

whether the bases are added or deleted

• Mutation Repair Mechanisms

o Light Repair: When a thymine dimer (thymines that are right  next to each other bind together) is created it causes a  mutation in the strand when the strands have to replicate, in  light repair the enzyme photolyase gets energy from the sun  and breaks the bonds of the dimers to reverse the strand to  its normal DNA sequence.

o Base-excision Repair: enzymes (Glycosylases) remove a  section of the DNA that has an error in it and DNA polymerase  I and ligase fills in the gap  

o Nucleotide Excision Repair/Dark Repair: enzymes cut out a  short section of the DNA strand (about 12 nucleotides) that  contains a dimer and DNA polymerase I and DNA ligase repair  the gap with help of the undamaged complementary strand  

o Mismatch Repair: enzymes scan newly synthesized DNA  strands (they can tell which ones are new and which are old  because the new are non-methylated for a short period of  time) for mismatched bases and remove and replace them

o SOS Response: Prokaryotic cells with a lot of DNA damage  use a variety of processes to induce DNA polymerase to  

replicate the damaged strand, replication of this strand may  introduce new and possibly fatal mutations but it does allow a  few offspring to survive.  

*THIS IS THE BEGINNING OF MATERIAL FOR EXAM 3*

• Effects of Mutations

o Cause nonfunctional proteins = could be harmful and even  fatal

o Beneficial mutations = organisms can quickly adapt, survive  and reproduce (these mutations cause change in populations) ▪ Any change that is advantageous during selection  

pressure will be kept by the population

• Genetic Recombination and Transfer

o Genetic Recombination: when an organism gets & expresses  genes from another organism, is an exchange of nucleotide  sequences between 2 DNA molecules, usually involves  

segments that are identical or nearly identical  

▪ Three forms of Genetic Recombination in Bacteria:

???? Conjugation: transfer of a plasmid from donor cell  

(gram-negative cell donor is a cell with a fertility  

plasmid/F+ cell) to recipient cell (cell without a  

fertility plasmid/F- cell) with a direct connection

(pilus – tube where DNA passes through). Once  the F- cell gets the plasmid from the F+ cell it  becomes a F+ cell too.  

• In cells where the plasmid transferred  doesn’t stay in the cytosol but instead is  incorporated into the cellular chromosome  = High Frequency Recombination Cells (HFR  cells)

o In these cells when they perform  

conjugation a portion of the  

chromosome and a portion of the  

fertility plasmid are transferred to the  

recipient.  

???? Transformation: organism takes up DNA from its  environment, DNA fragments from lysed cells are  accepted by the recipient cell and the genetic  code is acquired by the recipient

• Donor and recipient cells can be unrelated,  is a useful tool in recombinant DNA  

technology  

???? Transduction: when a bacteriophage injects its  DNA into the host cell & degrades the host cells  DNA, when creating new phages if one takes up  part of the host cells DNA instead of its own it is  called a transducing phage and when it injects its  DNA into a new cell the old host’s DNA is  incorporated into the new cells DNA.

• Two Types:

o Generalized Transduction: random  

fragments of host DNA are picked up  

during phage assembly, any gene can  

be transmitted this way  

o Specialized Transduction: A specific  

piece of the host genome is  

incorporated into the virus

▪ Here the phage goes into  

lysogeny & is incorporated into  

the bacteria cell’s genome,  

something happens to make it  

reenter the lytic cycle. The  

excised phage DNA contains  

some host DNA and when new  

phages are produced it has  

some bacterial DNA. All phages  

can function as phages and  

when a new recipient cell is  

infected the bacterial DNA is  

transferred. The DNA (with  

bacterial DNA and phage DNA)  

can enter and do lysogeny or  

just the bacterial DNA can be  

incorporated into the cells  

genome.  

o Transposition: Mutation in which a genetic segment is  

transferred from one location in the genome to another via a  Transposon. This is transfer of genetic information within one  cell.

▪ Transposons are the segments of DNA that are able to  move from one location to another in the genome.  

“Jumping Genes”  

???? Causes rearrangement of the genetic material  

(like a frameshift mutation)  

???? Can occur between plasmids and chromosomes  

and within and among chromosomes  

???? Can be beneficial or harmful, is a way to get  

variation in genomes

???? They go to another location but also stay in their  

original location  

Controlling Microorganisms

• Targets are microorganisms that can infect or cause spoilage. Ex:

o Vegetative bacterial cells & endospores

o Fungal hyphae and spores, yeast  

o Protozoan trophozoites & cysts

o Worms

o Viruses

o Prions

• Ranking in terms of Most Resistant to Least Resistant to  chemical controls  

o Prions – part of microbes, pathogenic proteins that are  tolerant to steam

o Bacterial endospores

o Mycobacteria

o Cysts of Protozoa

o Active-stage protozoa (trophozoites)

o Most Gram-negative bacteria – have outer membrane as well  as the peptidoglycan layer  

o Fungi

o Non-enveloped viruses

o Most gram-positive bacteria

o Enveloped viruses – capsid is not as rigid because of  membrane, if it were more tightly packed it would be more  resistant  

