Microbio

What is a microbe

A living organism you need a microscope to see (the general definition)— microbial cells range from millimeters (rare) to 0.2 micrometers (typical in bacteria and fungi)

viruses can be 10 times smaller—

some microbes consist of a single cell(such as bacteria)

Fungi are multicellular— why there microbes: because there is a microscopic form/aspect to their life cycle as they form spores which is how they reproduce karyogamy etc

every microbe has the capacity in its genome (genome is its genetic blue print) to reproduce its own kind—definition of microbe

Types of microbes

Various types: Cyanobacteria are microbes and can make oxygen: are 20 microns

2nd image is bacteria trying to get into stomata

3rd is eukaryotic protist: they are bigger than other

D is a bacteria

E is a mutualistic relationship: its a lichen is a fungus and symbiote

F is a virus and its very tiny compared to bacteria: can be around 50 nanometers

What is a microbe

The simple definition of a microbe leaves us with contradictions

Examples of microbes

Supersize microbial cells: Thiomargarita namibiensis: A giant sulfur eating bacterium can grow up to a millimeter wide and can be seen with the naked eye—breaks the rule that we need a microscope to see it size of fruitfly head

Microbial communities: Biofilms: Microbes living in complex coordinated communities like dental plaque or slime on a rock acting together—

bacteria are single celled microorganisms: they reproduce but they can also communicate and build communities called biofilms

can be single or multispecies biofilms and are a community of organisms collaborating together through division of labor:

produce protect matrix—some involved in metabolism providing food for biofilm: so every microbe or bacteria can participate in different aspects—form a division of labor this is called biofilm

Viruses: Mimivirus: Massive viruses rivaling small bacteria—however they can’t reproduce on their own without hijacking a host cell—infect amoebas (single-celled organism that can change its shape by stretching parts of its body outward)

Microbes include members of the three domains of life

Basically microbes are found across all three domains of life:

Bacteria and Archaea are prokaryotes with are simpler cells with no nucleus

Eukarya: Organisms with complex cells with a nucleus includes microbes like protists and microscopic fungi like yeasts along with algae

bacteria use to be grouped with archaea than we found out they’re different through a common ancestor becoming their own domain of life are still prokaryotes (archaea)

Microbial genomes are sequenced

A genome is the total genetic information in an organism’s chromosomal DNA (The DNA that does this is inside the chromosome but DNA is found everywhere)

(Basically a genome is a set of instructions or DNA with all the genetic information needing for the microbe to rebuild itself function survive and reproduce)

First method of DNA sequencing was made by Fred Sanger—1980 nobel prize in chemistry winner

in 1995 scientists completed first genome sequence of a cellular microbe the bacterium called Haemophilus influenzae

Metagenome: A collection of sequences taken directly from the environment

its very quick to get bacteria genome—back than you had to plate them and separate and than plot genome—hard to do not every bacteria is growable—captures everythinig there that might not be culturable

Microbes shape human history

Microbes affect food availability: They destroy crops but preserve food

Microbes preserve and create: Bread, Wine, Cheese, Chocolate

Microbial disease also changes history

Bubonic plague, smallpox in Americas, Irish Potato Famine (caused mass migration to new world to find place were they didn’t need potatoes), Covid-19

Microbial Disease Devastates Human Populations

More soldiers have died in microbial infections than of wounds in battle

British nurse Florence Nightingale discovered how significant disease was in warfare she lived from (1820 to 1910)

She founded the science of medical statistics which shows the deaths of soldiers due to various causes- she made the “polar area chart”—found more soldiers died from bacteria than from battle

Polar area chart is a circular statistical graphic looks like a pie chart but the radius changes based off the size of the data—used in Crimean war to prove infectious disease kill soldiers

Image attached is polar area chart—heres people we thought died from bullet wound but this chunk actually died from the bacteria

Discovery of Microbes

A light microscope was invented in the 1600s and the quality kept improving ( it used visible light and glass lenses to magnify things)

in the mid 1600s Robert Hooke made the first compound microscope and observed small eukaryotes

in 1676 Van Leeunwenhoek was the first to observe single cell organisms and discovered bacteria

Do microbes have parents—spontaneous generation

spontaneous generation: The theory that living creatures could arise without parents

Francesco Redi in the 1660s showed that maggots in decaying meat were the offspring of flies—disproved that maggots can just appear out of nowhere through a spontaneous generation

Lazzaro Spallanzani in the 1760s showed that a sealed flask of meat broth sterilized by boiling failed to grow microbes—Showed that microbial life can’t just spontaneously generate and needs a good environment to grow

if you seal flask after boiling microbes to death, no more will get in or spawn out of no where

Louis Pasteur in the 1860s

He discovered the microbial basis of fermentation

He devised swan-neck flasks

Showed that after boiling the contents remain free of microbial growth despite access to air

the father of modern day microbiology: showed that just because theirs oxygen you won’t get microbes: got around criticism there’s no oxygen

s curve excludes dust and microbes but if you turn it microbes can enter and reproduce so its not about the oxygen but microbes reproducing

Germ theory of Disease by Pasteur continued

Pasteur’s theory was that transmission of germs causes disease

What all scientific Theories do

Explain many known observations: For example transmission of rabies

Provide framework for understanding: Where do diseases come from

Can be tested further: Do germs cause anthrax

A scientific theory is NOT a guess its based on facts

we didn’t know why we got sick at first so this helps us understand and break the transmission cycle

Medical Microbiology

Germ theory of disease: Many diseases are caused by microbes

Robert Koch who is a German physician is the founder of the scientific method of microbiology

He applied his methods to numerous lethal diseases around the world

one of the founders of scientific method so we use his method to classify diseases

growth of microbes in pure culture

Working with Anthrax, Koch demonstrated an important principle of epidemiology: The chain of infection or transmission of a disease

To prove a particular bacterium caused a specific disease, pure culture of microorganisms were needed—we needed a matrix to spread and grow different microbes but we needed a way to get broth into it to grow them

So basically scientists figured out how to grow microbes by themselves in lab as pure cultures

People who figured out how to get that broth and oxygen in to grow microbes

Angelina and Walther Hesse: Figured how to use agar to make a solid growth medium instead of just a liquid broth- helps separate different types of microbes

found out awther is best to use to grow microbes

Julius Petri: Invented the double dish container: Called the petri dish

basically we needed to learn how to grow microbes ourself

Koch’s postulates

Criteria for establishing a causative link between an infectious agent and a disease

1.Microbe is always present in a diseased host and absent in a healthy host

2. The microbe is grown in a pure culture and No other microbe is present

3. Introduce pure microbe into healthy host: Individual becomes sick

4. Same microbe reisolated from now sick individual (basically catch it again from sick host)

koch’s postulates defining the causative agent of a disease

This is an example of Koch’s postulate

1. First the healthy rabbit has no microbes for the disease but found in the sick rabbit that has the disease

2. Scientists take microbe from sick rabbit grow it by itself in petri dish

3. They inject the pure microbe into a healthy rabbit and it gets sick with the exact same disease

4. They take a sample from the newly sick rabbit and isolate the same exact strain of microbe from it—which shows the microbe caused the illness

Immunization prevents Disease

In the eighteenth century, smallpox infected a large fraction of the European population

Mary montagu introduced smallpox inoculation in Europe 1717

Edward Jenner than infected patients on purpose with matter from cowpox lesions— practice of cowpox inoculations was called vaccination (Vacca meant cow)

inoculation and vaccinations are almost the same: Innocculation is introducing infectious material to a healthy person to make them immune

vaccination is the type of inoculation made by edward jenner using cowpox to protect people against small pox

Small pox vaccination

Different people got vaccinated

Immunization prevents Disease

Louis Pasteur developed the first vaccines based on attenuated (weakened) strains

Fowl cholera and rabies were used

Immunization: It is the stimulation of an immune response by deliberate inoculation with an attenuated pathogen

Antiseptics

In 1847 Ignaz Semmelweis ordered doctors to wash their hands with chlorine which is an antiseptic agent: This caused mortality rates to fall

In 1865 Joseph Lister developed carbolic acid to treat wounds and clean surgical instruments

In the twentieth century, aseptic surgery was developed

Environments completely microbe-free

Antibiotics

Alexander Fleming discovered that penicillium mold generated a substance that kills bacteria in 1929

In 1941 Howard Florey and Ernst Chain purified Penicillin: The first commercial antibiotic to save human lives

The discovery of viruses

In 1892 Dmitri Ivanovsky studied the tobacco mosaic disease

The agent of transmission could pass through a porcelain filter that blocked all know microbes

Martinus Beijernick: The agent of tobacco mosaic disease is not a bacterium, it passes through a filter that retains bacteria

The filterable agent was purified and crystallized by Wendell Stanley (the scientist):

This proved it was Tobacco mosaic virus (TMV)

Tobacco mosaic virus

Capsid proteins are protein building blocks that wrap around and protect a virus’s genetic material

Microbial ecology

Microbes cycle the many minerals essential for all life, including the global N2 and much of the O2 of the world

less than 0.1% of microbial species can be cultured in the lab

The remainder make up the majority of Earth’s entire biosphere

Only the outer skin of Earth supports complex multicellular life

Microbes Support Natural Ecosystems

Sergei Winogradsky (1856–1953) A Russian scientist who was among the first to study microbes in natural habitats

Discovered lithotrophs - organisms that eat inorganic materials like rocks instead of organic food

Developed enrichment cultures - grow microbes by giving nutrients

Built the Winogradsky column - Sealed glass column naturally seperating and growing layers of different microbes with different metabolisms based on Light, oxygen and nutrients.

A wetland model ecosystem containing regions of enrichment for microbes of diverse metabolism

The winogradsky coloum

Cyanobacteria at the type, than purple,green sulfur bacteria. Sulfate reducing bacteria at bottom.

Geochemical cycling

This is showing the global nitrogen cycle

The problem is N2 makes up most air but plants and animals can’t use it unless converted to NH4+ and NO3- by bacteria

Winogradsky discovered bacteria drive geochemical cycling

Nitrogen fixation: Soil bacteria and bacterial symbionts living on plant roots (like legumes) convert atmospheric nitrogen gas into ammonium NH4+

Nitrification & Transformation: Other groups of bacteria convert ammonium into nitrite NO2- and then into nitrate NO3-

A way to remember: Nitrification is when you add oxygen to the mix

Plants absorb these nitrogen compounds from soil to grow and animals eat the plants driving the food web

Industrial fixation: Humans manufacture commercial fertilizers to add reactive nitrogen into the soil.

Bacterial Endosymbiosis with Plants and Animals

Endosymbionts are microbes livings symbiotically inside a larger organism

Rhizobia are enodymbiotic bacteria that induce roots of legumes (like beans or peas) to form special nodules to facilitate bacterial nitrogen fixation

(Nitrogen fixation happens inside nodules) and plant provides nutrients to bacteria

Endosymbiotic microbes also make essential nutritional contributions to host animals that host can’t produce on own

Colonic bacteria such as Escherichia coli and Bacteroides species grow as biofilm and help digest food synthesize vitamins and protect against harmful pathogens

Endosymbiont Theory-Lynn Margulis

Explored how Eukaryotes arose

Their DNA is similar to archaea’s

Mitochondrial, chloroplast DNA: Similar to bacterial DNA

Led to endosymbiont theory

Mitochondria use to be the bacteria

Chloroplasts were the cyanobacteria

they were infected or eaten by other species

ended up living together inside through endosymbiosis

Eukaryotes Evolved through Endosymbiosis

The five kingdom system was modified dramatically by Lynn Margulis

She said eukaryotic organelles such as mitochondria and chloroplasts:

evolved through endosymbiosis from

prokaryotic cells that were engulfed by pre-eukaryotes

so basically the prokaryotic cells got eaten by pre eukaryotes leading to eukaryotic organelles evolving