• Terminology and Methods of Control

o Sterilization: process that destroys ALL viable microbes, this  includes viruses and endospores

o Disinfection: process that destroys vegetative pathogens but  not endospores (this is used on inanimate objects, not the  body)  

o Antiseptic: disinfectants that are applied directly to the body  o Sanitization: cleansing techniques that mechanically remove  microbes, this destroys some things  

o Degermation: use mechanical means to reduce the number of  microbes, example would be hand washing with soap and  water

• Microbial Death – how do we tell a living microbe from a dead one?

o Difficult to detect this because microbes don’t reveal vital  signs to begin with, can tell w/membrane functions  sometimes

o One way to tell a microbe is dead is if it has permanent loss  of reproductive capability, even when in optimum growth  conditions  

o Suffixes:

▪ -cide = the death of something  

▪ -static = bacteria are not growing and are also not  dying, there is no change.  

o Factors that Affect Death Rate (how effective an agent is,  is determined by several factors)

▪ Number of Microbes

▪ Nature of Microbes in the population – ex. spores are  harder to kill, enveloped viruses are easier to kill than  naked  

▪ Temperature and pH of the environment - warm  disinfectants work better than cool ones (chemicals  react faster at higher temperatures), acidic conditions  enhance the antimicrobial effect of heat, some  

household disinfectant are more effective at a lower pH  ???? This affects the denaturing of proteins

▪ Concentration or dosage of the agent

▪ Mode of action of the agent – cellular targets of physical  and chemical agents  

???? The Cell Wall: wall becomes fragile and cell lyses,  done by some antimicrobial drugs, detergents, &

alcohol.  

???? The Cell Membrane: membrane loses integrity by  influencing the stability of the phospholipid  

bilayer, detergent surfactants do this.  

???? Protein and Nucleic Acid Synthesis: prevent  

replication, transcription, translation, formation of  peptide bonds, and protein synthesis. This is done  by chloramphenicol, ultraviolet radiation, and  

formaldehyde.

???? Disruption or Denaturing of Proteins: Done by  alcohols, phenols, acids, and heat.  

▪ Presence of solvents, organic matter, or inhibitors o Decimal Reduction: the time it takes to kill 90% of a  population, if start with a big number even a 90% reduction  would still leave a significant amount.  

o Methods of Physical Control  

▪ Heat: denatures proteins, interferes with the integrity o  of the membrane and walls, disrupts of the function and  structure of Nucleic Acids.  

???? Moist: Commonly used to disinfect, sanitize,  

sterilize, pasteurize, kills cells by denaturing  

proteins & destroying cytoplasmic membranes.  

More effective than dry heat because water  

conducts heat better than air.

• Boiling: kills vegetative cells of bacteria &  

fungi, trophozoites of protozoa & most  

viruses within 10 min.  

o Boil at 100°C for 30 minutes to  

destroy non-spore forming pathogens  

• Pasteurization: use heat to kill pathogens &  

reduce number of spoilage microorganisms  

in food & drinks  

o A way to control bacterial population  

so yeast is the dominating population  

without damaging the grapes

o 63°-66°C for 30min – Batch method

that kills a lot of microbes (old  

method)

o 71.6°C for 15 seconds – Flash Method

(New method, controls a lot of  

microbes)

o Not Sterilization – shelf stable milk – 

hit with high temperatures and 

changes flavor

• Autoclaving (like a pressure cooker):

sterilize chemicals & things that can tolerate  moist heat, prevents the escape of steam increasing the pressure, applying pressure  sterilizes because the temperature that  

water boils at increases as pressure  

increases. Destroys all microorganisms in  15 min.  

???? Dry: used for powders and oils, things that can’t  be sterilized with steam or boiling, Denatures  proteins, fosters oxidation of metabolic & structural chemicals. Requires higher temps for  longer times than moist heat because dry heat penetrates slower.  