Highly controversial endosymbiosis theory

because it implied polyphyletic ancestry of living species and not the long held assumption that:

species evolve only by divergence from a common ancestor (monophyletic ancestry)

The microbial Family tree

Archaea are not bacteria

They have a similar size and shape but very different biochemistry

For example:

Different membranes

Archaeal ribosomes are similar to eukaryotic ribosomes: More similar to eukaryotic than bacterial ribosomes

Many archaea live in harsh environments

16s rRNA gene sequence

is found in all creatures including Archaea

Archaea are a separate domain: Uses 16S rRNA gene as molecular clock to map out microbial family trees:

Shows archaea are separate domain of life

Archaea Differ from Bacteria and Eukaryotes

Carl Woese was studying recently discovered prokaryotes that:

- Live in hot springs

- Produce methane

Analysis of the 16S rRNA revealed that these prokaryotes were a distinct form of life - He called them archaea

Cell Biology Techniques

Electron microscopy: The observations of cell composition you see eukaryotic organelles and membrane

Ultracentrifuge: Separation of cell components

Study of biochemistry of organelles

n Fluorescence microscopy: Identification of cell components and Subcellular location of individual proteins

Genetics and DNA Revolution

Molecular genetics depends on bacteria especially E.Coli

Concept of “ gene ” proposed for bacteria

Rosalind Franklin- discovered the double helix along with James Watson, Francis Crick, Maurice Wilkins

Genetic code, Transcription, translation: Show us how how life works at the molecular level

Tools used for genetic Engineering

Restriction enzymes: Molecular scissors used to cut DNA at specific sequences

Recombinant DNA: Combining DNA from different sources

Cloning: Making identical copies of genes or organisms.

PCR reaction: A technique to rapidly copy and amplify DNA segments.

E. coli has best understood genome

n Molecular genetics depends on bacteria ¨ Concept of “ gene ” proposed for bacteria

¨ DNA structure- Rosalind Franklin- discovered the double helix; James Watson, Francis Crick, Maurice Wilkins

¨ Genetic code

¨ Transcription, translation

¨ Restriction enzymes

¨ Recombinant DNA ¨ Cloning ¨ PCR reaction ¨ E. coli has best understood genome

Start of lectures 3 and 4

3 and 4

Structural differences between Prokaryotes and Eukaryotes

Prokaryotes have no defined organelles

¨DNA is not bounded by a membrane

Prokaryotes are generally much smaller in size

¨Equivalent to a mitochondrion in eukaryotes

Eukaryotes are the biggest and have a nucleus, than prokaryotic cell and virus is the smallest

Eukaryotes have order and function to each part

DNA not bounded by nuclear membrane in prokaryote like the eukaryotic has

Cell structure

In eukaryote there is membrane around chromosomes

Prokaryotes don’t have this

Ribosomes are out in the cytoplasm in prokaryote and have dna genes coding for messenger RNA allowing transcription and translation to change very fast

Know prokaryote organelles have no membrane bound organelles, condensed form nucleoid and parts. Condensed because theres a single circular chromosome, DNA is bunched up and tightly packed

Prokaryote Size

Prokaryote Sizes Microbiology:

Small size of Prokaryotes affects biological properties

Large surface area/volume ratio means more rapid transport rates

¨ Excretion of waste products

¨ Import of nutrient

Bacteria like cyanobacteria are big and live in ocean and are photosynthetic

large surface area means large transport rate

Bacteria Cell Shapes

Many have rod shape (Bacilli) so you can’t identify them just from how they look

can come in

Cocci = spheres – in bunches

Bacilli = sticks, rods – alone

Vibrios = bent rods

Spiral

Irregular

in chains

Morphogenesis: change in shape

Monomorphic: one shape; observed in most pure cultures of bacteria

• Pleomorphic: multiple shapes

- Changes during growth

Cells become smaller as they age -

Response to environmental cues

Sporulation (nutrient limitation)

Sometimes they can change shape if they are under nutrient or environment stress

The Bacterial Cell

Cytoplasm surrounded by cell envelope

¨Cytoplasm contains DNA in the nucleoid- a system of looped coils of DNA

heres the chromosome and most bacterial genomes are circular

surrounded to cell envelope which are specific to bacteria as only bacteria have cell envelopes which helps with making antibiotics targeting this

The Bacterial Nucleoid

Single loop of double-stranded DNA- most bacteria have a circular chromosome made of one continuous double-stranded DNA molecule

¨ Single molecule of DNA ¨

~4x10^6 bp (base pairs) in most bacteria comparadly humans only have 3 million so they have far smaller and more compact genomes

¨ Compacted via supercoiling (instead of using histone proteins bacteria twist and coil the DNA tightly on itself in a compact shape)

Attached to cell envelope

¨ No membrane separates DNA from cytoplasm allowing transcription and translation to happen simultaneously

Replicates once for each cell division: Both cells get a complete clone copy of each other

Dna origin (oriC) is the start of DNA replication and guides cell division during binary fission

the two origins move toward opposite sides of the cell. helping organize the cell so that when it splits via binary fission, each daughter cell gets one complete chromosome.

• Cell envelope-

Very important to bacteria and target for antibiotics

envelope is made of different layers

we have cell membrane aka inner membrane, than cell wall which is rigid and forms its structure

Gram-Negative: Has a three-layer setup: Inner membrane than Cell wall than Outer membrane.

Gram-Positive (Not Gram-Negative): Has a two-layer setup: Inner membrane Cell wall (with no outer membrane)

if not gram negative same but no outer membrane

Comprised of the cell membrane + the cell wall + the outer membrane (if there is one)

• Keep in mind the cell envelope is different between Gram+ and Gram- bacteria

Helps us identify and classify bacteria in clinical setting when thinking of clinical solutions to infection

Cell Membranes

It is a phospholipid bilayer

it seperates cytoplasm from outer word and controls what goes into cell

and collection of membranes dictates what goes in and out through different transporters

dictated by the type of proteins that bacteria have

they can help anchor membrane to envelope and sense what’s going on

and can be transporters and channels for things like glucose to come into and waste to go out of cell

ATPase makes ATP for the cell

Made of lipid bilayer

¨ Double layer of phospholipids: two layers of fat molecules (phospholipids) arranged with their hydrophilic (water-loving) heads facing outward and hydrophobic (water-fearing) tails facing inward

Separate cytoplasm from outside world

Permeability barrier

Proteins embedded in membrane

¨ Anchor membranes to envelope They help link the membrane to other layers of the cell envelope.

¨ Sense the outside world detect changes in the external environment (like nutrients, toxins, or temperature).

¨ Transport materials into cell

Phospholipid Bilayer

Hyddrophobic inside and hydrophilic outside and it has fatty acids and glycerol

Forms general structure of biological membranes

Composed of fatty acids (hydrophobic) and glycerolphosphate (hydrophilic

Cell Transport

Alot of transport the cell allows like oxygen and water that can passively move in and out through transporters

Small or uncharged molecules (like oxygen and water) can slip right through the lipid bilayer or use unassisted channels to move along their concentration gradient (from high concentration to low concentration)

passive transport doesn’t cost ATP

some dictated by type of transporter/protein some specific to type of sugar

alot are active need ATP energy or through proton motor force and is cheap energy from electron transport chain: When the cell needs to pull in nutrients when there is already a high concentration of them inside, it has to force them against their gradient (from low to high concentration)

active transport needs energy either from ATP or PMF

Bacteria stores them in cell wall and they are a good source of energy to drive motor force of ATPsynthase

Transporters pass material into/out of cell

¨Passive transport follows gradient of material

¨Active transport of some sugars

PMF drives ATP synthase to make ATP

Pumps use energy

¨ATP or PMF ¨Move material against their gradient

Cell Transport. cont

Atp snthase is collection of proteins in cell to make atp

inexpensive biproduct of electron transport chain with protons to drive ATP chain and they have energetic potential turning motor of ATP synthase to make ATP

ATP made through ATP motor force and synthase with biproduct of glycolysis and electron transport chain

Proton pumps push protons out of cell

¨Generates proton-motive force (PMF)

• Osmotic force tries to push protons back into cell

• electrical force tries to push protons back into cell

• PMF is used to create ATP ¨ATP synthase uses PMF for energy

Bacterial Cell Walls

Rigid structural component that lies just outside the cytoplasmic membrane Enables bacteria to withstand turgor pressure due to dissolved solutes in cell

2 major types: 1)Gram positive (thick) and 2)Gram negative (thin)

The cell wall is the rigid layer on top of the membrane and helps dictate the shape and the size of bacterial cell and is specific to bacteria so others don’t have it

can withstand terminal pressure of too much salt or water coming in and maintaining osmotic pressure, etc

gram positive or negative named after scientist gram: gram negative have thin cell wall gram positive have thick cell wall made of peptidoglycan

Peptidoglycan- polymer that makes up the bacterial cell wall

• Peptidoglycan synthesis is unique to bacteria

• Makes up a large % of the cell mass (≈ 1%)

• Often a target for antibiotics (penicillin)

Peptidoglycan makes up the cell wall and is easily accessed and is a target for antibiotics

the process of building cell wall can be interrupted by penicillin which is a cell wall inhibitor which stops cell wall from being made which kills the bacteria

The Cell Wall Is a Single Molecule

The cell wall confers shape and rigidity to the cell, and helps it withstand turgor pressure (internal water pressure trying to make cell swell and explode, rigid cell wall stops cell from bursting).

• The bacterial cell wall, or the sacculus, consists of a single interlinked molecule.The cell wall is made of a repeating polymer called peptidoglycan (sugar chains cross-linked by short amino acid peptides)

You can extract a cell wall or sacculus also called and image it with electron microscope and image is sacculus

repeat polymer of peptido glycan and is a hotdog shaped cell single linked molecule and interrupting this can cause cell to lice

The Bacterial Cell Wall

• Sacculus made of peptidoglycan (murein)

• ¨Sugar chains wrapped in circles around cell

“glyco” = “sweet” The wall is built from long repeating chains of two sugar units (specifically, N-acetylglucosamine [NAG] and N-acetylmuramic acid [NAM])

¨Sugar chains linked to each other by short polymers of amino acids

• amino acid = “peptide”

peptido glycan is peptides and sugars linked together to make this long chain becoming peptidoglycan

so the wall is called peptidoglycan and its made of NAG and NAM

Monomeric units of peptidoglycan (murein) in bacteria

Peptido = peptide links

Four amino acids are linked together by peptide bonds.

Glycan (Sugar component) = NAM+ NAG disaccharide

NAG: N-Acetylglucosamine

NAM: N-Acetylmuramic acid

Monomers are made of NAG and NAM and have peptides that are tiny proteins and are amino acids linked to each other linked to sugar chain

a monomer is a single, small molecule that can chemically bond with other similar molecules to form a larger chain, which is called a polymer

Differences between Gram negative and Gram positive peptidoglycan crosslinks

Direct cross-linking

Thin layer Gram -

Peptide interbridge

Thick layer Gram +

in gram negatives We have sugar backbone and peptides and in gram negative layer is thin and linked to one another and is very close to each other giving thin layer of peptidoglycan

with gram positives we have sugarback bone and peptides but instead of being linked theres a peptide interbridge linking peptides creating thicker peptidoglycan layer

The Gram-Positive Envelope

Capsule (not all species): Called a Polysaccharide: A slippery, protective outer layer made of sugars (polysaccharides) found in some bacteria: helps them hide from immune system

S Layer (not all species): ¨Made of protein: Rigid armor like for structural support