• Dry Oven: takes longer than an autoclave  (15 min. vs. 16 hours), 150°-180°C  

coagulates proteins

• Incineration: fosters oxidation, alters  protein structure, ultimate means of  

sterilization, ex. Heating a loop in a Bunsen  burner

???? Thermal Death Measurements

• Thermal Death Time (TDT): shortest length  of time to kill all microbes at a certain  

temperature

• Thermal Death Point (TDP): lowest  

temperature to sterilize something within 10  minutes

• Thermal Death Times of Endospores  

o Moist Heat (121°C):  

▪ Bacillus subtilis – 1 min

▪ Clostridium botulinum – 10 min

o Dry Heat (121°C and 120°C)

▪ Bacillus subtilis – 120 min

▪ Clostridium botulinum – 120  

min

▪ Cold Temperatures: slows microbial growth ???? Refrigeration 0-15°C and freezing below 0°C – preserves microbes, used to preserve food, media  and cultures

???? Microbiostatic: slows the growth of microbes ▪ Desiccation: a way to preserve microbes, a gradual  removal of water from cells which leads to metabolic  inhibition – the cells can’t do anything but they are not  dead, preserve them by freeze drying,  

???? Can mail them this way & they don’t grow while  like this.  

???? Not an effective method of microbial control  because they can grow again once water is  

reintroduced.  

???? Lyophilization: freeze drying; preservation  ▪ Radiation: release of high-speed subatomic particles or  waves of electromagnetic energy from atoms ???? Ionizing: electromagnetic radiation  

w/wavelengths shorter than 1nm are ionizing  because when they hit molecule have enough  energy to eject electrons from atoms create ions  • A form of cold sterilization

• This disrupts h-bonding, oxidizes double  

covalent bonds, create highly reactive  

hydroxyl radicals.

• Ions denature other molecules and cause  fatal mutations & cell death.

• Ex: cathode rays, gamma rays, some x-rays • Used to sterilize medical supplies and food  products that can’t take heat (like plastics)  

???? Non-ionizing: electromagnetic radiation  

w/wavelength greater than 1nm, not enough  energy to force electrons out of orbit, excites  electrons to force creation of new covalent bonds  which affects structure of proteins and nucleic  acids.

• Ex: UV light, visible light, infrared radiation,  

radio waves  

o UV light used in microbial control by  

making thymine dimers that prevent  

replication of genetic material & cause  

cells to die. Does not penetrate well,  

disinfects air, transparent fluids, &  

surfaces

▪ Filtration: passage of fluid through a sieve that traps  particles (cells or viruses) and separates them from the  fluid.  

???? sterilize heat sensitive materials & estimate the  number of microbes in a fluid (count how many  

on filter after pouring certain amount through)  

???? HEPA filters used in labs, filter the air in the hoods  so the air in the hood is sterile and the exhaust of  the hood is filtered too

• Pressure system is used by these to control  

the air flow and control what leaves

o Chemical Microbial Control

▪ Types of Chemicals  

???? Disinfectants: do not guarantee all pathogens are  eliminated, used on inanimate objects, don’t kill  

endospores or some viruses, more concentrated  

than antiseptics and can be left on surfaces for  

longer  

???? Antiseptics: chemical used on skin or other tissue ???? Sterilants: chemicals that sterilize, denature  

proteins and DNA by cross-linking organic  

functional groups

???? Degermers: remove microbes but don’t kill them  ???? Preservatives

▪ Desirable qualities of chemicals:

???? Rapid action at a low concentration

???? Toxic to other things, not you (low toxicity)

???? Doesn’t stain, not corrosive

???? Affordable & readily available

???? Water soluble/Alcohol soluble

???? Stable  

▪ Levels of Chemical Decontamination

???? High-level Germicides: kills endospores, use on  something that needs to be sterile but can’t be  heated

???? Intermediate-level: Doesn’t kill endospores, will  kill fungal spores (tuberculosis, virus), disinfects  not sterilize, good for things that come into  

contact with mucous membrane  

???? Low-level: will only kill vegetative cells & normal  fungal cells not spores, ok if just touching but not  used for things that touch mucous membrane &  are invasive

▪ Factors that Affect Germicidal Activity of Chemicals  ???? Nature of material being treated – some things  can’t handle certain chemicals  

???? Degree of contamination – where things are going  is important, are they going to be in contact with  the mucous membrane etc.

???? Time of Exposure – Ex: Alcohol sanitizer needs to  be in contact with your hands for 10 seconds,  time microbes are exposed to chemicals affects  how effective the chemical is.  