Thick cell wall in gram positive: gives the cell high structural strength

¨9-amino acid crosslinks in peptidoglycan:Gram-positive cell walls use specific peptide cross-links (such as 9-amino acid bridges): use ¨Teichoic acids for strength in gram positive

Thin periplasm in gram positive (space between the plasma membrane and the cell wall)

Thick blue cell wall and there is a space between cell membrane and outer membrane which is were all those protons are stored in periplasm

sometimes they have capsules on outside with sugar

2 layers cell wall and membrane

Gram Negative Cell Envelope

A thin layer of peptidoglycan

Outer membrane and Inner Membrane

The Gram-Negative Envelope

Capsule (not all species) ¨Polysaccharide

Outer Membrane ¨Lipopolysaccharide: In outer leaflet only: acts like endotoxin triggering strong immune responses

Thin cell wall : 4-amino acid crosslinks in peptidoglycan

Thick periplasm

Plasma membrane

inner and outermembrane and thin cell wall and can have extra sugar decorations on outside helping build biofilms and causing disease and virolence and bacteria interactions

lipopolysaccharides which is unique to gram negatives

The difference

Link makes it thicker

in gram negative you have thin peptidoglycan layer

Lipopolysaccharide

Outer membrane made of lipids and sugar (Lipopolysaccharides)

sugar part on outside and no other organism makes it

our eukaryotic systems have evolved to think that’s foreign triggering immune response

The Gram Stain

Developed by Hans Christian Gram in 1884

The most widely-used stain in microbiology

Differential Stain: Does not stain all cells equally, Based on differences in composition of cell wall

Gram Positive: blue Gram Negative: pink

We have rod shaped bacteria from patient sample and this will decide what type of antibiotics to give

use differential stain it doesn’t stain equally based on differences of chemistry of peptidoglycan thin vs thick

it stains to gram positive cells but nothing happens to gram negative until later on in the reaction

How to stain

Get pure culture and grow it make sure its nice and not dead

1.Put some in microscope slide to see and add methanol to stick cells to surface and air dry and here they are all stuck (why cells don’t die from this: its such a short time that ethanol doesnt kill them )

2.than stain them with crystal violet stain which is super blue and pipet the stain and let it sit for one minute and is time specific now everything is blue

3.Add iodine which will stain At this point, both Gram-positive and Gram-negative cells still look purple.

4. than wash with ethanol for 20 seconds and stains are removed from gram negative cells gram positive stay dark purple. colorless cells are practically invisible under a standard light microscope so add safranin to tell

5. gram positive cells remain dark purple so add safranin and counterstain to be able to tell the difference of what’s positive and negative. positive stay purple and negative turn pink or red.

Outer Coverings

Glycocalyx: network of polysaccharides extending from cell surface, ¨ survival, attachment, virulence

Capsule: well organized glycocalyx; difficult to remove

S-layers – Gram positive bacteria ¨ layers of protein or glycoprotein ¨ Common among Archaea: basically protein armor: S-layer is a rigid, crystalline sheet made entirely of interlocking proteins or glycoproteins

Glycocalyx are sugars coating the bacteria and is sticky helping them stick to surfaces and survive

can be called glycocalyx, capsule, and gram positive bacteria have S-layers

all of these are coverings of the cell

not all have this and others can produce/not produce as it takes a lot of energy to make this layer

remember some have outer slimy coverings on outside

The Bacterial Nucleoid

Single loop of double-stranded DNA, Single molecule of DNA ¨~4x106 bp in many bacteria ¨Compacted via supercoiling

Attached to cell envelope ¨No membrane separates DNA from cytoplasm

Replicates once for each cell division

No nuclear membrane and is a condensed and organized

is bidirectional DNA replicaqtion 2 circles orginating from one

bp= base pairs

Nucleoid organization

Lice cell you disrupt DNA and a bunch of it spills out

The nucleoid forms about 50 loops of chromosome called domains

Within each domain, the DNA is supercoiled and partly compacted by DNA-binding proteins

Gene Expression

RNA Polymerase transcribes DNA to mRNA

Ribosome translates RNA to Protein

¨Processes occur simultaneously

Enabled by polymerase transcribing it into mRNA

MRNA transcribed from DNA

can happen simultaneously transcription and translations so are coupled and ribosomes can hop onto that RNA before its even transcribed

From one you can get many

RNA polymerase

In bacteria, making mRNA (transcription) and building protein (translation) happen at the same time in the same

Important: RNA Polymerase (Light Blue): Reads the DNA template and builds a growing single strand of RNA (Nascent RNA

Secondary Channel (ATP, GTP, CTP, UTP): Allows free ribonucleotides (the building blocks of RNA) to enter the enzyme to build the strand

Ribosome (Pink / Red): While the RNA strand is still being made by RNA polymerase, a ribosome latches onto it and immediately starts reading it to make a protein

30S & 50S Subunits: The two bacterial ribosomal subunits that form the functional ribosome

Transfer RNA (tRNA): Delivers incoming amino acids corresponding to the genetic code on the mRNA

Peptide Chain & Exit Channel: As amino acids link together, the growing polypeptide chain feeds out through the exit channel

Correctly Folded Protein: Once completed, the chain folds into a functional 3D protein

Lecture 4

Start

Cell Division

Cell elongates as it grows ¨ Adds new wall at cell equator

DNA replicates to make 2 chromosomes ¨ DNA replicates bidirectionally ¨ Can begin next replication before cell divides

Cell undergoes septation ¨ Usually at equator ¨ Each daughter has same shape

Binary fision turns one cell into 2 identical cells

during that time dna is tethered to cell envelope

this is were dna replication begins and his hooked by the inner cell wall (cell envelope)

theres a collection of proteins 2 of them starting dna replication (ori site )

ropel zones go around and hit termination side: cell grows and expands as origins of replication detatch slightly

each of 2 circles go into daughter cells

bacteria does this quickly and transcript and transcribe at same time

sometimes next round can start before even completing first cycle

in prokaryotes replication begins at ori sight and Enzymes pull the double-stranded DNA apart, creating two "replication forks"

each strand in parental dna used as template to build new matching strain

replisome perform replication function: (contains multiple copies of DNA polymerase (the enzyme that builds the new DNA) along with other helper enzymes, copying both directions at the same time.)

each contains 2 dna polymerase, enzymes etc

2 copies of dna origin replication attatch to envelope and move along expanding cell

2 progency helices get made

replication happens on 2 ends until the termination site: They keep copying until they meet at the exact opposite side of the circle, known as the termination site.

this causes septum to grow inwards to sides of cell constricting and sealing off called septation

inward growth eventually meets in the middle, sealing off the two halves completely. leading to 2 daughter cells

Bacterial Cytoskeleton

Shape-determining proteins

- FtsZ = forms a “Z-ring” in spherical cells

- MreB = forms a coil inside rod-shaped cells

- CreS “crescentin” = forms a polymer along the inner side of crescent-shaped bacteria

actin contributes to cytoskeleton

recent discoveries show they do have cytoskeletons improtant to cell processes

different proteins determine shape

Ftsz forms one ring along center of cell and makes spherical cells so whole cell is spherice

MReB forms a coil inside rod shaped cells and runs along the length of cell and is rodshaped so MreB and FtsZ combined make a rod shaped

CreS crescentin makes a crescent shape and is only on one side of cell causing bend so they have ftsz mreb and crescentin present here

Specialized Structures-Cell Attachment

Fimbriae and pili attach cells to surfaces ¨ Thin filaments of protein “pilin” that extend from cell surface

¨ Conjugation – sex pili ¨ Essential for bacterial pathogenicity

¨ Receptors for viruses

• Stalks attach cells to surfaces – Extension of cell cytoplasm

jewlery of what bacteria were

has to do with attachment helping bacteria to attach to cells

most bacteria attatch to some bacteria in their life cycle

fimbriae are hairlike structures on ends of cell

fimbriae are shorter and are sticky and made of pillin

specialized pili going beyond attatchement and transfer of dna between bacteria is conjugation which is their sex pili

Dna is transfered from one to another mediating dna transfer-horizontal dna transfer

how bacteria exchange genes very quickly can go from brother to brother etc and not just from mother to son

bacteria can do that when ever they want and can pick up genes from environment or from other bacteria which could be related to antibiotics etc gives them fitness edge

this all happens through sex pili

specialized structure are essential for bacterial pathogenicity

stalk enables bacterium to attatch to surfaces with strong hydrodynamic flow such as rivers and can attatch to a rock for example-strongest discovered moleculare glu

Flagellar Ultrastructure:

3 main components:

-Filament: flagellin

-Hook: hook protein

-Basal Body: consists of rings

short things enable attatchment and they use flagellin to swim to were they want to be

prokaryotes have flagellin but different structure than eukaryotes

flagellum is made of flagellin and all this is important in this

the hook protein helps with torque of system

basal body has rings that turn inside and rotate flagellum filament

all powered by proton motor force which is where the bacteria harbor protons in periplasmis space and use them for energy to create atp

or energy to drive motor

inexpensive energy lying around and use it to power flagellum motor

Flagella and Motility

Monotrichous (Polar) Flagella ¨Single flagellum attached to one end n Lophotrichous Flagella ¨A group (tuft) of flagella is attached to one or both ends n Peritrichous Flagella ¨Flagella are arranged on all sides

were are focusing on peritrichous and ecoli have this

Movement of Polar Flagella

n Reversible ¨ CCW rotation leads to forward swimming ¨ CW leads to reversal of direction n Unidirectional ¨ CW leads to forward movement ¨ Cell stops, reorients ¨ CW rotation commences, leading to forward movement

flagella aqueously enable movement

some flagella are reversible in the way they rotate so they can go forward or backwards

if a reversible is going ccw its forward

if clockwise its going backwards

some flagella can only rotate in one direction so clockwise if cell wants to reorient and move backwards it can stop rotating and stop and reorient itself and thats called a biased random walk

polar i unidirectional flagella

can send signal to flagella when it sees food it needs to go to or if theres a toxin and it needs to stop rotating

the flagellum is enabled by sensors coupled to flagella through signal conduction cascade

Movement of Peritrichous Flagella

n Counterclockwise (CCW) rotation of multiple flagella leads to formation of a bundle ¨ Smooth swimming—more or less in a straight line ¨ Swim forward n Clockwise (CW) rotation leads to disaggregation ¨ Tumble—allows reorientation

how ecoli move

lots of flagella are directing body

they form a braid and its bundeld enable ccw rotation

if they need to stop moving the tumble flagella are pushed apart and bundle is disssaggregated and rotation of flaggella is stopped as they are pushed apart

flagella is just floating around until ready to go forward again and than they bundle form

called biased random walk and is based on chemotactic receptors enabling bacteria to sense theres something like a vitamin or amino acid they want

enables them to counterclockwise rotate with bundle

if off course they disbundle and float around but are biased to that attractant

they will remake bundle when they are in position and go forward

so when they disbundle they float until they are in position to go to attractant

Chemotaxis

n Runs + tumbles cause “random walk” ¨Receptors detect attractant concentrations n Sugars, amino acids ¨Attractant concentration increases and prolongs run n Biased random walk n Net movement of bacteria toward attractants

your taxing or moving to a chemical attractant or away chemotaxis receptors enable movement to attractant

Chemotaxis

n The ability of organisms to move towards or away from specific chemicals n Random cell movement in absence of attractant n Attractant causes directed movement up-gradient towards the attractant – prolonged runs

Motility and Chemotaxis

n Movement in response to a chemical gradient n Attractants: Cells move up a gradient of these molecules (nutrients) ¨ Suppress tumbles, so runs become longer n Repellents: Cells move down a gradient of these molecules (wastes or toxic chemicals)

can also run away from something like antibiotic using same bundles and disaggregation tactic

Flagellum motor video notes

biomechanical motor is in flagellur motor powering flagellum in prokaryotes

hydrogen ions/protons in outer layer in high concentration and low protons on inside

energy can be made using this high protons on outside low protons on inside used as gradient by motor to turn itself

protein comes and binds to motor making it turn clockwise to stop

lectures 5 and 6

Bacterial Culture, Growth,

and Developmen

Introduction

The adage “To eat well is to live well” is

as true for microbes as it is for humans.

n Over eons, bacteria have evolved

ingenious strategies to find, acquire, and

metabolize a wide assortment of food

sources.

n - This owes to the remarkable plasticity

of microbial genomes.