???? Strength and Chemical Action of the Germicide  ▪ Examples of Chemicals

???? Halogens  

• Chlorine: Cl2, hypochlorites (Chlorine  

Bleach), chloramines (safer than chlorine),  

one of the most highly used  

o Method: denatures proteins by  

disrupting disulfide bonds  

o Intermediate Level

o Unstable in sunlight, inactivated by  

organic matter – if something is super

dirty it might not be as effective  

against it

o Use: decontaminate water, sewage,

wastewater, inanimate objects

• Iodine: I2, iodophors (betadine)

o Method: interferes with disulfide  

bonds of proteins

o Intermediate Level  

o Use: milder medical and dental  

degerming agents, disinfectants,  

ointments  

o *Staining is a downside

???? Phenolics

• Method: disrupts cell walls & membranes,  precipitates proteins  

o At high concentrations – disrupts cell  walls and proteins

o At low concentrations – disrupts  

critical enzyme systems  

• Low to intermediate-level – Bactericidal,  fungicidal, virucidal, not sporicidal (Strong antimicrobial but not sporocidal)  

• Because of high toxicity don’t use them as  antiseptics anymore

• Ex: Lysol, Triclosan – antibacterial additive  to soaps  

• Use: in lab use phenols to remove proteins  from preps  

• Lister used carboxylic acid (a types of  phenol) to clean skin but was really harsh  skin

???? Chlorohexidine – used instead of phenols, low  toxicity

• Method: surfactant & protein denaturant  w/broad microbicidal properties

• Low to intermediate level – good against  bacteria, variable against viruses & fungi,  Not good on endospores

• Use: skin degerming agents for  

preoperative scrubs, skin cleaning & burns  • Ex: Hibiclens & Hibitane (used to clean skin)  ???? Alcohols

• Method: acts as surfactants dissolving  membrane lipids & coagulating proteins of  vegetative bacterial cells & fungi  

• Intermediate Level – No impact on  

endospores, does impact fungal spores

• Ex: Ethyl, isopropyl in solutions of 50-95% - 70% is more effective than 100% because  100% is dehydrating.  

???? Hydrogen Peroxide

• Method: Produces highly reactive hydroxyl free radicals which damage protein & DNA  while decomposing to oxygen gas – toxic to  anaerobes

• At low concentrations: Antiseptic

• At high concentrations: Sporicidal  

???? Aldehydes

• Method: Glutaraldehyde & formaldehyde kill  by alkylating protein & DNA  

• High level – Glutaraldehyde in 2% solution  (Cidex) is used as a sterilant for heat  

sensitive instruments  

o 2nd chemical to use as a sterilant

• Intermediate level – Formaldehyde is a  disinfectant & preservative, limited use due  to toxicity, works fast

o Formalin – 37% aqueous solution  

???? Gases and Aerosols

• Method: strong alkylating agents

• High Level

• Use: sterilize & disinfect plastics &  

prepackaged devices, foods  

o Used in a chamber w/temperature,  

pressure & oxygen controlled  

o 19-3 hours are needed to aerate,

sterile air for hours after  

o gas is explosive & can harm our  

bodies & cause cancer  

• Ex: ethylene oxide and propylene oxide  ???? Detergents and Soaps  

• Method: quaternary ammonia compounds  (quats) work as surfactants (polar  

molecules that have hydrophobic and  

hydrophilic parts), change some fungi &  bacteria membrane permeability

• Very low level  

• Use: soaps are a method of degerming,  mechanically remove soil & grease that  contains microbes  

o Good against viruses = Anti-viral

o Most common in Bactene

o Non-germicidal soaps – cause  

inhabitant microbes to be pulled out  

of skin and after days of scrub water  

sitting the number of microbes are  

higher with these soaps (which is the  

category that antibacterial soaps are  

under) than with Germicidal Soap

o Germicidal Soaps – with prolonged  

used the number of bacteria  

decreases.  

???? Heavy Metals

• Method: low concentrations of silver and  mercury solutions kill vegetative cells by  inactivating proteins

o Oligodynamic action: bind to the final  

groups of proteins to inactivate them  

o Have antimicrobial properties

• Low level  

• Ex: Merthiolate, silver nitrate, silver  

???? Dyes as Antimicrobial Agents

• Method: aniline dye very active against  

gram-positive species of bacteria and  

various fungi  

• Low level, narrow spectrum of activity

• Use: antisepsis & treat wounds

???? Acids & Alkalis

• Low level

• Organic Acids: prevent spore germination,  

bacterial growth, fungal growth

• Acetic Acid: inhibits bacterial growth

• Propionic Acid: slows molds – this is the  

thing that propionic bacteria makes that  

gives cheese its flavor  

• Lactic Acid: prevents anaerobic bacterial  

growth – lactobacillus ferments glycogen  

and decreases pH = keeps the vagina acidic

• Benzoic Acid: inhibit yeast

Antibiotics  

• Principles of Antimicrobial Therapy  

o Give a drug to an infected person, it destroys the infective  agent without harming the host’s cells  

▪ Therapeutic Index is good when antimicrobial has an  

increased toxicity to microbes and decreased toxicity to  

us/humans

???? Therapeutic index: the ratio of the dose of the  

drug that is toxic to humans as compared to its  

minimum effective dose (Ratio= smallest effective  

dose/amount that is toxic to humans) ???? want #  

on bottom to be smaller  

• Higher index is desirable

o Antimicrobial drugs are produced naturally or synthetically  ▪ *Side Note: The mortality rate in some countries is the same as before antibiotics because of high rates of  

infant mortality due to lack of access to healthcare  

• Characteristics of an Ideal Antimicrobial  

o Selectively Toxic

o Microbicidal – kills microbes

o Relatively soluble – functions when highly dilute, makes sense  because we are made of mostly water

o Remains potent long enough – not broken down or excreted  prematurely, with penicillin this was a problem early on  because people were peeing it out  

o Doesn’t lead to resistance – this is difficult

o Complement or assists the activities of the host defenses o Active in tissues and body fluids  

o Can get to site of infection

o Affordable  

o Doesn’t negatively affect host’s health…allergies or other  infections

• Domagk – showed that red dye called prontosil could be active  against bacteria – this was the first sulfur drug

• Paul Erlich – some dyes dye the microbes and not the tissue, he  came up with an early chemotherapy for syphilis

• Terminology

o Prophylaxis: Action taken to prevent disease, with a specified  means against a specified disease

o Chemotherapeutic drug: drugs that act against diseases o Antimicrobial: any compound used to treat infectious disease,  may also function as intermediate-level disinfectant  

o Antibiotic: antimicrobial chemicals produced naturally by  microorganisms  

o Synthetic/Semi-Synthetic: Antimicrobials that are completely  synthesized in the lab

o Narrow Spectrum: Drugs that work against only a few kinds  of pathogens, target a specific cell component that is only  found in certain microbes  

o Broad Spectrum: Drugs that work against many different  kinds of pathogens, target cell parts that are common to most  pathogens (ribosomes)  

o Spectrum: range of activity of a drug  

• For antibiotics to work must work against the differences in the cells  o For example antimicrobial or antibiotics works against the  peptidoglycan in bacteria

• Antibiotics come from one genus, are natural in origin – we are not  very good at creating drugs on our own  

• Sporulation (Life cycle of streptomycin)

o Exospore ???? Spore Germination ???? Vegetative Hyphae Growth  (digs into the plate) ???? Aerial Hyphae Growth (microbe grows  up and out of the media, produces antibiotic as it grows aerial  hyphae, when it does this it breaks down substrate hyphae  for nutrients and the nutrients go into the soil) ???? Septation  (growth subdivides and twists) ???? Spores maturation

• Theory for why organisms make Antibiotics  

o Competitive advantage idea

o Sub-inhibitory levels of antimicrobials cause response to  things around it  

o Junk Mechanism  

o Evolutionary leftover mechanism ???? this is the reason we have  resistance ???? prokaryotes are the main producers of  

antibiotics, they can kill other things in the same genus, these  microbes have a mechanism of resistance ???? can’t get rid of  the resistance  

• Interactions Between Drug & Microbe

o Antimicrobial drugs should be selectively toxic – drugs should  kill or inhibit microbial cells without damaging the host tissue  ▪ When the characteristics of the infectious agent become  similar to the host cell selective toxicity becomes  

difficult to achieve = more side effects  

• Mechanisms of Drug Action

o Inhibition of Cell Wall Synthesis

▪ Β-lactams  

???? Basic Structure: beta-lactam rings

???? Mode of Action: inhibit peptidoglycan formation by  binding to the enzymes that cross-link NAM  

subunits (attacks between the NAG/NAM  

subunits). This causes the bacterial cells to have  

weakened cell walls as they grow and they are  

not resistant to osmotic pressure, as water moves  into the cell, the membrane bulges through the  

weakened part of the cell wall & the cell  

eventually lyses.  

• For something that attacks to the forming  

bonds we need something to be growing  

rapidly for this to work (aka cause cells to  

lyse)  

???? Effectiveness:

• Penicillinase or B-lactamase

???? Drugs within this group:

• Penicillins – Penicillin chrysogenum is a  

major source  

o Consists of three parts:

▪ Thiazolidine Ring

▪ Beta-lactam ring

▪ Variable side chain dictating  

microbial activity – affect the  

ability to get across the outer  

membrane  

o Subgroups and Uses of Penicillins

▪ Penicillin G and V most  

important natural forms  

▪ Drug of choice for Gram-Positive  

cocci (streptococci), some  

Gram-Negative bacteria  

(meningococci and syphilis  

spirochete)

▪ Semisynthetic Penicillins –

ampicillin, carbenicillin,  

amoxicillin broader spectra –

Gram-Negative infections  

▪ Penicillinase-resistant –

methicillin, nafcillin, cloxacillin  

(resistant to enzyme that  

breaks down penicillin)