Microbiology: An Evolving Science

© 2009 W. W. Norton & Company, Inc. Copyri

they have ability to change genomes very quickly allowing microbes to inhabit every aspect in the planet

Importance of Bacteria to Our Lives

Greatest metabolic diversity of all life forms: major agents in the recycling of C, N, S, Fe, and other elements in the environment n Provide the genetic source and cellular scaffold for recombinant DNA biotechnology

microbes have the greatest metabolic diversity out of every lifeform

major recycling component in planet for nutrients and plants and animals can use them

Bacteria are scafold for DNA microtechnolgy which means taking gene and making it generate a protein, etc based on micrbiology and biochem we have learned

Microbial Nutrition

Essential nutrients are those that must be supplied from the environment. § Macronutrients - Major elements in cell macromolecules - C, O, H, N, P, S -

Cations necessary for protein function § Micronutrients - Trace elements necessary for enzyme function

Macro nutrients are carbon based and you need a lot of it

micronutrients you need in smaller amounts for nutrients

How Microbes Build Biomass

All of Earth’s life-forms are based on carbon, which they acquire in different ways.

- Autotrophs fix carbon dioxide and assemble into organic molecules (mainly sugars).

- Heterotrophs use preformed organic molecules

If you expose bacteria to alot of copper they will die so you can spray copper on plant itll kill bacteria

all of earths lifeforms are carbon based and get it in different ways. Autotrophs fix carbon from atmosphere that’s not usable and convert it to glucose

bacteria can also use energy from inorganic minirals to drive autotrophy: taking co2 from environment converting it into usable enrgy like sugar

heterotrophs acquire it from premade organic forms of energy

How Microbes Obtain Energy

§ All organisms require an energy source. ̵

Phototrophs obtain energy from chemical reactions triggered by light. ̵ Chemotrophs obtain energy from oxidationreduction reactions. ̵

Lithotrophs use inorganic molecules as a source of electrons.

̵ Organotrophs use organic molecules.

they convert carbon back to energy: photo trophs get energy from sun or light

chemotrophs get it from oxidation reduction reactions in envrionment

lithotrophs use inorganic molecules as source of energy and break down inorganic compounds

Organic trophs use organic molecules as energy

How Microbes Obtain Energy Part 2

§ In short, microbes are classified based on their carbon and energy acquisition as follows: - Autotrophs - Photoautotrophs - Chemolithoautotrophs (or lithotrophs) - Heterotrophs - Photoheterotrophs - Chemoheterotrophs (or organotrophs)

classified by how they get carbon and their energy:

autrophs can be photoautotrophs or chemolithoauto trophs

lithotrophs take inorganic compounds sas energy and degrade them into energy and use that to fix CO2

Hetertrophs can be photoheterotrophs or chemoheterotrophs: Can also have sunlight as energy to help them break stuff down and can also use redux reactions as metabolisms to

these metabolisms allow them to colonize extreme and non extreme environment

The Carbon Cycle

Driven by glucose production becoming polymerase to polysachhrides that heterotrophs break down back into glucose

you need autrophs to take co2 convert to organic and hetrrotrophs break it down and produce co2 as biproduct going back to autotrophs

Energy Is Stored for Later Use

A membrane potential is generated when chemical energy is used to pump protons outside of the cell. § The H+ gradient plus the charge difference form an electrochemical potential called the proton motive force. § The potential energy stored can be used to transport nutrients, drive flagellar rotation, and make ATP by the F1FO ATP synthase

energy can be used for later use and they do these by creating membrane potential that is storage of protons in periplasm stored in gradient creating electrochemical potential called proton motor force

when you have more of something going down its gradient to create energy to convert electrochemical energy through proton motor force

bacteria funnels energy through and make ATP synthase: funnel through damn to create electricity and energy

they store protons and funnel them through protein complexes like ATP synthase produce atp through phosphorisation

Nutrient Uptake

n Passive diffusion ¨Some gases pass freely through membranes n O2, CO2 ¨Follows gradient of material n Forms of Transporters ¨Facilitated diffusion n Transporters pass material into/out of cell n Follows gradient of material n Does not require energy ¨Active Transport n Requires energy

gases and things they need to live can pass through membranes and depends on gradient

Different forms of transporters dictate where proton can live and what membrane can bring in and send out

transporters can be through facilitated diffusion and follow gradient of other compound facilitated diffusion means its faccilated by something but doenst use energy like phosphate

Active transport is higher level and are dedicated transport but need energy

Facilitated diffusion

n Cannot move a molecule against a concentration gradient n Does not use cellular energy, relies on diffusion across a gradient n Example-glycerol transporter

cannot move molecule against a concentration gradient

does not use cellular energy and relies on diffusion

Three Classes of Active Transport Systems

Simple transport: Simplest form driven by protons can

Nutrient Uptake—Simple Active Transport

n Symport and Antiport ¨ Gradient of one molecule transports another ¨ Transports material against its gradient - In symport, the two molecules travel in the same direction. - In antiport, the actively transported molecule moves in the direction opposite to the driving ion. Symport: Gradient of pumps in same direction Antiport: Gradient of pumps in opposite direction

symport 2 molecules go in same direction and antiport mean they go in opposite direction through same transport

source of energy for simple active transport from proton transport chain

Nutrient Uptake— Active Transport

n ABC Transporters n ATP-binding cassette superfamily ¨Use ATP energy to pass material into cell ¨Transport material against gradient

solute binding protein binds to solute binds to transporter causing change using ATP energy transport things against gradient requires a lot of energy