▪ Primary problems: allergies to  

penicillin and resistant strains of  

bacteria  

• Cephalosporins: Targets the building of  peptidoglycan, 1/3 of all antibiotics  

administered

o Relatively broad spectrum, resistant  to most penicillinases, cause fewer  

allergic reactions  

o Some given orally, many parentally  o Generic Names have root – cef, ceph,  kef

o 4 Generations Exist (each group more  effective against gram-negatives than  the previous one, better dosing  

schedule and less side effects) –

become more broad range as  

progress:

▪ First Generation: cephalothin,  

cefazolin – most effective  

against gram-positive cocci and  

few gram-negative  

▪ Second Generation: cefaclor,  

cefonacid – more effective  

against gram-negative  

▪ Third Generation: cephalexin,  

ceftriaxone – broad-spectrum

activity against enteric bacteria  

w/beta-lactamases

▪ Fourth Generation: cefepime,  

widest range, both gram

negative & gram-positive  

• Carbapenems: Imipenem – broad spectrum  drug, used for infections w/aerobic &  

anaerobic pathogens, low dose,  

administered orally, few side effects  

• Monobactams: Aztreonam – narrow  

spectrum drug, used for infections by gram

negative aerobic bacilli, used by people  

allergic to penicillin

▪ Non beta-lactam Cell Wall Inhibitors  

???? Vancomycin: Narrow-spectrum, used for  

staphylococcus infections when there is resistance  to penicillin & methicillin or if patient is allergic to  penicillin, is toxic and hard to administer, use is  restricted

• Disrupts formation of G-positive cell wall by  interfering with alanine-alanine crossbridges  linking NAG subunits

• Used to treat MRSA

• Now some things are resistant to this  

???? Bacitracin: narrow-spectrum, made by strain of  Bacillus subtilis, used in topical ointments  

• Blocks NAG/NAM secretion from the  

cytoplasm

• Main ingredient in neosporin

???? Isoniazid (INH): interferes w/mycolic acid  synthesis, treats infections w/Mycobacterium  tuberculosis

• Disrupts the formation of arabinogalactan mycolic acid by mycobacteria  

• Used in a triple therapy to treat  

Tuberculosis

o Breakdown of cell membrane structure or function: cell dies  from disruption in metabolism or lysis  

▪ Cant carry out chemical rxns without intact membrane ▪ These drugs have Specificity for particular microbial  group based on differences in types of lipids in  

membranes  

▪ Polymyxins: interact w/phospholipids, cause leakage,  specifically in gram-negative bacteria

▪ Amphotericin B and Nystatin: make complexes with  sterols (ergosterol) on fungal membranes ???? disrupts  the membrane and causes lysis = leakages (fungicidal)  

???? The difference between fungal cells and your cells  are the sterols, membranes without cell walls  

have sterols to maintain rigidity.  

???? Human cells somewhat susceptible because  

cholesterol is similar to ergosterol (but don’t bind  as well)

o Inhibition of nucleic acid synthesis, structure or function ▪ Block synthesis of nucleotides, inhibits replication, stops  transcription  

▪ Chloroquine: binds and cross-links the double helix,  inhibit DNA helicases  

▪ Antiviral drugs, analogs of purines and pyrimidines  insert in viral nucleic acids, prevents replication  

▪ Rifampin: bind and inhibit the action of RNA polymerase  during synthesis of RNA from DNA, binds more readily  to prokaryotic than eukaryotic so more toxic to those.  o Drugs that Act on DNA or RNA  

▪ DNA Gyrase Inhibitors  

???? Fluoroquinolones: synthetic drugs that are active  against bacterial DNA, work by binding to DNA  

gyrase (enzyme necessary for correct coiling and  uncoiling of replicating bacterial DNA) and  

topoisomerase IV, may act against replication of  

mitochondrial DNA in some Eukaryotes.  

• Broad spectrum effectiveness

• Concerns w/overuse of quinolone drugs –

recommend careful monitoring to avoid  

ciprofloxacin-resistant bacteria

o Inhibiting Protein Synthesis – Ribosomes of euk. differ from  prok. Antimicrobics selective action against prokaryotes but  can also damage the euk mitochondria

▪ Aminoglycosides: ex. Streptomycin (treats TB),  gentamycin. These insert on sites on the 30S subunit,  cause misreading of mRNA  

▪ Tetracyclines: 4 ring structure, block attachment of  tRNA on A acceptor site & stop further synthesis, used  against lime disease, Typhus, some STD’s, has side  effects

▪ Chloramphenicol: phenol ring in structure, affects  peptide bonding, attaches to 50S subunit and prevents  peptide bond formation  

???? Broad spectrum  

???? Nitrobenzene type structure

???? Harms bone marrow

???? Used for rickettsia and chlamydia  

▪ Macrolides – Erythromycin: binds to 50S subunit  blocking proper movement of mRNA through ribosome,  synthesis stops  

???? Used prophylactically – before someone has an  infection

???? Used for penicillin resistant organisms

o Blocks key metabolic pathways  

▪ Sulfonamides & Trimethoprim block enzymes required  for tetrahydrofolate synthesis that is needed for DNA  and RNA synthesis  

???? Methods

• Competitive inhibition: the drug competes  

with normal substrate for the enzyme’s  

active site

• Synergistic Effect: drugs work better  

together than on their own

▪ *Remember that the problem with broad spectrum  antibiotics is that they kill normal microbiota too, which  can be good/helpful to us

• Agents to Treat Fungal Infections  

o Fungal cells eukaryotic, drug toxic to fungal cells also toxic to  human cells = hard to treat, bad side effects, remember  antibiotics don’t work on fungal organisms  

o Five Antifungal Groups:

▪ Macrolide polyene  

???? Amphotericin B: mimic lipids, most versatile &  

effective, topical and systemic treatments  

???? Nystatin: topical treatment and oral swishes

• Again the function of these make complexes  

w/sterols (ergosterol) on fungal membranes  

???? disrupts the membrane & causes lysis =  

leakages (fungicidal)  

▪ Griseofulvin: used for stubborn cases of dermatophyte  infections, is nephrotoxic (damaging to the kidneys)

▪ Synthetic azoles: broad spectrum, ketoconazole,  

clotrimazole, miconazole  

▪ Flucytosine: analog of cytosine, used against cutaneous  mycoses (disease of the hair, skin, and nails) or used  with Amphotericin B for systemic mycoses  

▪ Echinocandins: damages cell walls, used against  

capsofungin, these are the reason they stopped  

antifungal research because they are very good against  dominant fungal infections.

• Antiparasitic Chemotherapy

o Antimalarial drugs: quinine, chloroquinine, primaquine,  mefloquine

o Antiprotozoan drugs: metronidazole (Flagyl), quinicrine,  sulfonamides, tetracyclines

o Antihelminthic drugs: these immobilize, disintegrate, inhibit  metabolism

▪ Mebendazole, thiabendazole –broad spectrum- inhibit  function of microtubules, interfere w/the usage of  

glucose & disables them

▪ Pyrantel, piperazine – paralyze muscles  

▪ Niclosamide – destroys scolex

• Antiviral Chemotherapy

o Selective toxicity hard because of obligate intracellular  parasitic nature of viruses

o Methods

▪ Inhibition of virus entry or release (interfere with fusion  of virus to the membrane)

???? Fuzeon: blocks HIV infection

???? Amantidine: blocks influenza virus

???? Tamiflu and Relenza: stops actions of influenza  

neuramidase required to enter the cell  

▪ Block replication, transcription, or translation of viral  genetic material – Inhibition of nucleic acid synthesis

???? Nucleotide analogs  

• Acyclovir – used against herpes virus

(terminates DNA replication)

• Ribavirin – a guanine analog used against  

RSV, hemorrhagic fevers  

• AZT – thymine analog used against HIV

(HIV is an RNA based virus, if block reverse  

transcriptase, which converts RNA to DNA,  

then can’t produce viral DNA to enter the  

host DNA)  

▪ Prevent maturation of the viral particles – Inhibition of  Effective Viral Assembly and Release  

???? Protease inhibitors – used against HIV (inserts  

into HIV protease an enzyme that clips viral  

proteins into functional pieces)  

• Interferons (INF) – human-based glycoproteins, made mostly by  fibroblasts and leukocytes  

o Therapeutic benefits:

▪ Decrease healing time and complications of infections – antiviral & anticancer properties

▪ Prevents/reduces symptoms of cold & papilloma virus  

▪ Slows progress of some cancers, leukemia, &  

lymphomas

▪ Treats hepatitis C, genital warts, Kaposi’s sarcoma

Drug Resistance – an adaptive response when microbes begin to tolerate  an amount of drug that would normally kill them, because of genetic  versatility or variation (can be intrinsic or acquired)

• Acquired Resistance

o spontaneous mutations in critical chromosomes

o get new genes/sets of genes from resistance factors (R  plasmids) encoded w/drug resistance, transposons

▪ Antibiotic Resistance Transfer – based on R plasmids  

(plasmids carrying resistance genes)  

• Mechanisms of Drug Resistance  

o Mechanism 1: limiting access of the antibiotic due to  

decreased permeability to drug/increased elimination of drug  from cell – acquired/mutation

▪ Outer membrane porins 

▪ Active Efflux: resistant cells can pump the antimicrobial  out of the cell before the drug can act. Some cells are  

able to pump more than one antimicrobial from the cell.  