Nutrient Uptake—Active Transport 2

n Phosphotransferase System (PTS) ¨ Uses energy from phosphoenolpyruvate (PEP) to pass material into cell ¨ Modifies material as it enters cell n Gradient is maintained, pushing material into cell glucose enters cell and is phosphorylated. As a result, gradient of pushes more glucose inside. (glucose-6-phosphate) cannot pass out of cell

It capitalizes on fact it can maintain concentration gradient and uses energy

uses phoptransport system it doesn’t use ATP it uses phosphate and phosphoenolpyruvate

glucose becomes glucose 6 phosphate and cannot pass out of cell so inside of cell is different than outer glucose so it can’t leave

Nutrient Uptake—Active Transport 3

PEP transfers through a series of proteins

mannitol has a different enzyme 2 complex they pass phosphate through enzyme 1 and HDR than to enzyme to becoming glucose 6 phosphate

Video:Phosphotransport system use PEP energty to transport glucose into cell PEP is intermediate of glycolosys with high energy phosphate group

transfered to enzyme 1 transfered to HPR histidine protien than transfered to ENZyme 2: A to 2B to glucose from 2C

glucose converted into glucose 6 phosphate included mannos and mannitol can be transported

enzyme 2 is substrate specific

mannitol and mannos use unique enzyme 2 to transport Rewatch take notes for better understanding

each has own 2c complex

Culturing and Counting Bacteria

Microbes in nature exist in complex, multispecies communities, but for detailed studies they must be grown separately in pure culture. § After 120 years of trying, we have succeeded in culturing less than 1% of the microorganisms around us. - The vast majority has yet to be tamed.

We culture bacteria and manipulate them to isolate and study them

we can only culture 1 percent of all bacteria

sometimes they need things from other microbes, etc

we don’t know enough to grow all types of bacteria

Bacteria Are Grown in Culture Media

Bacteria are grown in culture media, which are of two main types: 1. Liquid or broth - Useful for studying the growth characteristics of a pure culture 2. Solid (usually gelled with agar) - Useful for trying to separate mixed cultures from clinical specimens or natural environments

passing something through ecoli

media can be liquid or broth we know what’s in them and use them to grow pure culture

in solid media we like to use that to separate bacteria and get them into pure culture

Bacteria Are Grown in Culture Media 2

We can be interested in a specific type of bacteria so we have to get that one away to study it in pure culture

Dilution Streaking and Spread Plates

How to get pure culture

§ Pure colonies are isolated via two main techniques: 1. Dilution streaking - A loop is dragged across the surface of an agar plate. 2. Spread plate - Tenfold serial dilutions are performed on a liquid culture. - A small amount of each dilution is then plated.

diluting them across solid matrix to grow them into new colonies we can consider pure could have been a mixed colony etc, so you have to do all steps

subculture trhough streaking or spreading into another plate

Dilution Streaking and Spread Plates 2

dilution Streaking is using metal with loop pass through plane to sterilize it in Bunsen burner

than go back through colony take little peace and streak it unto plate by moving loop back and forth across petri dish if you let it grow itll look like a smear can’t see individual colonies because theyre so concentrated so its not useful

to fix we dilute them so take loop put it back to flame to sterilize and pass it once or twice through first pass to pick up a few cells spreading them across plate

2nd pass we start to get single colonies but they are close and touching

do it 3rd time, sterilize in flame, pass 1 or 2 more times and spread on clean part of dish again so 3 steps

we finally get single colonies so we put Petri dish in incubator and we see individual colonies

the rule of thumb is that one colony represents being from a single cell spread of plate they are clonal so every cell grew from one cell so they are all brothers

all came from same cell from binary fission

some exceptions for bacteria that live in chains or clumps so separating is tricky because they are colony forming unit can be single cell or group of cells

Streaking video

inoculating loop is sterilized under flame which collects drop of liquid from bacteria culture

drop of liquid streaked across petri dish and organisms rub off loop: is sterilized and spotted again to be spreaded in another spot of dish

single cell multiples in colonies and pure culture obtained by touching single colony with loop

that colony loop dipped in the broth again

Dilution Streaking and Spread Plates 3

tells you number of living cells experiment biased towards living cells

take original flask, setup series of tubes with blank media and put 9mls take 1mL out of flask to create 1 to 10 dilution over and over so multiply by 10 every time

so 10 to 100 to 1000 to 1:100,000 to 1:1 million

don’t want two little or too many colonies between 10 and 50 is reliable range which is something you can count

311 is too many because you might miss some while counting we plated out a tenth of a milliliter from tube

so we have flask we take out 1mL than mix it around in other flask and mix out out over and over now we have a lot of tubes

different amounts and dilution gives you different amounts 1 to 10^5 is the ratio to use here it gets you 27 which is ideal

Spread plating again

Dilution streaking vs spread plating

Dilution streaking is a metal or plastic loop held against agar and you streak the surface

spread plating is moving a colony or liquid from one place to another so we are trying to spread liquid around the plate

you can use a slightly smaller volume like 100 microliters: use L shaped machine that spreads the liquid for you so it spreads sit instantly

we wanna know how much bacteria is in flask

when you start: put 9 ml of liquid media into tubes or 10 ml this becomes 10^-1 dilution

do that again 10^-2,10^-3, etc and you are diluting out bacteria

than put 100 microliters on petri dishes and seperate colonies we want a number 10 to 50, we got 27

so do 27 to 10%5 times 10

on exam: Info given certain dilution number of colonies you plated it out for X microliters so do math so you have X number