▪ Reduced uptake across cytoplasmic membrane  

o Mechanism 2: Drug Inactivation due to acquired enzymatic  activity – penicillinases – acquired mutation

▪ B-lactamases: enzymes break the beta-lactam rings of  penicillin & similar molecules making them inactive

???? In Penicillin the efficacy of it as an antibiotic is  

dependent on the Lactam Ring and penicillinases  

breaks that ring to make it inactive  

▪ Modifying Enzymes  

o Mechanism 3: Modification or protection of target -

acquired/mutation

▪ Resistant cells may alter the target of the drug so that  the drug either cannot bind to it or binds less effectively ???? change in drug receptors

o Mechanism 4: Change in metabolic Patterns – mutation of  enzyme  

▪ Alter the metabolic chemistry or abandon sensitive  metabolic step altogether, cell could become more  

resistant to a drug by producing more enzyme  

molecules for the metabolic pathway & reducing the  

power of the drug

▪ Trymethoprin and Sulfonamides

• Development of Resistance  

o Spontaneous Mutation

o Transfer of Resistance – 3 Methods

▪ Transduction: when a bacteriophage injects its DNA into  the host cell and degrades the host cells DNA, when  

creating new phages if one takes up part of the host  

cells DNA instead of its own it is called a transducing  

phage and when it injects its DNA into a new cell the  

old host’s DNA is incorporated into the new cells DNA.

▪ Transformation: organism takes up DNA from its  

environment, DNA fragments from lysed cells are  

accepted by the recipient cell and the genetic code is  

acquired by the recipient

▪ Conjugation: transfer of a plasmid from donor cell  

(gram-negative cell donor is a cell with a fertility  

plasmid/F+ cell) to recipient cell (cell without a fertility  plasmid/F- cell) with a direct connection (pilus – tube  where DNA passes through).

• Factors that Contribute to Resistance  

o Natural Selection

▪ Big populations of microbes are likely to have drug  resistant cells from prior mutations or transfer of  

plasmids, there is no growth advantage to these until  they are exposed to a drug.

▪ Once exposed sensitive cells die & resistant cells survive

▪ Population becomes resistant from selective  

pressure/natural selection

▪ Indiscriminate use of antimicrobials worldwide has led  to many drug resistant microorganisms.  

o Health care mentality

o Agriculture

o Worldwide Resistance

• How to Limit Drug Resistance

o Drug Usage

▪ Physicians – accurate diagnosis, give right drug

▪ Patients – comply w/guidelines

▪ Combined Therapy – more than 1 antibiotic works to kill  off microbes, remember dead cells can still give other  cells nucleic acids

o Drug Research

▪ Develop shorter duration, higher dose antimicrobials ▪ Find drugs whose structures are not inactivated by  enzymes & not readily circumvented

o Long-term  

▪ Educate Healthcare workers – reduce the abuse of  antibiotics

▪ Restrict use of antibiotics

▪ Stop using antibiotics in animal feed

▪ Come up w/programs that have effective therapy  

available to low income populations

▪ Vaccine where possible – there is nothing out there  supporting a link between autism & vaccines

???? Some components of vaccines work better with  

others, they are synergistic so vaccines work  

better if taken together

• Interactions Between Drug & Host

o ~5% of everyone taking antimicrobials will experience serious  adverse reaction/side effects  

o Major Side Effects:

▪ Damage to tissue because of drug toxicity

???? If use Tetracycline when child is developing =  

tooth discoloration

???? Flagyl - hairy tongue is a side effect of this  

antibiotic because it kills good microbes that were  

stopping that before

▪ Allergic Reactions

▪ Disrupts balance of normal flora = superinfections  

possible (Secondary infections that are caused by  

treatment of antibiotics which are killing microbes and  opening niches)  

• What to Consider when Selecting a Drug

o Identify microorganism causing infection – restrict the use of  anything that might work against an ideal organism

▪ Identify as soon as possible

▪ Specimens should be taken before starting  

antimicrobials  

o Test microorganism’s susceptibility to drugs in vitro when  indicated

▪ Essential for bacteria that are commonly resistant  

▪ Kirby-bauer disk test: create lawn of microbe and  

inoculate with a disk of antibiotic, read the zone of  

inhibition to determine if the microbe is sensitive or  

resistant

???? Resistant: If something is resistant then we can’t  

get the antibiotic to a high enough concentration  

in the patients to safely kill the microbes

???? Intermediate: Can use in patients but will have  

side effects

???? Sensitive: a low enough concentration of the  

antibiotic kills the microbe that it could be used in  

a patient

▪ E-test diffusion test

▪ Dilution Test: conducted to find the MIC (minimum  inhibitory concentration) – the smallest concentration of  a drug that will visibly inhibit the microbes growth  

▪ Provide profile of drug sensitivity